CA2481372A1

CA2481372A1 - Method to inhibit cell growth using oligonucleotides

Info

Publication number: CA2481372A1
Application number: CA002481372A
Authority: CA
Inventors: Mark S. Eller; Mina Yaar; Barbara A. Gilchrest
Original assignee: Individual
Current assignee: Boston University
Priority date: 2002-04-12
Filing date: 2003-04-11
Publication date: 2003-10-23
Also published as: EP2210956A3; CN1659288B; US20100222419A1; KR20050007455A; MXPA04010000A; AU2003262191A1; EP2210956A2; JP2010031051A; CN1659288A; US20030032610A1; NO20044503L; IL164314A0; WO2003087411A8; IL164314A; BR0309203A; EP1499746A1; US8183222B2; US20100249218A1; NZ535702A; IL202597A

Abstract

Described are methods for treating hyperproliferative disorders, including cancers, by administering to the affected mammal (e.g.,human) an effective amount of a composition comprising pTT or a composition comprising one or mo re oligonucleotides which share at least 50% nucleotide sequence identity with the human telomere overhang repeat. Methods of treatment or prevention of hyperproliferative diseases or pre-cancerous conditions affecting epithelial cells, such as psoriasis, atopic dermatitis, or hyperproliferative diseases of other epithelia and methods for reducing photoaging, or oxidative stress or for prophylaxis against or reduction in the likelihood of the development of skin cancer, are also disclosed.

Description

METHOD TO INHIBIT CELL GROWTH USING OLIGONUCLEOTIDES
RELATED APPLICATIONS
This application is a continuation-in-part of Application No. 10/122,630 filed April 12, 2002, which is a continuation-in-part of International Application No.
PCT/LJSOl/10162, which designated the United States and was filed on March 30, 2001, published in English, which is a continuation-in-part of Application No.
09/540,843 filed March 31, 2000, which is a continuation-in-part of Application No.
09/048,927 filed March 26, 1998, now U.S. Patent No. 6,146,056, which is a continuation-in-part of the U.S. National stage of International Application No.
PCT/LTS96/08386 filed June 3, 1996, published in English, and assigned U.S.
Application No. 08/952,697, now abandoned, which is a continuation-in-part of Application No. 08/467,012 filed June 6, 1995, now U.S. Patent No. 5,955,059.
The entire teachings of the above applications are incorporated herein by reference.
BACKGROUND OF THE INVENTION
Mammalian cells have a complex response to DNA damage, as well as a tightly regulated program of replicative senescence, all suggested to be fundamental defenses against cancer [Campisi, J. (1996). Cell 84, 497-500]. In mammals, cell senescence is precipitated by critical shortening of telomeres, tandem repeats of the DNA sequence TTAGGG that cap the ends of chromosomes [Greider, C.W. (1996) Anrau Rev Biochena 65, 337-365] and become shorter with each round of DNA
replication.. In germline cells and most cancer cells, immortality is associated with maintenance of telomere length by telomerase, an enzyme complex that adds TTAGGG repeats dues to the 3' terminus at the chromosome ends [Feng, J., et al.
Science 269, 1236-1241; Harrington, L., et al., (1997) Science 275, 973-977;
Nakamura, T.M., et al., (1997) Science 277, 955-957]. The catalytic subunit of telomerase is generally not expressed in normal somatic cells [Greider, C.W.
(1996) Annu Rev Biochem 65, 337-365], and after multiple rounds of cell division critically shortened telomeres trigger either replicative senescence or death by apoptosis, _2_ largely dependent on cell type [de Lange, T. (1998) Science 279, 334-335], although the detailed mechanism is unknown. The mechanism by which telomeres participate in DNA damage responses has been less clear.
The frequency of cancer in humans has increased in the developed world as the population has aged. Melanoma and other skin cancers have increased greatly among aging populations with significant accumulated exposure to sunlight. For some types of cancers and stages of disease at diagnosis, morbidity and mortality rates have not improved significantly in recent years in spite of extensive research.
Cancers are currently often treated with highly toxic therapies. Alternative therapies are needed that could take advantage of the natural mechanisms of the cells to repair environmental damage.
SUMMARY OF THE INVENTION
One embodiment of the invention is a method for treating a hyperproliferative disorder in a human, the method comprising administering to the human an effective amount of a composition comprising one or more oligonucleotides which share at least 50% nucleotide sequence identity with the human telomere overhang repeat. Herein, where it is said that an oligonucleotide is homologous to the telomere overhang repeat, it is meant that the oligonucleotide shares at least 50% nucleotide sequence identity with the human telomere overhang repeat. In this method, the oligonucleotides can be at least 2 nucleotides long, for example 2-200 nucleotides long, or at least 3 nucleotides long, for example, 3-oligonucleotides long, and preferably can be 2-20 nucleotides long, and more preferably are 5-11 nucleotides long. Oligonucleotides having a 5' phosphate group are preferred. Where the 5' phosphate is a part of the oligonucleotide, it is indicated, where the sequence is given, by preceding the 5' to 3' nucleotide sequence with a "p." Preferred oligonucleotides in the treatment of hyperproliferative disorders are pTT, pGAGTATGAG (SEQ ID N0:1), pGTTAGGGTTAG (SEQ ID NO:S), pGGGTTAGGTT (SEQ ID N0:13), and pTAGATGTGGTG (SEQ ID N0:14). The methods of the invention can be applied to mammals, but are applied to humans, in more particular embodiments.

A part of the above method is a method for inhibiting the growth of cancer cells in a human, comprising administering to the human an effective amount of a composition comprising one or more oligonucleotides which share at least 50%
nucleotide sequence identity with the human telomere overhang repeat. The cancer cells can be derived from any cell type, but in particular embodiments are lymphoma, osteosarcoma, melanoma, leukemia or carcinomas.
Another part of the invention is a method for promoting differentiation of malignant cells in a mammal, the method comprising administering to the mammal an effective amount of a composition comprising one or more oligonucleotides which share at least 50% nucleotide sequence identity with (TTAGGG)".
A further method, useful in the treatment of cancers, is a method for enhancing the expression of one or more surface antigens indicative of differentiation of cancer cells in a human, the method comprising administering to the human an effective amount of one or more oligonucleotides which share at least 50% nucleotide sequence identity with the human telomere overhang repeat. The cells in this method can be, for example, melanoma, and the antigen can be, for example, MART-1, tyrosinase, TRP-1 or gp-100. The cells can be breast cancer cells and the antigen can be estrogen receptor a. Another oligonucleotide to be used in this method is pTT; a combination of one or more telomere-homologous oligonucleotides and pTT can also be used. pGTTAGGGTTAG (SEQ m NO:S) is one oligonucleotide applicable in this method.
Oligonucleotides which share at least 50% nucleotide sequence identity with the human telomere overhang repeat sequence have been found to decrease expression of genes which are normally primarily or exclusively expressed in embryonic development, and not expressed or minimally expressed in normal, differentiated cells, but are expressed in cancer cells. For example, pGTTAGGGTTAG (SEQ ID NO:S) reduced the expression of livin/ML-IAP in a melanoma cell line, as described in Example 44.
Further, the invention is a method for inducing apoptosis in cancer cells in a human, said method comprising administering to the human an effective amount of a composition comprising one or more oligonucleotides which share at least 50%

nucleotide sequence identity with the human telomere overhang repeat. This method can be applied, for example, to melanoma and lymphoma.
A further effect on cancer cells of the oligonucleotides described herein as sharing at least 50% sequence identity with the telomere overhang repeat is the induction of senescence. As illustrated in Examples 41, 43 and 48, breast cancer cells, ovarian cancer cells and fibrosarcoma cells treated with oligonucleotides responded with the altered morphology and expression of (3-galactosidase typical of senescence. Thus, another method of the invention is a method for inducing senescence in cancer cells in a mammal (e.g., a human), the method being to administer to the mammal an effective amount of a composition comprising one or more oligonucleotides sharing at least 50% squence identity with the nucleotide sequence of the mammalian telomere overhang repeat.
Also a part of the invention is a method for inhibiting the growth of cancer cells in a human, the method being independent of the presence or activity of telomerase in the cancer cells, in which the method includes the step of administering to the human an effective amount of a composition comprising one or more oligonucleotides which share at least 50% nucleotide sequence identity with the human telomere overhang repeat.
A further aspect of the invention is a method to inhibit the growth of cancer cells in a human, the method not requiring the presence or activity of p53 gene product in the cancer cells, the method comprising administering to the human an effective amount of a composition comprising one or more oligonucleotides which share at least SO% nucleotide sequence identity with the human telomere overhang repeat.
A further aspect of the invention is a method to inhibit the growth of cancer cells in a human, the method resulting in S-phase arrest in said cells, the method comprising administering to the human an effective amount of a composition comprising one or more oligonucleotides which share at least 50% nucleotide sequence identity with the human telomere overhang repeat. The oligonucleotide to be used can be various lengths, but in one embodiment the oligonucleotide can be less than 6 nucleotides long.

Herein is also described a method for preventing spongiosis, blistering or dyslceratosis in the skin of a man meal, following exposure to ultraviolet light, the method comprising applying to the skin an effective amount of a composition comprising one or more oligonucleotides which share at least 50% nucleotide sequence identity with the human telomere overhang repeat. The method can employ, for example, pGAGTATGAG (SEQ m NO:l), or the oligonucleotide pTT, or a combination of oligonucleotides.
Also described herein is a method for reducing the occurrence of skin cancer in a human, the method comprising applying to the skin an effective amount of a composition comprising one or more oligonucleotides which share at least 50%
nucleotide sequence identity with the human telomere overhang repeat. Such oligonucleotide can be, for example, pGAGTATGAG (SEQ m NO:1). pTT can also be used in the method, or a combination of oligonucleotides can be used.
A special aspect of the above methods to reduce the occurrence of skin cancer in a human is a method for reducing the occurrence of skin cancer in a human with xeroderma pigmentosum, or other genetically determined cancer predisposition, the method comprising applying to the skin an effective amount of a composition comprising one or more oligonucleotides which share at least 50% nucleotide sequence identity with the human telomere overhang repeat. In one aspect, the oligonucleotide is pGAGTATGAG (SEQ m NO:1).
Another particular aspect of the invention is a method for reducing the occurrence of skin cancer in a human with xeroderma pigmentosum or other genetically determined predisposition, the method comprises applying to the skin an effective amount of a composition comprising pTT.
Also included in the invention is a method for enhancing repair of ultraviolet irradiation-induced damage to skin in a human, in which the method includes applying to the slcin an effective amount of a composition comprising one or more oligonucleotides which share at least 50% nucleotide sequence identity with the human telomere overhang repeat. A particular oligonucleotide that can be used is pGAGTATGAG (SEQ m NO:1). Another that can be used in the method is pTT.

Also included as an aspect of the invention is a method for reducing oxidative damage in a mammal, the method comprising administering to the mammal an effective amount of a composition comprising one or more oligonucleotides which share at least 50% nucleotide sequence identity with the human telomere overhang repeat. One such oligonucleotide is pGTTAGGGTTAG
(SEQ ID NO:S). pTT can also be used in the method. In a particular aspect of this method, the composition can be administered to the skin.
It is also an object of the invention to provide a method for treating melanoma in a mammal, comprising administering to the mammal an effective amount of a composition comprising one or more oligonucleotides that share at least 50% nucleotide sequence identity with the human telomere overhang repeat. The method is applicable to humans. Oligonucleotides to be used in the method include the telomere-homologous oligonucleotide pGTTAGGGTTAG (SEQ ID NO:S). pTT
can also be used. Various combinations of oligonucleotides can also be used in the method.
It is also an object of the invention to provide a method for reducing proliferation of lceratinocytes in the skin of a human, the method comprising applying to the skin an effective amount of a composition comprising one or more oligonucleotides that share at least 50% nucleotide sequence identity with the human telomere overhang repeat. W particular applications of the method, the hiunan to be treated has seborrheic keratosis, actinic keratosis, Bowen's disease, squamous cell carcinoma, or basal cell carcinoma. In a particular aspect of the method, the composition comprises pGTTAGGGTTAG (SEQ ID NO:S). A related method is a method for reducing proliferation of keratinocytes in the skin of a human, said method comprising applying to the skin an effective amount of a composition comprising pTT.
Another embodiment comprises increasing DNA repair in epithelial cells, comprising contacting said cells with an effective amount of a composition comprising at least one oligonucleotide, wherein the oligonucleotide comprises a base sequence selected from the group consisting of SEQ ID NO: 1, SEQ ll~ NO:

2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ

_7_ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 12, and a contiguous portion of any of the foregoing sequences. Another embodiment comprises inhibiting proliferation of epithelial cells, comprising contacting said cells with an effective amount of a composition comprising at least one oligonucleotide, wherein the oligonucleotide comprises a base sequence selected from the group consisting of SEQ >D NO: 1, SEQ m NO: 2, SEQ m NO: 3, SEQ m NO: 4, SEQ m NO: 5, SEQ m NO: 6, SEQ
m NO: 7, SEQ m NO: 8, SEQ m NO: 1 l, SEQ m NO: 12, and a contiguous portion of any of the foregoing sequences.
Also included in the invention are truncated versions of the above oligonucleotides identified above by sequence. The oligonucleotides of the present invention may be truncated by one or more nucleotides on the 5' erid, the 3' end, or both the 5' end and the 3' end, so long as at least two contiguous nucleotides of the untruncated oligonucleotide remain. Preferably the truncated oligonucleotides have 10, 9, 8, 7, 6, 5, 4, 3 or 2 contiguous nucleotdides found in the untruncated nucleotides.
Also a part of the invention are compositions comprising one or more oligonucleotides in a physiologically acceptable carrier, wherein~the oligonucleotide comprises base sequence SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 11, 12, 13, 14, 16, 17 or 18. Further, the invention can be a composition comprising one or more oligonucleotides in a physiologically acceptable carrier, wherein the oligonucleotide consists of base sequence SEQ m NO:1, 2, 3, 4, 5, 6, 7, 8, 11, 12, 13, 14, 16, 17 or 18.
Preferred for applications in which it is desired to inhibit cell proliferation are oligonucleotides comprising SEQ ID NO: 1 or SEQ ID NO: 6, or oligonucleotides consisting of SEQ ID NO: 1 or SEQ ID NO: 6. Preferred in applications in which apoptosis is desired are oligonucleotides comprising base sequence SEQ m NO: 5, or the oligonucleotide consisting of base sequence SEQ m NO: 5.
Also provided as part of the invention is a composition comprising an oligonucleotide and a physiologically acceptable carrier, wherein the oligonucleotide is pGAGTATGAG (SEQ >D N0:1), pCATAC (SEQ ID N0:6), pGTTAGGGTTAG

_g_ (SEQ ID NO:S), pGGGTTAGGTT (SEQ ID N0:13), pTAGATGTGGTG (SEQ ID
N0:14), pTTAGAGTATGAGTTA (SEQ. ID N0:16.), pTTAGAGTATGAG (SEQ
ID NO: 17) or pGAGTATGAGTTA (SEQ ID N0:18).
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a graphic representation of the cell growth rate of human squamous carcinoma cells dosed with water (diluent), 100 ~,M pTpT (TZ) or 100 ~,M
pdApdA (AZ), where day 0 is before dosage and days 1, 3, 4 and 5 are days after dosage.
Figure 2 is a graphic representation of the cell growth rate of normal human fibroblasts dosed with water (diluent) or 100 ~,M pTpT (TZ), where day 0 is before dosage and days 1, 3, 4 and 5 are days after dosage, and where values represent averages + standard deviations of duplicate cultures.
Figure 3 is a graphic representation of the cell growth rate of human cervical carcinoma cells dosed with either water (diluent) or 100 ~.M pTpT (TZ), where day 0 is before dosage and days 1, 4 and 6 are days after dosage.
Figure 4 is a graphic representation of the cell yield of human melanoma cell lines dosed with either diluent or 100 ~,M pTpT (TZ).
Figure 5 is a graphic representation of the cell growth rate of normal human keratinocytes dosed with water (diluent) or 100 ~M pTpT (TZ), where day 0 is before dosage and 8, 24, 48 and 72 are hours after dosage and where values represent averages + standard deviations of duplicate cultures.
Figure 6 is a graphic representation of the average cell number of human neonatal fibroblasts dosed with either water, TZ or A2.
Figure 7 is a graphic representation of the average cell number of human neonatal fibroblasts dosed with either water, TZ or Az.
Figure 8 is a graphic representation of the cell growth rate of normal human fibroblasts dosed with water (diluent) or 100 ~,M pTpT (TZ), where day 0 is before dosage and where values represent averages + standard deviations of duplicate cultures.

Figure 9 is a graphic representation of the cell growth rate of p53-null H1299 lung carcinoma cells dosed with water (diluent) or 100 ~,M pTpT (T2), where day 0 is before dosage and 1, 2, 3 and 4 are days after dosage, and where values represent averages + standard deviations of duplicate cultures.
Figure 10 is a graphic representation of enhancement of DNA repair of a reporter plasmid in human lceratinocytes treated with pTpT, where open boxes represent sham-irradiated control plasmid and filled boxes represent W-irradiated plasmid.
Figure 11 is a graphic representation of enhancement of DNA repair of a reporter plasmid in human fibroblasts treated with pTpT where open boxes represent sham-irradiated control plasmid and filled boxes represent UV-irradiated plasmid.
Figure 12 is melanin content of Cloudman S91 cells treated with 100 ~,M of the indicated oligonucleotide or an equal volume of diluent for 5 days, where data are shown as averages of duplicate cultures calculated as a percentage of diluent-treated controls.
Figure 13 shows a densitometric analysis of p21 expression detected by Northern blot analysis of SCC12F cells treated with 100 ~,M of the indicated oligonucleotide or an equal volume of diluent for 48 hours.
Figure 14 shows cell yields of the samples in Figure 13, as mean ~ standard deviation.
Figure 15 shows melanin content of Cloudman S91 cells treated with 100 ~,M of the indicated oligonucleotide or an equal volume of diluent for 5 days as a percent of diluent-treated controls (mean ~ standard deviation) for 3 independent experiments.
Figure 16 shows melanin content of Cloudman S91 cells treated with 100 ~M of the indicated oligonucleotide or an equal volume of diluent as described for Figure 12, where the values represent three independent experiments and where * = p< 0.004, **~ < 0.03, two-tailed Student's t-test.
Figure 17 shows melanin content of Cloudman S91 cells treated with the indicated oligonucleotide.

Figure 18 shows melanin content of Cloudman S91 cells treated with the indicated oligonucleotide.
Figures 19A-19H show FACS (fluorescence activated cell sorter) analysis of propidium iodide stained Jurl~at cells (immortalized T
lymphocytes), treated with diluent (Figures 19A and 19E); 40 ~M l lmer-1 pGTTAGGGTTAG
(SEQ m NO:S) (Figures 19B and 19F); 40 ~.M l lmer-2 pCTAACCCTAAC
(SEQ m NO:9) (Figures 19C and 19G); 40 ~M l lmer-3 pGATCGATCGAT
(SEQ m NO:10) (Figures 19D and 19H). Jurl~at cells were treated with the stated reagents for 48 hours before analysis (Figures 19A-19D) or 72 hours (Figures 19E-19H).
Figures 20A-20F are profiles showing the results of fluorescence activated cell sorting, for the following additions to the cells: Figure 20A, diluent; Figure 20B, 0.4 ~,M l lmer-1; Figure 20C, 0.4 ~,M l lmer-1-S; Figure 20D, diluent; Figure 20E, 40 ~,M l liner-1; Figure 20F, 40 ~,M l liner-1-S.
Figures 21A-21G are profiles showing the results of fluorescence activated cell sorting, for the following additions to the cells: Figure 21A, diluent; Figure 21B, 10 ~,M llmer-l; Figure 21C, 10 ~,M l liner-1 and 1 ~M
l liner-1-S; Figure 21D, 10 ~M l liner-1 and 5 ~M l liner-1-S; Figure 21E, 10 ~M l liner-1 and 10 ~,M llmer-1-S; Figure 21F, 20 ~,M llmer-1-S; Figure 21G, 10 ~M l liner-1-S.
Figure 22 is a bar graph showing the melanin content (in pg/cell) of cells treated with diluent, pTpT or pTspT.
Figure 23 is a bar graph showing the melanin content (in pg/cell) of cells treated with diluent, l liner-1 or 1 liner-1-S.
Figure 24 is a bar graph showing the melanin content (in pg/cell) of cells that have been sham-treated (no irradiation, no oligonucleotides), or treated with ultraviolet light (UV), or unirradiated but given pTspT, or irradiated with W
and given pTspT.
Figure 25 is a graph in which viable cell count, as a percent of the cell count for the respective sham-irradiated control, is shown as a function of time after ultraviolet irradiation for diluent-treated (squares) or pTpT-treated (diamonds) cells of squamous carcinoma cell line SCC12F.
Figure 26 is a bar graph in which the repair capacity of normal human fibroblasts, after WA-induced damage and treatment with pTpT, is shown as the luciferase activity generated from expression of a gene in the non-replicating vector pCMV-Luc. Luciferase activity, as a percent of a sham-irradiated control, is plotted for diluent-treated (blaclc bars) and pTpT-treated (white bars) for fibroblasts irradiated for 10 or 20 minutes. See Example 24.
Figure 27 is a bar graph showing the results of an assay for caspase-3 activity in Jurkat cells treated with (bars, as shown left to right): diluent, oligonucleotide homologous to telomere sequence, oligonucleotide complementary to telomere sequence, or an unrelated oligonucleotide. Caspase-3 activity (in pmol pNA/hr/~g protein resulting from the assay) is plotted for 48, 72 and 96 hours of incubation of the cells with the oligonucleotides.
Figure 28 is a bar graph depicting the average volume of primary tumors that resulted in SCID (severe combined immunodeficiency) mice injected with MM-AN cells pre-treated with diluent, "complement" oligonucleotide with sequence pCTAACCCTAAC (SEQ ID N0:9), or "T-oligo" with sequence pGTTAGGGTTAG (SEQ ID NO:S).
Figure 29 is a bar graph depicting the average volume of metastatic tumors that resulted in SCID mice injected with MM-AN cells pre-treated with diluent, "complement" oligonucleotide, or "T-oligo." See Example 30.
Figures 30A-30H are profiles of fluorescence intensity as determined by fluorescence activated cell sorting, for the following additions to Jurkat cells:
Figures 30A and 30E, diluent; Figures 30B and 30F, 10 ~,M pGGGTTAGGGTT
(SEQ m N0:13); Figures 30C and 30G, 10 ~,M pTAGATGTGGTG (SEQ ID
N0:14); Figures 30D and 30H, 10 ~,M pCGGGCTTATTG (SEQ ID NO:15).
Cells were collected and processed for FAGS 72 hours after addition of oligonucleotides (Figures 30A 30B, 30C and 30D) or 96 hours after addition of oligonucleotides (Figures 30E, 30F, 30G and 30H). See Example 31.

Figures 31A-31E are profiles of fluorescence intensity as determined by fluorescence activated cell sorting, for the following additions to Jurlcat cells:
Figure 31A, diluent; Figures 31B and 31D, pTT at 20 ~M and 5 wM, respectively; Figures 31C and 31E, pGTTAGGGTTAG (SEQ m NO:S) at 20 ~M and 5 ~M, respectively. See Example 31.
Figures 32A-32D are profiles of fluorescence intensity as determined by fluorescence activated cell sorting, for the following additions to preconfluent normal neonatal human fibroblasts: Figure 32A, diluent; Figure 32B, pGTTAGGGTTAG (SEQ m NO:S) at 40 ~.M; Figure 32C, pCTAACCCTAAC
(SEQ m NO:9) at 40 ~M; Figure 32D, pGATCGATCGAT (SEQ m N0:10) at 40 ~.M. Cells were analyzed by FACS 24 hours after addition of oligonucleotides or diluent.
Figures 33A-33H are profiles of fluorescence intensity as determined by fluorescence activated cell sorting, for the following additions to (Figures 33D) SCC12F cells or (Figures 33E-33H) Saos-2 cells: Figures 33A and 33E, diluent; Figures 33B and 33F, pGTTAGGGTTAG (SEQ m NO:S); Figures 33C
and 33G, pCTAACCCTAAC (SEQ m N0:9); Figures 33D and 33H, pGATCGATCGAT (SEQ m NO:10). Cells were analyzed by FAGS 48 hours after addition of 40 ~,M oligonucleotides.
Figures 34A-34H are profiles of fluorescence intensity as determined by fluorescence activated cell sorting, for the following additions to (Figures 34D) normal control fibroblasts or (Figures 34E-34H) NBS fibroblasts: Figures 34A and 34E, diluent; Figures 34B and 34F, pGTTAGGGTTAG (SEQ m NO:S); Figures 34C and 34G, pCTAACCCTAAC (SEQ m N0:9); Figures 34D
f and 34H, pGATCGATCGAT (SEQ m NO:10). Cells were analyzed by FAGS
48 hours after addition of 40 ~,M oligonucleotides or diluent.
Figure 35 is a diagram of a proposed mechanism for induction of senescence, cell cycle arrest, adaptive differentiation or apoptosis by exposures of the single-stranded telomere DNA sequence. The 3' telomere overhang is normally sequestered within a loop structure stabilized by TRF2, forming a "capped" or silent telomere. Destabilization of this loop structure by DNA

damage due to IJV irradiation or chemical adducts, expression of TRF2DN, or gradual erosion during aging is hypothesized to expose this single-stranded DNA
(repeats of TTAGGG; SEQ m NO:l 1), "uncapping" the telomere.
Displacement of the TRF2 protein might or might not accompany loop disruption under physiological conditions. This single-stranded DNA is then detected by an as yet unidentified sensor molecule. Interaction of this sensor with the 3' overhang initiates a cascade of events that includes ATM
activation, followed by p53 activation and p95/Nbsl modification leading to cell cycle arrest. Depending on cell type and/or intensity and duration of the signal, these events might lead to the eventual induction of senescence, adaptive differentiation or apoptosis. DNA oligonucleotides homologous to the overhang sequence could be recognized by the same sensor molecule, triggering the cascade in the absence of telomere disruption.
Figure 36 is a bar graph representing averages of cell population doublings after additions, with standard error of mean, for duplicate cultures of Saos-2 cells treated with oligonucleotides for 36 hours, as described in Example 40. ' Figure 37 is a graph showing yield of MCF-7 cells following a single dose treatment with T-oligo (filled diamonds), control oligonucleotide (filled squares) or diluent (filled triangles), left in contact with the cells over a 4-day period. See Example 41.
Figure 38 is a graph showing yield of BT-20 cells following a single dose treatment with T-oligo (filled diamonds), control oligonucleotide (filled squares) or diluent (filled triangles), left iri contact with the cells over a 4-day period. See Example 41.
Figure 39 is a graph showing yield of MCF-7 cells following doses of treatment with T-oligo (filled diamonds), control oligonucleotide (filled squares) or diluent (filled triangles) on days 1 and 3. See Example 41.
Figure 40 is a profile of fluorescence intensity as determined by fluorescence activated cell sorting, for MCF-7 cells maintained for 72 hours following addition to the cultures of T-oligo, control oligonucleotide, or diluent.
See Example 41.
Figure 41 is a profile of fluorescence intensity as determined by fluorescence activated cell sorting, for BT-20 cells maintained for 96 hours following addition to the cultures of T-oligo, control oligonucleotide, or diluent.
See Example 41.
Figure 42 is a bar graph depicting the percentage of cells staining positive for [i-galactosidase activity in cultures of MCF-7 cells treated with 40 ~,M
pGTTAGGGTTAG (SEQ ID NO:S) and maintained in culture one week before staining. See Example 41.
Figure 43 is a bar graph showing cell yield for MCF-7 cells maintained in culture and supplemented with diluent T-oligo, cisplatin, or ICI 182,780 for 1, 2, 3, 4 and 5 days after treatment. See Example 41.
Figure 44 is a bar graph showing cell yield for BT-20 cells maintained in culture and supplemented with diluent, T-oligo, cisplatin or ICI 182,780 for 1, 2, 3, 4 and 5 days after treatment. See Example 41.
Figure 45 is a graph depicting cell yield for cultures of HPAC cells treated with 40 ~M pGTTAGGGTTAG [T-oligo; SEQ ID NO:S (filled squares)], 40 ~,M pCTAACCCTAAC [control oligonucleotide; SEQ ID NO:9 (filled trianges)] or diluent alone (filled diamonds). See Example 42.
Figure 46 is a graph depicting the number of cells in cultures of OVCARS cells subjected to treatment on day 0 with 40 wM T-oligo (filled squares), complement oligo (filled triangles), or diluent (filled diamonds).
Cells were counted on days 3, 4 and 5 after treatment. See Example 43.
Figure 47 is a graph depicting the number of cells in cultures of MCF-7 cells subjected to treatment on day 0 with 40 ~M T-oligo (filled squares), complement oligo (filled trianges), or diluent (filled diamonds). Cells were counted on days 3, 4 and 5 after treatment. See Example 43.
Figure 48 is a profile showing the results of FAGS analysis of MM-AN
cells treated with oligonucleotides or diluent, as shown. See Example 44.

Figure 49 is a profile showing the results of the TUNEL assay on MM-AN cells treated with oligonucleotides or diluent, as shown. See Example 44.
Figure 50 is a profile showing the results of the TUNEL assay on MM-AN cells and melanocytes treated with oligonucleotides or diluent, as shown, after 24 hours and 96 hours of treatment. See Example 44.
Figure 51 is a bar graph showing the average tumor volume for tumors that developed in SCID mice 40 days after injection of MM-AN cells previously treated with T-oligo, complementary oligonucleotide, or diluent. See Example 44.
Figure 52 is a bar graph showing the average number of tumors that developed in SCID mice 40 days after injection of MM-AN cells previously treated with T-oligo, complementary oligonucleotide, or diluent. See Example 44.
Figure 53 is a diagram of oligonucleotides of nucleotide sequence SEQ
m NO:S which were synthesized with phosphorothioate linkages. See Example 46.
Figure 54 is a bar graph showing the results of testing the effects of phosphorothioate oligonucleotides 1, 2, 3 and 4 depicted in Figure 53 in causing senescence in cultures of normal neonatal human fibroblasts, indicated by the cells staining positive for ~i-galactosidase activity. Oligonucleotide "11-1"
in Figure 54 indicates fibroblast cultures treated with pGTTAGGGTTAG (SEQ ID
NO:S) synthesized entirely with phosphodiester linkages. "Dil" in Figure 54 indicates fibroblast cultures treated with diluent not containing oligonucleotide.
See Example 46.
Figure 55 is a graph showing the results of an experiment in which XPC+~- mice received 5 consecutive cycles (150 days) of one week daily topical pTT (or diluent alone) followed by 3 weelcs of daily UV irradiation. Data are plotted as the percentage of the pTT- or diluent-treated mice that remained free of tumors, over time following initiation of the experiment. See Example 49.
Figure 56 is a graph showing the results of an experiment in which XPC+~ mice received 5 consecutive cycles (150 days) of one week daily topical pTT (or diluent alone) followed by 3 weeks of daily UV irradiation. Data are plotted as the average number of tumors per XPC~~- mouse that were observed over time following initiation of the experiment. See Example 49.
Figure 57 is a bar graph that depicts the results of an experiment in which XPC+~- mice received 5 consecutive cycles (150 days) of one weelc daily topical pTT (or diluent alone) followed by 3 weeks of daily UV irradiation, as in Example 49. The number of tumors per mouse is shown for mice treated with diluent or pTT. (AK = actinic leeratoses; SCC = squamous cell carcinomas) Figure 58 is a bar graph that shows the results of an experiment in which tumors induced by LTV irradiation were inj ected on day 0 with pTT. Tumor volume is plotted versus day relative to day 0. See Example 50.
Figure 59 is a graph showing the results of an assay indicating DNA
repair on plasmid pCMVluc following ultraviolet B irradiation of the plasmid and transfection of the irradiated plasmid into fibroblasts. Cells were treated two days before transfection, using 40 ~,M (~) T-oligo pGTTAGGGTTAG (SEQ ID
NO:S), (0) control oligonucleotide pCTAACCCTAAC (SEQ ID NO:9), (D) unrelated oligonucleotide pGATCGATCGAT (SEQ ID NO:10), or (0) diluent.
Figure 60 is a graph showing the results of an assay measuring DNA
photoproducts present in fibroblasts pretreated with 40 ~,M oligonucleotide or diluent and then UVB-irradiated. Symbols are as above for Figure 59.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is based on the discovery that treatment of cells with DNA fragments, oligonucleotides or similar compounds can inhibit cell proliferation, or induce DNA repair or elicit a protective response to subsequent exposure to UV-irradiation or carcinogenic chemicals. pTpT evokes a melanogenic (tanning) response in skin (U.S. Patent 5,643,556, the teachings of which are incorporated herein in their entirety), a protective response to UV
irradiation.

More specifically, the invention pertains to the use of compounds such as DNA fragments, polynucleotides, oligonucleotides, deoxynucleotides, dinucleotides, or dinucleotide diners, or similar compounds, for the inhibition of cell proliferation or induction of DNA repair. As used herein, inhibition of cell proliferation includes complete abrogation of cell division, partial inhibition of cell division and transient inhibition of cell division as measured by standard tests in the art and as described in the Examples. The invention also pertains to the prevention and/or treatment of hyperproliferative diseases, including, but not limited to, cancer and pre-cancerous conditions, wherein the hyperproliferative disease affects cells of any organ and any embryonic origin. Metastatic tumors and cancers that have regrown or relapsed after treatment, as well as primary tumors, can be treated by the methods of the invention. In particular embodiments, the diseases and conditions to be treated include skin diseases such as psoriasis and hyperproliferative, pre-cancerous or UV-induced dermatoses in mammals, particularly in humans.
The invention further pertains to use of the compounds of the present invention to reduce photoaging (a process due in part to cumulative DNA
damage), and to reduce oxidative stress and oxidative damage. The invention also pertains to prophylaxis against, or reduction in the likelihood of, the development of skin cancer in a mammal. Iri addition, the compounds of the present invention can be used to induce apoptosis in cells such as cells that have sustained genetic mutation, such as malignant or cancer cells or cells from an actinic keratosis. The invention further provides compositions comprising said compounds.
All types of cells, and in particular embodiments, epithelial cells, are expected to respond to the methods of the present invention as demonstrated by the representative ih vitro and in vivo examples provided herein. Epithelial cells suitable for the method of the present invention include epidermal cells, respiratory epithelial cells, nasal epithelial cells, oral cavity cells, aural epithelial cells, ocular epithelial cells, genitourinary tract cells and esophageal cells, for example. Gastrointestinal cells are also contemplated in methods of the invention as described herein.
Cells that contain damaged or,mutated DNA include, for example, actinic lceratosis cells, cancer cells, cells that have been irradiated, as with W, and cells that have been exposed to DNA damaging chemicals or conditions. As described herein, allergically mediated inflammation includes conditions such as atopic dermatitis, contact dermatitis, allergic rhinitis and allergic conjunctivitis.
In one embodiment, the compositions of the present invention comprise DNA oligonucleotides approximately 2-200 bases in length, which can be administered to a mammal (e.g., human) in an appropriate vehicle. In another embodiment, the DNA oligonucleotides are about 2 to about 20 nucleotides in length. In still another embodiment, the oligonucleotides are about 5 to about nucleotides in length. In yet another embodiment, the DNA oligonucleotides are about 2-5 nucleotides in length. As used herein, "DNA fragments" refers to single-stranded DNA fragments, double-stranded DNA fragments, or a mixture of both single- and double-stranded DNA fragments.
It is understood that other base-containing sequences can also be used in the present invention, where bases are, for example, adenine, thymine, cytosine, guanine or uracil. In one embodiment, the oligonucleotides of the present invention comprise a 5' phosphate. A combination of one or more of oligonucleotides of the present invention can also be used.
As shown in the Examples, certain DNA fragments, oligonucleotides and dinucleotides of the present invention caused inhibition of proliferation, melanogenesis, TNFa production and induction of apoptosis in cells, when the cells were contacted with the DNA fragments, oligonucleotides and nucleotides of the present invention. For example, thymidine dinucleotide (pTpT) inhibits proliferation of several human cell types including squamous cell carcinoma, cervical carcinoma, melanoma, neonatal keratinocytes and normal neonatal fibroblasts (Examples 1-5, respectively). pTpT also reduced epidermal proliferation in vivo in a guinea pig model (Example 6). Furthermore, pTpT
treatment of cells resulted in the nuclear localization of p53 (Example 7) and the induction of p53-regulated genes (Example 8) such as genes involved in DNA
repair. Pretreatment of cells with pTpT enhanced their ability to repair DNA
damaged by UV irradiation and by the chemical carcinogen benzo[a]pyrene (Examples 8 and 9). pTpT up-regulated the levels of p53, PCNA and the XPA
protein 2 to 3-fold within 2 days of treatment (Example 9). Of note XPA is known not to be p53 regulated, demonstrating that some enhancement of DNA
repair capacity may also occur in cells lacking p53. Pretreatment of mouse skin with pTpT also resulted in a reduced level of LTV-induced DNA damage (thymine dimers) if2 vivo (Example 14).
Thymidine dinucleotide, pTpT, mimics some effects of LTV light, including inducing melanogenesis and stimulating keratinocyte production of TNFa (Example 4). pTpT also induces TNFa and reduces contact hypersensitivity in vivo (Example 10). LTVB radiation is a potent inlubitor of the inductive phase of contact hypersensitivity (CH), and TNFa is a mediator of this suppressive effect. Thymidine dinucleotides (pTpT), a substrate for LTV-induced thynine dimer formation, stimulate several effects, including increased tyrosinase expression and melanin content in cultured melanocytes and slcin tanning in guinea pigs. As shown in Example 10, the compounds of the present invention also mimic the suppressive effect of LJVB on contact hypersensitivity in a mouse model. As demonstrated by the present invention, intracutaneous inj ection or topical application of pTpT can inhibit the induction of contact hypersensitivity and can activate the TNFa gene ifZ vivo.
Example 10 also demonstrates that pTpT induces production of IL,-10 mRNA and protein which is active in inhibiting T cell proliferation in allogenic mixed lymphocyte assay. In hmnan skin, IL-10 as well as TNFa induces specific tolerance for contact hypersensitivity and delayed-type hypersensitivity reactions.
Therefore, the DNA fragments, oligonucleotides, deoxynucleotides, dinucleotides and dinucleotide dimers of the present invention are reasonably expected to have immunosuppressive effects ifa vivo, e.g., to inhibit contact hypersensitivity and delayed-type hypersensitivity.

In further examples, a nine-nucleotide oligomer, GAGTATGAG (SEQ
m NO:1) stimulated melanogenesis in human melanocytes and induced the expression of p21/Waf/Cip 1, a growth inhibitory gene product, in a squamous cell carcinoma cell line. Furthermore, TAGGAGGAT (SEQ m N0:2; 5/9 identity with telomere overhang repeat), and truncated versions of the original 9-mer, AGTATGA (SEQ m N0:3) and GTATG (SEQ m N0:4), also stimulated melanogenesis in human melanocytes (Example 11). In addition, the sequence pGTTAGGGTTAG (SEQ m NO: 5) stimulated pigmentation in Cloudman S91 melanoma cells (Example 12) and induced apoptosis in a human T-cell line (Example 13). As demonstrated herein, pGTTAGGGTTAG (SEQ m NO: 5) induced human T cells to undergo apoptosis, while SEQ m NOs: 9 and 10 (pCTAACCCTAAC and pGATCGATCGAT) did not significantly increase apoptosis in these cells (Example 13). Oligonucleotides with sequences SEQ m NOs: 6-12 demonstrated at least some ability to induce melanogenesis (Examples 11-13).
As demonstrated herein, oligonucleotides as small as dinucleotides (e.g.
pTpT) and oligonucleotides of about 20 nucleotides in length can also be used, as it has been demonstrated that oligonucleotides of these lengths are taken up into the cells. In another embodiment, oligonucleotides of about 11 nucleotides can be used. In still another enibodimerit, oligoriucleotides 5 nucleotides in length can be used to penetrate the skin barrier and effectively induce melanogenesis, inhibit cell growth and induce immunosuppression. A 9-mer was shown to protect the skin of mice from photodamage in Example 25.
These results demonstrate that the ifz vitro effects of these compounds also occur in vivo upon contacting the cells or tissue of interest with the compounds of the present invention. For example, as demonstrated herein, for the effect of inhibition of cell proliferation, TNF-a production and melanin production, ih vitro induction of these activities by the compounds of the present invention is predictive of the ability of these compounds to produce the same effects ira vivo. Any suitable method of administering the compounds of the present invention to the organism, such that the compound contacts the cells or tissues of interest, is reasonably expected to be effective. The effects can be optimized using routine optimization protocols.
The compounds of the present invention are therefore useful in methods of inhibiting cell proliferation, preventing cancer, photoaging and oxidative stress by enhancing DNA repair, and, in the slcin, by enhancing pigmentation through increased melanin production. Melanin is known to absorb photons in the UV range and therefore its presence reduces the risk of cancer and photoaging.
The DNA fragments, oligonucleotides, deoxynucleotides, dinucleotides, and dinucleotide dimers can be obtained from any appropriate source, or can be synthetically produced. To make DNA fragments, for example, salmon sperm DNA can be dissolved in water, and then the mixture can be autoclaved to fragment the DNA. In one embodiment, the DNA fragments, oligonucleotides, deoxynucleotides, dinucleotides or dinucleotide dimers contain a 5' phosphate.
The compounds of the present invention also play a protective role in UVA-induced oxidative damage to the cell (Example 15). As described in Example 15, primary newborn fibroblasts treated with pTpT for 3 days and then stimulated with 5 x 10-5 or 5 x 10-4 HZOZ had higher cell yields compared to diluent treated controls. Analysis of mRNA and protein revealed that in pTpT
treated cells, Cu/Zn superoxide dismutase was elevated. This enzyme participates in the process of oxygen radical quenching. Thus, in one embodiment of the present invention, the compounds of the present invention are administered to cells to protect against oxidative damage. In one embodiment, these compounds are topically administered to the epidermis of an individual.
An "agent that increases activity of p53 protein," as used herein, is an agent (e.g., a drug, molecule, nucleic acid fragment, oligonucleotide, or nucleotide) that increases the activity of p53 protein and therefore results in increase in an DNA repair mechanisms, such as nucleotide excision repair, by the induction of proteins involved in DNA repair, such as PCNA, XPB and p21 proteins. The activity of p53 protein can be increased by directly stimulating transcription of p53-encoding DNA or translation of p53-specific mRNA, by increasing expression or production of p53 protein, by increasing the stability of p53 protein, by increasing the resistance of p53 mRNA or protein to degradation, by causing p53 to accumulate in the nucleus of a cell, by increasing the amount of p53 present, by phosphorylating the serine -15 residue in p53, or by otherwise enhancing the activity of p53. A combination of more than one agent that increases the activity of p53 can be used. Alternatively or in addition, the agent that increases the activity of p53 can be used in combination with DNA
fragments, deoxynucleotides, or dinucleotides, as described above.
Ultraviolet irradiation produces DNA photoproducts that when not promptly removed, can cause mutations and skin cancer. Repair of W-induced DNA damage requires efficient removal of the photoproducts to avoid errors during DNA replication. Age-associated decrease in DNA repair capacity is associated with decreased constitutive levels of p53 and other nuclear excision repair (NER) proteins required for removing UV-induced photoproducts. As demonstrated herein, compounds of the present invention induced NER proteins in human dermal cells when these cells were treated with these compounds before UV irradiation (Example 16). While there were age related decreases in NER proteins, NER proteins in cells from donors of all ages from newborn to 90 years were induced by 200-400%. A significant decrease in the rate of repair of thymine dimers and photoproducts occurs with increased age of the cell sample;
however, cells that were pre-treated with compounds of the present invention, then UV irradiated, removed photoproducts 30 to 60 percent more efficiently.
Thus, the treatment of cells with small DNA oligonucleotides partially compensates for age-associated decreases in DNA repair capacity. In light of the in vivo efficacy of the compounds of the present invention, it is reasonable to expect that treatment of human skin with the compounds of the present invention enhances endogenous DNA repair capacity and reduces the carcinogenic risk from solar UV irradiation. This method is especially useful in older individuals who likely have reduced cellular DNA repair capacity.
DNA fragments, oligonucleotides, deoxynucleotides, dinucleotides or dinucleotide dimers, to be applied to the skin in methods to prevent the sequelae of UV exposure or to reduce the occurrence of skin cancer, to reduce oxidative damage, or to enhance repair of UV-induced damage, can be administered alone, or in combination with physiologically acceptable Garners, including solvents, perfumes or colorants, stabilizers, sunscreens or other ingredients, for medical or cosmetic use. They can be administered in a vehicle, such as water, saline, or in another appropriate delivery vehicle. The delivery vehicle can be any appropriate vehicle which delivers the DNA fragments, oligonucleotides, deoxynucleotides, dinucleotides, or dinucleotide dimers. In one embodiment, the concentration of oligonucleotide can be 10 ~,M - 100 ~M.
To allow access of the active ingredients of the composition to deeper-lying skin cells, vehicles which improve penetration through outer layers of the skin, e.g., the stratum corneum, are useful. Vehicle constituents for this purpose include, but are not limited to, ethanol, isopropanol, diethylene glycol ethers such as diethylene glycol monoethyl ether, azone (1-dodecylazacycloheptan-2-one), oleic acid, linoleic acid, propylene glycol, hypertonic concentrations of glycerol, lactic acid, glycolic acid, citric acid, and malic acid. In one embodiment, propylene glycol is used as a delivery vehicle. In a preferred embodiment, a mixture of propylene glycol:ethanol:isopropyl myristate (1:2.7:1) containing 3% benzylsulfonic acid and 5% oleyl alcohol is used.
In another embodiments a liposome preparation can be used. The liposome preparation can comprise liposomes which penetrate the cells of interest or the stratum corneum, and fuse with the cell membrane, resulting in delivery of the contents of the liposome into the cell. For example, liposomes such as those described in U.S. Patent No. 5,077,211 of Yarosh, U.S. Patent No.
4,621,023 of Redziniak et al. or U.S. Patent No. 4,508,703 of Redziniak et al.
can be used. The compositions of the invention intended to target skin conditions can be administered before, during, or after exposure of the skin of the mammal to UV or agents causing oxidative damage. Other suitable formulations can employ niosomes. Niosomes are lipid vesicles similar to liposomes, with membranes consisting largely of non-ionic lipids, some forms of which are effective for transporting compounds across the stratum corneum.

Other suitable delivery methods intended primarily for skin include use of a hydrogel formulation, comprising an aqueous or aqueous-alcoholic medium and a gelling agent in addition to the oligonucleotide(s). Suitable gelling agents include methylcellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, carbomer (carbopol), hypan, polyacrylate, and glycerol polyacrylate.
In one embodiment, the DNA fragments, oligonucleotides, deoxynucleotides, dinucleotides, dinucleotide dimers, or composition comprising one or more of the foregoing, is applied topically to the skin surface. In other embodiments, the DNA fragments, oligonucleotides, deoxynucleotides, dinucleotides, dinucleotide dimers, or composition comprising one or more of the foregoing, is delivered to other cells or tissues of the body such as epithelial cells. Cells of tissue that is recognized to have a lesser barrier to entry of such substances than does the skin can be treated, e.g., orally to the oral cavity;
by aerosol to the respiratory epithelium; by instillation to the bladder epithelium; by instillation or suppository to intestinal (epithelimn) or by other topical or surface application means to other cells or tissues in the body, including eye drops, nose drops and application using angioplasty, for example. Furthermore, the oligonucleotides of the present invention can be administered intravenously or injected directly into the tissue of interest intracutaneously, subcutaneously, intramuscularly or intraperitoneally. In addition, for the treatment of blood cells, the compounds of the present invention can be administered intravenously or during extracorporeal circulation of the cells, such as through a photophoresis device, for example. As demonstrated herein, all that is needed is contacting the cells of interest with the oligonucleotide compositions of the present invention, wherein the oligonucleotides contacting the cells can be as small as dinucleotides.
The DNA fragments, oligonucleotides, deoxynucleotides, dinucleotides, dinucleotide dimers, agent that promotes differentiation, agent that increases p53 activity, or composition comprising one or more of the foregoing, is administered to (introduced into or contacted with) the cells of interest in an appropriate manner. The "cells of interest," as used herein, are those cells which may become affected or are affected by the, hyperproliferative disease or precancerous condition, or cells which are affected by oxidative stress, DNA-damaging conditions such as UV: irradiation or exposure to DNA damaging chemicals such as benzo[a]pyrene. Specifically encompassed by the present invention are epithelial cells, including melanocytes and lceratinocytes, as well as other epithelial cells such as oral, respiratory, bladder and cervical epithelial cells. As demonstrated herein, methods and compositions of the present invention can inhibit growth, induce melanogenesis and induce TNFa production in epithelial cells from numerous sources.
The oligonucleotides, DNA fragments, deoxynucleotides, dinucleotides, dinucleotide dimers, agent that promotes differentiation, agent that increases p53 activity, or composition comprising one or more of the foregoing, is applied at an appropriate time, in an effective amount. The "appropriate time" will vary, depending on the type and molecular weight of the oligonucleotides, DNA
fragments, deoxynucleotides, dinucleotides, dinucleotide dimers, or other agent employed, the condition to be treated or prevented, the results sought, and the individual patient. An "effective amount," as used herein, is a quantity or concentration sufficient to achieve a measurable desired result. The effective amount will depend on the type and molecular weight of the oligonucleotides, DNA fragments, deoxynucleotides, dinucleotides, dinucleotide dimers, or agent employed, the condition to be treated or prevented, the results sought, and the individual patient. For example, for the treatment or prevention of psoriasis, or for hyperproliferative, cancerous, or pre-cancerous conditions, or UV-induced dermatoses, the effective amount is the amount necessary to reduce or relieve any one of the symptoms of the disease, to reduce the volume, area or number of cells affected by the disease, to prevent the formation of affected areas, or to reduce the rate of growth of the cells affected by a hyperproliferative disorder.
The concentration can be approximately 2-300 ~M. In a another embodiment, the concentration of agent (e.g., oligonucleotide) is about 50-200 ~,M; in another embodiment, the concentration is about 75-150 ~,M. For some applications, the concentration of oligonucleotide can be about 10-100 wM.

In one embodiment of the present invention, oligonucleotides, DNA
fragments, such as single-stranded DNA fragments, deoxynucleotides, dinucleotides, or dinucleotide diners, agent that increases p53 activity, agent that promotes differentiation, or a composition that can comprise one or more of the foregoing, is administered, in an appropriate delivery vehicle, to the cells of interest in the mammal in order to treat or prevent a hyperproliferative disease affecting epithelial cells. The DNA fragments, oligonucleotides, deoxynucleotides, dinucleotides, dinucleotide diners, agent that. promotes differentiation, agent that increases p53 activity, or composition comprising one or more of the foregoing, can be administered systemically, can be administered directly to affected areas, or can be applied prophylactically to regions commonly affected by the hyperproliferative disease.
In another embodiment, the DNA fragments, oligonucleotides, deoxynucleotides, dinucleotides, dinucleotide diners, agent that increases p53 activity, agent that promotes differentiation, or composition comprising one or more of the foregoing, is administered to the epidermis for the treatment or prevention of oxidative stress or for the treatment or prevention of hyperproliferative, cancerous, or pre-cancerous conditions, or UV-responsive dermatoses.
In still another embodiment, DNA fragriients, oligonucleotides, deoxynucleotides, dinucleotides, dinucleotide diners, agent that promotes differentiation, agent that increases p53 activity, or a composition comprising one or more of the foregoing, can be administered, either alone or in an appropriate delivery vehicle, to the epidermis for reduction of photoaging, or prophylaxis against or reduction in the likelihood of development of skin cancer.
The DNA fragments, oligonucleotides, deoxynucleotides, dinucleotides, dinucleotide diners, agent that promotes differentiation, agent that increases p53 activity, or composition comprising one or more of the foregoing, can be administered topically or by intracutaneous injection at an appropriate time (i.e., prior to exposure of the skin to UV irradiation). The DNA fragments, oligonucleotides, deoxynucleotides, dinucleotides, dinucleotide diners, agent that promotes differentiation, agent that increases p53 activity, or composition comprising one or more of the foregoing, can be applied before, during or after exposure to a carcinogen such as UV irradiation. They can be applied daily or at regular or intermittent intervals. In one embodiment, the DNA fragments, oligonucleotides, deoxynucleotides, dinucleotides, dinucleotide dimers, agent that promotes differentiation, agent that increases p53 activity, or composition comprising one or more of the foregoing, can be administered on a daily basis to skin which may be exposed to sunlight during the course of the day.
In a further embodiment of the invention, the DNA fragments, oligonucleotides, deoxynucleotides, dinucleotides, dinucleotide dimers, agent that promotes differentiation, agent that increases p53 activity, or composition comprising one or more of the foregoing, is administered, in an appropriate delivery vehicle, to an individual (e.g., epithelial cells or other cells of an individual) for the treatment or prevention of hyperproliferative, cancerous or pre-cancerous conditions, or to repair or prevent DNA damage caused by DNA
damaging chemicals, such as benzo[a]pyrene.
As demonstrated herein, the compounds of the present invention are active in vitf°o and in vivo in their unmodified form, e.g., sequences of unmodified oligonucleotides linked by phosphodiester bonds. As used herein, the terms "oligonucleotide," "dinucleotide," "DNA fragment," etc., refer to molecules having deoxyribose as the sugar, and having phosphodiester linlcages ("phosphate backbone") as occur naturally, unless a different linkage or backbone is specified.
Furthermore, although not necessary for the ability to elicit the UV-mimetic effects of the present invention, the compounds of the present invention can be modified, derivative or otherwise combined with other reagents to increase the half life of the compound in the organism and/or increase the uptake of these compounds by the cells of interest. Modification reagents include, for example, lipids or cationic lipids. In one embodiment, the compounds of the present invention are covalently modified with a lipophilic group, an adamancy moiety. The compounds of the present invention can be modified to target _2 $_ specific tissues in the body. For example, brain tissue can be targeted by conjugating the compounds with biotin and using the conjugated compounds with an agent that facilitates delivery across the blood-brain barrier, such as anti-transfernng receptor antibody coupled to streptavidin.
S From the results of experiments described in Examples 17-20, it can be concluded that the activity of pTpT and the activity of the telomere overhang repeat homolog oligonucleotide 1 liner-1 (SEQ m NO: 5) require that they be hydrolyzable to induce apoptosis, cell cycle arrest, and melanogenesis. The non-hydrolyzable phosphorothioate form of these oligonucleotides has the ability to inhibit the activity of the hydrolyzable molecules and can be used to block the intracellular responses in vivo as well as in vitf°o.
The Examples, especially Example 31, demonstrate that telomere homolog oligonucleotides but not complementary or unrelated DNA sequences of the same length induce an S-phase arrest and apoptosis in an established human lymphocyte line and in a human melanoma cell line. The effects are not due to selective uptake of the telomere homolog, as the control sequences are taken up comparably in the present study. Oligonucleotides partially homologous to the 3' overhang sequence produce qualitatively the same responses but to a lesser degree or at a higher concentration. Thus, an 11 base oligonucleotide with 55% identity with the telomere overhang repeat sequence telomere is intermediate between the l liner-1 (100% nucleotide sequence identity with telomere repeat) and l liner-2 (complementary to telomere repeat) or l liner-3 (unrelated to telomere repeat) in producing apoptosis. pTT, corresponding to one-third of the TTAGGG (SEQ m NO:11) repeat sequence, duplicates the l liner-1 effect on cell cycle arrest but at a 4-fold higher concentration. In work comparing pTT to a 9 base oligonucleotide with 56%
sequence identity with (TTAGGG)n, the oligonucleotide having 56% sequence identity was found to have an ability comparable to pTT to induce p53 and p53-regulated genes at 40% the concentration, at 40 ~.M versus 100 ~M pTT.
The oligonucleotide pTT has been shown to induce and activate p53 and to transcriptionally upregulate a number of genes, many but not all of which are lcnown to be p53 regulated. Experiments with the 1 lmer-1 complete homolog demonstrate that the S-phase arrest induces results from phosphorylation of the p95/Nbsl protein, which also mediates S-phase arrest following ionizing radiation. Of note, this decreases proliferation of cells lacking functional p53.
Furthermore, as anticipated from the fact that p95/Nbsl is phosphorylated by the ATM lcinase (Gatei, M., et al., (2000), Nat Genet 25, 115-119; Wu, X.,et al., (2000), Nature 25, 477-482; Lim, D.S., et al., (2000), Nature 404, 613-617;
Zhao, S., et al. (2000), Nature 405, 473-477), the oligonucleotide effects also require ATM and are not observed in cells from patients with ataxia telangiectasia in whom the ATM kinase is mutated.
Experimental telomere disruption [I~arlseder, J., et al., 1999, 8cieyzce 283: 1321-1325] and cellular manipulations that precipitate premature senescence, such as transfection with the ras oncogene or exposure to oxidative stress, are known to digest the 3' telomere overhang and/or to shorten overall telomere length. In contrast, exposure of cells to telomere homolog oligonucleotides in the present studies increases mean telomere length (MTL).
While not wishing to be bound by a single mechanism, these data strongly suggest that the oligonucleotides can activate telomerase, presumably by inducing TERT, and imply that transient activation of telomerase may be a part of the physiologic telomere-based DNA damage response that also includes activation of the ATM lcinase with subsequent signaling through p53 and p95/Nbsl. The apparent ability of oligonucleotides to induce this response in the absence of DNA damage and telomere disruption offers the possibility of "rejuvenating" cells through'telomere elongation, as recently reported in human skin equivalent constructs containing fibroblasts transfected with TERT, without the enhanced risk of carcinogenesis observed even in even normal cells that ectopically express telomerase. Equally, the phenomenon suggests that the advantages of robust DNA damage responses of the type observed in p53 over-expressing mice could be separated from the premature senescence also observed 30, in the transgenic animals (Tyner, S.D., et al., 2002, Natuf°e 415:
45-523). Such "rejuvenation" or delay in acquiring the senescent phenotype associated with critical telomere shortening would be in addition to other benefits that might accrue from treatment with oligonucleotides partially or completely homologous to the TTAGGG (SEQ m N0:11) repeated sequence. Based on extensive worlc in vivo as well as in vitro, these are understood to include sunless tanning and related photoprotection, enhanced DNA repair capacity, cancer prevention and treatment, and immunomodulation.
Under normal conditions, the 3' telomere overhang DNA sequence is believed to be folded baclc and concealed in a loop structure stabilized by [Griffith, J.D., et al., (1999), Cell 97, 503-514]. However, this sequence might be exposed if the telomere were distorted, for example by ultraviolet (IJV)-induced thymine dimers or carcinogen adducts involving guanine residues (as with cisplatin or benzo[a]pyrene) that could render the loop-back configuration unstable. Exposure of the TTAGGG (SEQ m NO:11) repeat sequence could be the initial signal leading to a variety of DNA damage responses, dependent on cell type as well as intensity and/or duration of the signal. These responses include cell cycle arrest, apoptosis, and a more differentiated sometimes adaptive phenotype, for example, increased melanin production (tanning). See Figure 35.
The proposed model predicts that inability to repair damage to telomeric DNA would lead to exaggerated damage responses, such as p53 induction and apoptosis, as has been reported for UV-irradiated xeroderma pigmentosum cells that cannot efficiently remove DNA photoproducts (Dumaz, N., et al., 199, Carci~ogenesis 19: 1701-1704). This model is further consistent with the recent finding that transgenic mice with supra-normal p53 activity are highly resistant to tumors, yet age prematurely (Tyner~ S.D., et al., 2002, Nature 41 S: 45-523).
A DNA damage recognition mechanism might have evolved to contain predominantly thyrnidine and 'guanine bases. The TTAGGG (SEQ ~ NO:l 1) repeat sequence is an excellent target for DNA damage, as dithymidine sites most commonly participate in formation of UV photoproducts (Setlow, R.B. and W.L. Carrier. 1966, JMoI Biol 17: 237-254) and guanine is both the principal site of oxidative damage, forming ~-oxoguanine [Kasai, H. and S. Nishimura, 1991. "Oxidative Stress: Oxidants and Antioxidants," pp. 99-116 In H. Sies (ed.) (London, Academic Press, Ltd.)], as well as the base to which most carcinogens form adducts [Friedberg, E.C., et ccl., 1995, pp. 1-58 In E.C. Friedberg, G.C.
Wallcer and W. Siede, eds. (Washington, D.C.,, ASM Press)].
The postulated telomere-initiated signal transduction pathway would provide a single evolutionary point of departure for several distinct defenses against carcinogenesis in higher organsms: permanent loss of proliferative capacity (senescence) in cells expected on a statistical basis alone to have accumulated multiple mutations throughout the genome during prolonged environmental exposure and serial rounds of DNA replication and cell division;
transient cell cycle arrest to increase the time available for repair before resuming DNA replication; activation of a cell suicide program to remove cells from the tissue if damage exceeds the repair capacity, since acute telomeric damage would be expected to reflect the degree of DNA damage throughout the genome; and induction over several days of adaptive responses, such as taiming, to reduce DNA photoproduct formation and/or enhanced DNA repair capacity to prevent damage from similar insults in the future.
The invention includes methods for treating a hyperproliferative disorder in a manunal, in which the therapy includes administering to the mammal an effective amount of a composition comprising one or more oligonucleotides which share at least 50°/o nucleotide sequence identity with the vertebrate telomere overhang repeat. These methods can be applied especially to human subj ects. Hyperproliferative disorders can be characterized by benign growth of cells beyond a normal range, and which sometimes may result in a benign tumor or widespread epidermal thickening, as in psoriasis. Also among the various hyperproliferative disorders to be treated by these methods are cancer as it is manifested in various forms and arising in various cell types and organs of the body, for example, cervical cancer, lymphoma, osteosarcoma, melanoma and other cancers arising in the skin, and leukemia. Also among the types of cancer cells to which the therapies are directed are breast, lung, liver, prostate, pancreatic, ovarian, bladder, uterine, colon, brain, esophagus, stomach, and thyroid.

The oligonucleotides can be administered in the methods of treatment described herein as a single type of oligonucleotide or in a combination comprising with one or more different oligonucleotides. Oligonucleotides without a 5' phosphate can be used in any of the methods of therapy for treatment, or for the reduction of incidence of a disease or disorder described herein. However, oligonucleotides having a 5' phosphate are preferred, as it has been shown that the 5' phosphate improves uptal~e of the oligonucleotide into cells. The oligonucleotides can be at least 2 nucleotides in length, preferably 2-200 nucleotides, and more preferably from 2 to 20 nucleotides in length.
Oligonucleotides 5-11 nucleotides are more preferred.
The telomere overhang repeat sequence of vertebrates is (TTAGGG)".
The invention encompasses methods in which the oligonucleotides administered for therapy have at least 50% nucleotide sequence identity to the telomere repeat sequence. Preferred are oligonucleotides having at least 60% nucleotide sequence identity. More preferred are oligonucleotides having at least 70%
nucleotide sequence identity. Still more preferred are those oligonucleotides having at least 80% nucleotide sequence identity. More highly preferred are oligonucleotides having at least 90% nucleotide sequence identity. Most preferred are oligonucleotides having 100% nucleotide sequence identity to (TTAGGG)". The particular telomere repeat homologs pGAGTATGAG (SEQ
B? NO:l), pGTTAGGGTTAG (SEQ ID NO:S; also called "1 lmer-1" and "T
oligo" herein), pGGGTTAGGGTT (SEQ ID N0:13) and pTAGATGTGGTG
(SEQ m N0:14) are especially preferred where it is desired to use an oligonucleotide sharing at least 50% nucleotide sequence identity with the telomere repeat sequence. Other oligonucleotides having at least 50%
nucleotide sequence identity with the telomere overhang repeat sequence can be used as anti-proliferative agents, for example pTAGGAGGAT (SEQ m N0:2), pAGTATGA (SEQ ID N0:3), pGTATG (SEQ m N0:4), pTTAGAGTATGAGTTA (SEQ >D NO:16), pTTAGAGTATGAG (SEQ m N0:17), and pGAGTATGAGTTA (SEQ ID N0:18).

Oligonucleotides are relatively short polynucleotides. Polynucleotides are linear polyners of nucleotide monomers in which the nucleotides are linlced by phosphodiester bonds between the 3' position of one nucleotide and the 5' position of the adjacent nucleotide. Unless otherwise indicated, the "oligonucleotides" of the invention as described herein have a phosphodiester backbone.
To enhance delivery through tl~e slcin, the oligonucleotides of the invention may be modified so as to either mask or reduce their negative charges or otherwise alter their chemical characteristics. This may be accomplished, for example, by preparing ammonium salts of the oligonucleotides using readily available reagents and methods well known in the art. Preferred ammonium salts of the oligonucleotides include trimethyl-, triethyl-, tributyl-, tetramethyl-, tetraethyl-, and tetrabutyl- ammonium salts. Ammonium and other positively charged groups can be convalently bonded to the oligonucleotide to facilitate its transport accross the stratum comeum, using an enzymatically degradable linkage that releases the oligonucleotide upon arrival inside the cells of the viable layers of the epidermis.
Another method for reducing or masking the negative charge of the oligonucleotides includes adding a polyoxyethylene spacer to the 5' phosphate groups of the oligonucleotides and/or the inteW al phosphates of the oligonucleotides using methods and reagents well known in the art. This, in effect, adds a 6- or 12- carbon modifier (linker) to the phosphate that reduces the net negative charge by +1 and makes the oligonucleotides less hydrophilic.
Further negative charge reduction is achieved by addding a phosphoroamidite to the end of the polyoxyethylene linker, thereby providing an additional neutralizing positive charge.
The phosphodiester backbone of the oligonucleotides of the present invention can also be modified or synthesized to reduce the negative charge. A
preferred method involves the use of methyl phosphonic acids (or chiral-methylphosphonates), whereby one of the negatively charged oxygen atoms in the phosphate is replaced with a methyl group. These oligonucleotides are similar to oligonucleotides having phosphorothioate linkages which comprise a sulfate instead of a methyl group and which are also within the scope of the present invention.
The oligonucleotides of the present invention can also take the form of peptide nucleic acids (PNAs) in which the bases of the nucleotides are connected to each other via a peptide backbone.
Other modifications of the oligonucleotides such as those described, for example, in US Patent Nos. 6,537,973 and 6,506,735 (both of which are incorporated herein by reference for all of the oligonucleotide modifications described therein) and others will be readily apparent to those skilled in the art.
The oligonucleotides can also be "chimeric" oligonucleotides which are synthesized to have a combination of two or more chemically distinct backbone linkages, one being phosphodiester. In one embodiment are chimeric oligonucleotides with one or more phosphodiester linkages at the 3' end. In another embodiment are chimeric oligonucleotides with one or more phosphodiester linkages at the 3' and 5' ends.
As reported in Example 46, 11-mer oligonucleotides with sequence SEQ
ID NO:S were synthesized to contain phosphodiester linkages throughout, phosphorothioate linkages throughout, or a combination of linkages. One oligonucleotide had two phosphorothioate linlcages at the 5' end; another oligonucleotide had two phosphorothioate linkages at the 3' end; still another had two phosphorothioate linlcages at each end. Oligonucleotides with phosphodiester linkages at the 3' end were found to be the most effective at stimulating reactions associated with senescence in fibroblasts. Thus, enzymatic cleavage at the 3' end of the oligonucleotide may be a step in induction of the senescence response.
Sequence identity is determined by a best fit alignment of the oligonucleotide in question with (TTAGGG)". The sequences are compared at each position, and a determination of "match" or "no match" is made at each nucleotide position, and the percent of matches, without resorting to deletion or insertion in either sequence, is the percent identity of the sequences as counted along the oligonucleotides in question. Thus, by illustration, SEQ m NO:S
shares 100% sequence identity with (TTAGGG)". SEQ a7 NO:1 shares 5/9 sequence identity with (TTAGGG)". SEQ m N0:4 shares 3l5 sequence identity with (TTAGGG)". pTT shares 100% sequence homology with (TTAGGG)".
Examples of particular molecules found to have an anti-proliferative effect, and thus appropriate for use in methods to reduce proliferation of cells in a mammal, are the oligonucleotides,thymidine dinucleotide ("pTpT' or "pTT" or "TZ" ) and pGTTAGGGTTAG (SEQ m NO:S).
Another part of the invention is a method for promoting differentiation of malignant cells in a mammal, the method comprising administering to the mammal an effective amount of a composition comprising one or more oligonucleotides which share at least 50% nucleotide sequence identity with (TTAGGG)n. A differentiated state, can in many ways, be considered the opposite of a malignant state. Depending on the cell type, differentiation can 1 S involve the regulation of expression of a number of different genes to result in an increase or decrease in certain enzymatic activities, or cell surface proteins, for example. For example, melanocytes respond to oligonucleotides with an increase in tyrosinase expression.
Herein, an association has been found between the inhibition of growth of cancer cells, caused by the cells taking up oligonucleotides with sequence identity to the telomere repeat sequence, and an increase in the appearance on the cell surface of antigens typical of differentiated cells, rather than cancer cells.
Thus, a further method of the invention is to enhance the expression of one or more surface antigens indicative of differentiation of cancer cells in a mammal, said method comprising administering to the mammal an effective amount of one or more oligonucleotides as described herein, for example, one or more oligonucleotides which share at least 50% nucleotide sequence identity with the vertebrate telomere overhang repeat.
This inducement of the cells to a more differentiated state, or to take on one or more characteristics of differentiation, can be exploited in immunotherapy methods. The surface antigens associated with a differentiated state, fragments thereof, or synthetic peptides derived from the studies of the externally exposed loops of the surface antigens, can be incorporated into a vaccine to induce a cancer patient to produce cytotoxic T lymphocytes against the cells displaying the cell surface antigen. For example, in melanoma cells, the cell surface antigens MART-l, tyrosinase, TRP-1 or gp-100, or combinations thereof, can be made to increase on the cell surface when the cells talce up oligonucleotides sharing at least 50% nucleotide sequence identity with the telomere'overhmg repeat. See Example 29. These cell surface antigens can become targets for immunotherapy, for example by vaccinations with the isolated cell surface antigen or peptides having amino acid sequences derived from the surface loops of the antigens. See, for example, Jager, E. et al., Int. J. Cancer 66:470-476, 1996; I~awakami, Y. et al., J. Immunol. 154:3961-3968, 1995; and de Vries, T.J.
et al., J. Pathol. 193:13-20, 2001.
Telomerase has been a target for antiproliferative methods based on theories of using antisense oligonucleotides to bind to the RNA portion of the enzyme. However, the therapeutic methods described herein can be used independently of the presence or function of telomerase in the target cells.
The telomere repeat overhang homolog pGTTAGGGTTAG (SEQ ID NO:S) was seen to bring about S-phase cell cycle arrest in normal fibroblasts and in cells of the osteosarcoma cell line Saos-2, neither of wluch have telomerase activity.
See Examples 32 and 34. Thus, for cancer cells, most of which have telomerase activity, but some of which do not, the present method can be used regardless of telomerase activity. The inhibition of growth of the cancer cells is characterized by cell cycle arrest, apoptosis, and/or differentiation to a more differentiated state. A method for inhibiting the growth of cancer cells in a mammal (e.g., human), operationally independent of the telomerase (+) or telomerase (-) state of the cancer cells, is to administer to the mammal an effective amount of a composition comprising one or more oligonucleotides which share at least 50%
nucleotide sequence identity with the telomere overhang repeat.
The experiments described in Example 34 demonstrate that the function of a wild type p53 protein is also not necessary to bring about the S-phase cell cycle arrest in tumors or tumor cells treated with an oligonucleotide with at least 50% sequence identity to the telomere repeat sequence. A p53-null osteosarcoma cell line was shown to respond,to the addition of pGTTAGGGTTAG (SEQ m NO:S) by arresting in S-phase. Thus, the method for inhibiting the growth of cancer cells in a mammal (e.g., human), the method including administering to the mammal an effective amount of a composition comprising one or more oligonucleotides which share at least 50% nucleotide sequence identity with the telomere overhang repeat, can be carned out whether or not the target cells have normal p53 function.
- The invention further comprises a method for preventing the sequelae of exposure of the slcin of a mammal to ultraviolet light -- spongiosis, blistering or dyskeratosis, or any combination of these -- by administering to the skin an effective amount of a composition comprising one or more oligonucleotides which share at least 50% nucleotide sequence identity with the telomere overhang repeat. The steps or steps of this method can also be used in the reduction in the incidence of skin cancer in a human, and is particularly applicable to reduce the occurrence of skin cancer in patients with xeroderma pigmentosum or other genetic predisposition to skin cancer. The method of applying to the skin an effective amount of a composition comprising one or more oligonucleotides which share at least 50% nucleotide sequence identity with the telomere overhang'repeat is also a method for enhancing repair of ultraviolet irradiation-induced damage to skin. Any of the oligonucleotides described herein and characterized by having at least 50% sequence identity to the telomere repeat sequence found in humans -- the oligonucleotides pGAGTATGAG (SEQ m NO:l), pCATAC (SEQ m N0:6), pGTTAGGGTTAG (SEQ m NO:S), pGGGTTAGGGTT (SEQ m N0:13), or pTAGATGTGGTG (SEQ m N0:14), for example, can be used in the methods.
Further, the oligonucleotides pTT and pGAGTATGAG (SEQ m NO:1) can also be used in the methods.
Oxidative damage is characterized by the reaction products of reactions of molecules found in the cells with reactive oxygen species (ROS), such as hydrogen peroxide, hydroxyl radicals, and superoxide. Oxidative damage can result, for instance, from normal cellular metabolism, UVA irradiation, ionizing radiation, or exposure to a variety of chemicals. Reactive oxygen species can be measured in a number of ways. One assay employs a probe dichlorofluorescein diacetate (Molecular Probes, Inc.), a colorless reagent that is taken up by the cells and becomes fluorescent upon oxidation by ROS. The level of fluorescence correlates with the intracellular ROS level.
Applicants also described a method for reducing oxidative damage in a mammal, the method comprising administering to the mammal, especially to the skin of the mammal, an effective amount of a composition comprising one or more oligonucleotides which share at least 50% nucleotide sequence identity with the human telomere overhang repeat. Preferred are embodiments in which the oligonucleotide is pGAGTATGAG (SEQ m NO:l). Also effective in the method is pTT. See results in Examples 15, 21, 22, 23 and 52 suggesting that oligonucleotide treatment enhances the ability of cells to repair oxidative DNA
damage.
Applicants have further described a method for treating melanoma in a mammal, comprising administering to the human an effective amount of a composition comprising one or more oligonucleotides that share at least 50%
nucleotide sequence identity with the human telomere overhang repeat. In particular cases, the oligonucleotide can be pGTTAGGGTTAG (SEQ m NO:S);
pTT can also be used in the method. Applicants have shown the effectiveness of oligonucleotide therapy using human melanoma cells in a mouse model. See Examples 30, 49, 50 and 51.
Another aspect of the invention concerns a method for reducing proliferation of keratinocytes in the slcin of a human, in which the method comprises applying to the shin an effective amount of a composition comprising one or more oligonucleotides that share at least 50% nucleotide sequence identity with the human telomere overhang repeat. The method is applicable to disorders of the skin characterized by proliferation of keratinocytes in the skin, such as seborrheic lceratosis, actinic, keratosis, Bowen's disease, squamous cell carcinoma or basal cell carcinoma. pTT is effective in the method..
The present invention includes the method of treating a disease or disorder in a mammal, wherein the disease or disorder is characterized by abnormal proliferation of cells, including, but not limited to, cancers, solid ' tumors, blood-born tumors (e.g., leukemias), tumor metastases, benign tumors (e.g., hemangiomas, acoustic neuromas; neurofibromas, trachomas, and pyogenic granulomas), and psoriasis. Hyperproliferative disorders can also be those characterized by excessive or abnormal stimulation of fibroblasts, such as scleroderma, andhypertrophic scars (i.e., keloids).
The oligonucleotide or oligonucleotides to be used in therapies to alleviate hyperproliferative disorders such as cancer can be used in a composition in combination with a pharmaceutically or physiologically acceptable carrier. Such a composition may also contain in addition, diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. Cationic lipids such as DOTAP [N-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium salts may be used with oligonucleotides to eWance stability. Oligonucleotides may be complexed with PLGA/PLA copolymers, chitosan or fumaric acid/sebacic acid copolymers for improved bioavailability {where PLGA is [poly(lactide-co-glycolide)]; PLA is poly(L-lactide)}. The terms "pharmaceutically acceptable" and "physiologically acceptable" mean a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the Garner will depend on the route of administration.
A composition to be used as an antiproliferative agent may further contain other agents which either enhance the activity of the oligonucleotide(s) or complement its activity or use in treatment, such as chemotherapeutic or radioactive agents. Such additional factors and/or agents may be included in the composition to produce a synergistic effect with the oligonucleotide(s), or to minimize side effects. Additionally, administration of the composition of the present invention may be administered concurrently with other therapies, e.g., administered in conjunction with a chemotherapy or radiation therapy regimen.
The oligonucleotides as described herein can be used in combination with other compositions and procedures for the treatment of diseases. For example, a tumor may be treated conventionally with surgery, radiation, chemotherapy, or immunotherapy, combined with oligonucleotide therapy, and then oligonucleotides may be subsequently administered to the patient to extend the dormancy of micrometastases and to stabilize and inhibit the growth of any residual primary tumor.
The compositions of the present invention can be in the form of a liposome in which oligonucleotide(s) of the present invention are combined, in addition to other pharmaceutically acceptable Garners, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like.
Pharmaceutical compositions can be made containing oligonucleotides to be used in antiproliferative therapy. Administration of such pharmaceutical compositions can be carried out in a variety of conventional ways lcnown to those of ordinary skill in the art, such as oral ingestion, inhalation, for example, of an aerosol, topical or transdennal application, or intracranial, intracerebroventricular, intracerebral, intravaginal, intrauterine, oral, rectal or parenteral (e.g., intravenous, intraspinal, subcutaneous or intramuscular) route, or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection.
The route of administration can be determined according to the site of the tumor, growth or lesion to be targeted.
To deliver a composition comprising an effective amount of one or more oligonucleotides to the site of a growth or tumor, direct inj ection into the site can be used. Alternatively, for accessible mucosal sites, ballistic delivery, by coating the oligonucleotides onto beads of micometer diameter, or by intraoral jet inj ection device, can be used.

Viral vectors for the delivery of DNA in gene therapy have been the subject of investigation for a number of years. Retrovirus, adenovirus, adeno-associated virus, vaccinia virus and plant-specific viruses can be used as systems to package and deliver oligonucleotides for the treatment of cancer or other growths. Adeno-associated virus vectors have been developed that cannot replicate, but retain the ability to infect cells. An advantage is low immunogenicity, allowing repeated administration. Delivery systems have been reviewed, for example, in Page, D.T. and S. Cudmore, Df~ug Discovefy Toclay 6:92-1010, 2001.
Studies carried out using oligonucleotides on the theory of their inhibiting the function of a target nucleic acid (antisense oligonucleotides), most of these studies carried out with phosphorothioate oligonucleotides, have found effective methods of delivery to target cells. Antisense oligonucleotides in clinical trials have been administered in saline solutions without special delivery vehicles (reviewed in Hogrefe, R.L, Antisense and Nucleic Acid Drug Development 9:351-357, 1999).
Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the fluids of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or mufti-dose containers for example, sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid ca 'rner, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous inj ection should contain, in addition to oligonucleotide(s) of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art. The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives lcnown to those of slcill in the art.
Use of timed release or sustained release delivery systems are also included in the invention. Such systems are highly desirable in situations where surgery is difficult or impossible, e.g., patients debilitated by age or the disease course itself, or where the risk-benefit analysis dictates control over cure.
One method is to use an implantable pump to deliver measured doses of the formulation over a period of time, for example, at the site of a tumor.
A sustained-release matrix can be used as a method of delivery of a pharmaceutical composition comprising oligonucleotides, especially for local treatment of a growth or tumor. It is a matrix made of materials, usually polymers, which are degradable by enzymatic or acid/base hydrolysis or by dissolution. Once inserted into the body, the matrix is acted upon by enzymes and body fluids. The sustained-release matrix desirably is chosen from biocompatible materials such as liposomes, polylactides (polylactic acid), polyglycolide (polymer of glycolic acid), polylactide co-glycolide (co-polymers of lactic acid and glycolic acid) polyanhydrides, poly(ortho)esters, polyproteins, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, p~lyamino acids, amino acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone. A preferred biodegradable matrix is a matrix of one of either polylactide, polyglycolide, or polylactide co-glycolide (co-polymers of lactic acid arid glycolic acid).
The amount of oligonucleotide of the present invention in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone. For a human patient, the attending physician will decide the dose of oligonucleotide of the present invention with which to treat each individual patient. hzitially, the attending physician can administer low doses and observe the patient's response. Larger doses may be administered until the optimal therapeutic effect is..obtained for the patient, and at that point the dosage is not increased further. The duration of therapy using the pharmaceutical composition of the present invention will vary, depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual patient.
The invention is further illustrated by the following non-limiting Examples.
EXAMPLES
EXAMPLE 1: Application to Human Squamous Carcinoma Cells Human squamous carcinoma cell line SCC12F cells were maintained in primary keratinocyte medium (300 ml DME, 100 ml F-12 nutrient supplement, 50 ml 10x adenine, SO ml fetal bovine serum, 5 ml penicillin/streptomycin stock, and 0.5 ml of 10 ~,g/ml epidermal growth factor and hydrocortisone to final concentration of 1.4 ~,g/ml) and dosed with either water (diluent), 100 ~M
pTpT
(T2, Midland Certified Reagent Company, Midland, TX) or 100 ~M pdApdA
(AZ). Cells were harvested before dosing (day 0), and l, 3, 4, and 5 days after dosage, and were counted by CoulterTM counter. After harvesting, the cells were processed for total RNA isolation and were analyzed by Northern blot. Addition of pTpT to human squamous carcinoma cells resulted in marked decreases in cell growth rate, as shown in Figure 1. Addition of a control deoxyadenine dinucleotide (pdApdA), a compound very similar to pTpT but not readily dimerized by UV irradiation, and therefore rarely excised during the course of UV-induced DNA repair, has no effect.
In a second experiment, SCC12F cells were cultured as described above.
Two or three days after seeding, the preconfluent cultures were given fresh medium supplemented with either 100 ~,M Ta or diluent as a control. Cells were collected daily by trypsinization and counted by CoulterTM counter. The cell yield in cultures treated with Tz was reduced by 75% compared to that of paired control cultures after five days (Figure 2). This corresponds to 2.3 population doublings in this time for control cells, compared with 1 doubling for TZ-treated cells. These results further demonstrate that application of TZ DNA fragments inhibits cell proliferation, including proliferation of cancerous cells.
Tn a third experiment, it was demonstrated that addition of TZ to human squamous carcinoma cells for 24-72 hours resulted in upregulation of at least three genes: growth arrest and DNA damage (GADD 45), senescence-derived inhibitor (Sdi I), and excision repair cross-complementing (ERCC-3). Paired cultures of SCC12F cells were maintained in a Dulbecco's modified Eagle's Medium-based keratinocyte growth medium (DMEM; GIBCOBRL, Gaithersburg, MD) supplemented with 10% fetal calf serum (Hyclone Labs, Logan, UT) and epidermal growth factor as described (Hollander, M.C. et al., J.
Biol. Clzem. 265:328-336 (1992)). Pre-confluent cultures were given fresh medium supplemented with either 100 ~,M Tz, or an equal volume of diluent.
Cells were collected daily after additions, and processed for total RNA
isolation using the Tri-Reagent extraction method (Molecular Research Center, Cincimiati, OH) following the protocol of the manufacturer. Ten micrograms of RNA from each sample was gel electrophoresed, transferred to a nylon filter and probed as described previously (Nada, A. et al., Exp. Cell Res. 211:90-98 (1994)). The cDNA for GADD 45 was generated by PCR using primers based on the human GADD 45 gene sequence (Mitsudomi, T. et al., Oneogene 7:171-180 (1992)). The cDNA for ERCC 3 was purchased from the American Type Culture Collection (ATCC, Rockville, MD). The SDI 1 cDNA was a gift of Dr.
J. Smith and has been described previously (Walworth, N.C. and Bernards, R., Science 271:353-356 (1996)).
Compared to the diluent control, the mRNAs for GADD 45, ERCC 3 and SDI 1 were up-regulated in TZ-treated cells as early as 24 hours, and remained elevated for several days. Addition of the control AZ was less effective or ineffective in inducing these genes. Comparable data have been obtained in experiments with S91 melanoma cells, and normal human fibroblasts.

The time course of induction is,similar to that observed after UV
irradiation for the two genes for which this has been studied [GADD 45 and p21 (also called Sdi I)] and also similar to.the time course of induction of the tyrosinase gene by Tz in melanocytes and melanoma cells. Sdi I is known to be involved in cell cycle regulation and specifically in blocking cell division.
GADD 45 and ERCC-3, a human DNA repair enzyme, are known to be involved in repair of UV-induced DNA damage. The response toTz is identical to that observed after LTV irradiation of these cell lines, and is also similar to the response to various antimetabolites, such as methotrexate, that are clinically effective in the treatment of hyperproliferative skin disorders.
EXAMPLE 2: Application to Human Cervical Carcinoma Cells Human cervical carcinoma cells (HeLa cells) were maintained in DME +
10% calf serum and dosed with either water (diluent) or 100 wM TZ. Cells were collected 1, 4 and 6 days after dosage and counted by CoulterTM counter.
Addition of TZ to the human cervical carcinoma cells resulted in marked decreases in cell growth rate, as shown in Figure 3.
EXAMPLE 3: Application to Human Melanoma Cells Human melanoma cell lines CRL 1424, Malma, Sk Mel 2, and Sk Mel 28 were obtained from the American Type Culture Collection (ATCC). The cell lines were maintained in DME + 2% calf serum, and dosed with either water (diluent) with DME, or 100 ~,M Tz in DME. One weelc after dosage, cells were collected and counted by CoulterTM counter.
Addition of TZ to any of the four different human melanoma cell lines results in marked decreases in cell yields, as shown in Figure 4.
EXAMPLE 4: Application to Human Keratinocytes Normal human neonatal keratinocyte cells were cultured as described above in Example 1 for SCC12F cells, and treated with either 100 ~,M TZ or diluent as a control. Cells were harvested for cell counts. The cell yield in cultures treated with TZ was reduced by 63% compared to that of paired control cultures after three days (Figure 5). This corresponds to one population doubling in this time for control cells, while the number of TZ-treated cells remained the same. These results demonstrate that application of the DNA fragments inhibits cell proliferation.
Northern blot analysis of the normal human keratinocytes treated with TZ
for 24-72 hours that showed induction of the tumor necrosis factor alpha gene (TNFa) . This immunomodulatory cytolcine, known to be induced by UV
irradiation, may thus be induced by T2. Use of locally applied DNA fragments, deoxynucleotides, dinucleotides, or dinucleotide dimers is useful in irnmunomodulation of cutaneous reactions and in treatment or prevention of diseases or conditions involving immune mediators.
EXAMPLE 5: Inhibition of Cell Growth of Normal Neonatal Fibroblasts by DNA Fragments Normal human neonatal fibroblasts were plated in Falcon P35 culture dishes at a density of 9 x 10~ cells/dish. The culture medimn was DME + 10%
calf serum, 2 ml per plate. One day after plating, cultures were supplemented with either 100 ~,M TZ in DME or 100 ~M AZ in DME, or water (control). Two plates were collected and counted before the additions to give a starting, or "day 0," reading. Duplicate plates of each condition were harvested through five days after addition of the supplements and cell number determined. All cell counts were done by CoulterTM Counter. Results of two experiments, are shown in Figures 6 and 7. The results indicate that application of the DNA fragments inhibits cell proliferation.
In a second experiment, normal human neonatal fibroblasts were plated and cultured, as described above in Example 1 for SCC12F cells. Cultures were supplemented with either 100 ~,M TZ or water (control), and cells were harvested for cell counts. The cell yield in fibroblast cultures treated with TZ was reduced by 40% compared to that of paired control cultures after tluee days (Figure 8).
This corresponds to 4 population doublings in this time for control cells, compared with 3.6 doublings for:T2-treated cells. These results further demonstrate that contacting cells of interest with the DNA fragments of the present invention inhibits cell proliferation.
EXAMPLE 6: Effect of pTpT Applications, on Epidermal Cell Proliferation Guinea pigs received one or two daily topical applications of 100 ~,M
pTpT, or vehicle alone as control, for three days. On the fourth day, punch biopsies were obtained and maintained for 7 or 8 hours in primary lceratinocyte medium supplemented with 10 ~Ci/ml 3H-thymidine (specific activity 9.0 Ci/m mole, NEN). Proliferating cells are expected to incorporate the 3H-thyrnidine into newly synthesized DNA. Tissues were then rinsed with cold medium and fixed in 10% phosphate buffered formalin. After a series of dehydration steps, tissues were embedded in paraffin. 6 ~m sections were cut and mounted onto glass slides, dipped in NTB-2 Nuclear Track emulsion and kept in the darlc at 4°C for 7 days. Sections were developed in Kodak D-19 developer and stained with hematoxylin and eosin. Labeling index, a measure of DNA replication and therefore cell proliferation, was measured by calculating the percentage of labeled nuclei among 100 basal keratinocytes. Results are shown in Table 1.

Table 1 Labeling Index 2 daily applications Vehicle control pTpT
4 ~ 1.4 1.5 ~ 0.7 1 daily application Vehicle control pTpT
4.5 ~ 2.1 2 ~ 0 Mean values ~ SD are shown.
Labeling index (a measure of epidermal cell proliferation) is less in pTpT-treated skin than in vehicle-treated skin, (p < 0.03 paired T test) in both experiments.
These results demonstrate that contacting cells of interest with the DNA
fragments of the present invention inhibits cell proliferation.
EXAMPLE 7: Role of p53 in DNA Repair Both the GADD 45 and SDI 1 genes are lmown to be transcriptionally regulated by the tumor suppressor protein p53. After UV- and y-irradiation, as well as treatment of cells with DNA-damaging chemical agents, there is a rapid stabilization, phosphorylation and nuclear accumulation of p53 after which this protein binds to specific promoter consensus sequences and modulates the transcription of regulated genes. Recent data suggest that p53 can also be activated by the binding of small single-stranded DNAs, as well as certain peptides and antibodies, to a carboxyl terminal domain of this protein. W
order to determine whether the inhibitory effect of the dinucleotide pTpT on cell proliferation is mediated in part through p53, the growth response of a p53 null cell line, H1299 lung carcinoma cells, was examined. The p53-null H1299 cells (Sanchez, Y. et al., Science 271:357-360 (1996)) were maintained in DMEM
with 10% calf serum. Preconfluent cultures were given fresh medium supplemented with either 100 ~M pTpT or diluent. Cells were collected on consecutive days by trypsinization, and counted by CoulterTM counter. As shown in Figure 9, there was no inhibition of proliferation of pTpT-treated H1299 cells compared to diluent-treated controls.
fii another experiment, pTpT was found to induce the expression of SDI
1 mRNA in a p53-dependent manner. Preconfluent cultures of H1299 cells were transfected with an expression vector containing the wild type human p53 cDNA
under the control of the human cytomegalovirus promoter/enhancer (Dr. Bert Vogelstein, Johns Hopkins Oncology Center). Control transfections were performed using the vector from which the p53 cDNA was removed.
Transfections were carried out using the Lipofectin Reagent Kit (GIBCO/BRL).
One day after transfection, cells were collected for Western blot analysis using ~,g total protein as described (Yaar, M. et al., J. Clih. Invest. 94:1550-1562 (1994)). p53 was detected using mAb 421, anti-mouse Ig linked to horseradish peroxidase (Amersham, Arlington Heights, IL,) and an ECL-kit (Amersham), 15 following the directions of the manufacturer. At the time of protein collection, duplicate cultures of H1299 cells transfected with the p53 expression vector (designated "p53") or control vector ("Ctrl") were given either diluent (DMEM) or 100 ~M pTpT. After 24 hours, the cells were collected, processed for RNA
isolation and Northern blot analysis with an SDI 1 cDNA probe. The 20 autoradiograph was scanned using a'Maciutosh IIsi computer and Macintosh One Scanner, and the brightness and contrast were adjusted to display differences in autoradiographic signals maximally. The results indicated that p53-null H1299 cells express a very low level of the SDI 1 transcript and this level is not affected by addition of pTpT. Transfection of these cells with a wild-type p53 expression vector increased the level of SDI 1 and rendered this transcript inducible by addition of pTpT. Western analysis confirmed that H1299 cells normally express no p53 and that transfected H1299 cells expressed high levels of p53.
These data indicate that pTpT increases the transcriptional activity of p53.
The effect of pTpT on the level and intracellular distribution of p53 in normal neonatal fibroblasts was examined by immunoperoxidase staining using a p53-specific monoclonal antibody (mAb 421, Oncogene, Cambridge, MA).

Preconfluent cultures were treated with either 100 ~M pTpT or diluent for 24 hours before cell staining. Cells were first fixed for one minute in Histochoice fixative (Amresco, Solon, OH) followed by a five-minute rinse in PBS. p53 was detected using the Vectastain Elite ABC lcit (Vector Laboratories, Burlingame, CA) and the p53-specific monoclonal antibody mAb 421. Within 24 hours, an increase in intranuclear p53 was detected in pTpT-treated cells compared to diluent-treated cells, as has been reported after UV-irradiation. These results are consistent with the induction of the p53-regulated genes GADD 45 and SDIl (p21) in fibroblasts as well as in SCC12F cells, by pTpT.
EXAMPLE 8: Enhancement of DNA Repair Expression of a UV-damaged reporter plasmid containing the bacterial chloramphenicol acetyltransferase (CAT) gene under the control of SV40 promoter and enhancer sequences was previously shown to detect decreased DNA repair capacity in human lymphocytes associated with aging and early-onset skin cancers. This reporter plasmid was used to measure the DNA repair capacity of normal neonatal human skin-derived fibroblasts and keratinocytes.
Keratinocytes from newborns were established as described (Stanulis Praeger, B.M. and Gilchrest, B.A., J. Cell. Physiol. 139:116-124 (1989)) using a modification of the method''of Rheinwald and Green (Gilchrest, B.A. et al., J.
Invest. De~f~zatol. 101:666-672 (1993)). First-passage keratinocytes were maintained in a non-differentiating low Caz+ medium (K-Stim, Collaborative Biomedical Products, Bedford, MA). Fibroblasts were established from dermal explants as described (Rheinwald, 'J.G. and Green, J., Cell 6:331-343 (1975)) and maintained in DMEM supplemented with 10% bovine serum. Cells were treated with either 100 ~,M pTpT or an equal volume of diluent (DMEM) for five days prior to transfection. Duplicate cultures kept under each condition were transfected using the Lipofectin Reagent Kit (GIBCO/BRL) and 5 ~,g reporter DNA, pCAT-control vector (Promega, Madison, WI). Before transfection, the vector DNA was either sham irradiated or exposed to 100 mJ/cmz WB radiation from a 1 KW Xenon arc solar simulator (XMN 1000-21, Optical Radiation, Azuza, CA) metered at 285 + S nm using a research radiometer (model IL
1700A, International Light, Newburyport, MA), as described (Yaar, M. et al., J.
Invest. Derfnatol. 85:70-74 (1985)). Cells were collected 24 hours after transfection in a lysis buffer provided in the CAT Enzyme Assay System (Promega, Madison, WI) using a protocol provided by the manufacturer. CAT
enzyme activity was determined using the liquid scintillation counting protocol and components of the assay system lcit. Labeled chloramphenicol [50-60 mCl (1.85-2.22 GBq) rmnol] was purchased from New England Nuclear (Boston, MA). Protein concentration in the cell extracts was determined by the method of Bradford (Anal. Biochem. 72:248 (1986)). CAT activity was expressed as c.p.m./100 ~,g protein and is represented as percent activity of cells transfected with sham-irradiated, non-damaged, plasmid.
In preliminary experiments, exposure of the plasmid to a dose of solar-simulated irradiation (100 mJ/cmz, metered at 285 nm) prior to transfection was identified as resulting in approximately 75% reduction in CAT activity assayed in cell lysates 16-24 hours after transfection, compared to that of sham-irradiated plasmid transfected into paired cultures. However, keratinocytes (Figure 10) and fibroblasts (Figure 11) pretreated with 100 ~,M pTpT for five days before transfection displayed CAT activity more than 50% that of sham-irradiated transfected controls. Because the reporter plasW id was nonreplicating, the level of CAT activity directly reflects the degree of DNA repair of the UV-damaged CAT gene restoring its biological activity. These data indicate that pTpT
treatment of normal human fibroblasts and lceratinocytes more than doubles the capacity of cells to repair UV-induced DNA damage over a 24 hour period. The enhanced expression of LTV-irradiated plasmid in pTpT-treated cells did not result from a general increase in plasmid transcription in these cells, because the expression of the sham-irradiated plasmid was not higher in non-pTpT-treated cells.

EXAMPLE 9: Activation of p53 and Repair of Benzo[a]pyrene DNA Adducts.
Cell Culture.
,Newborn human lceratinocytes were established using a modification (Stanislus et al. J. Invest. Dermatol. 90: 749-754 (1998)) of the method of Rheinwald and Green (Cell 6:331-343 (1975)). First-passage keratinocytes were maintained in a non-differentiating medium containing a low concentration of calcium ion (K-Stim, Collaborative Biomedical Products, Bedford, MA).
The p53-null H1299 lung carcinoma cell line (American Type Culture Collection, ATCC, Rockville, MD) was maintained in Dulbecco's modified Eagle's medium (DMEM; GIBCO/BRL, Gaithersburg, MD) supplemented with 10% bovine serum (Hyclone Labs, Logan, UT).
Transfection of H1299 cells with a p53 expression vector.
Preconfluent cultures of H1299 cells were transfected with an expression vector containing the wild type human p53 cDNA under the control of the human cytomegalovirus promoter/enhancer (Dr. Bert Vogelstein, Johns Hopkins Oncology Center). Control transfections were performed using the same vector lacking the p53 cDNA. Transfections were carried out as described previously.
One day after transfection, cells were collected for western blot using 20 ~g total protein as described. p53 was detected using the monoclonal antibody DO-1 (Ab-6) known to detect both active and inactive forms of the protein (Oncogene, Cambridge, MA), anti-mouse ~Ig linked to horseradish peroxidase (Amersham, Arlington Heights, IL) and an ECL-kit (Amersham) following the direction of the manufacturer.
p53 assay using hGH reporter plasmid.
Normal human keratinocytes were transfected with the human growth hormone (hGH) reporter plasmid (pPG-GH) using the Lipofectamine Reagent Kit (GIBCO/BRL) as suggested by the manufacturer and 0.5 ~,g pPG-GH added to each p35 culture dish. pPG-GH contains the hGH coding region under the control of the thymidine kinase (TK) promoter and p53 consensus sequence, and hGH protein production is laiown, to be proportional to p53 activity (Fern et al., 1992). Transfection was performed in the presence of 100 ~M pTpT (Midland Certified Reagent Company, Midland, TX) or an equal volume of diluent. At the same time, the PSV-[i-galactosidase control vector (Promega, Madison, WI) was co-transfected to determine the transfection efficiency (Norton and Coffin, 1985). Four hours after transfection, the medium was removed and replaced with I~-Stim medium with or without 100 ~,M pTpT. Twenty-four hours after transfection and pTpT treatment, 400 ~.1 of the mediiun was harvested from each 35 mm culture dish, and 100 ~,1 of'ZSI-hGH antibody solution (Nichols Institute Diagnostics, San Juan Capistrano, CA) was added to detect secreted hGH as described below. The cells were harvested in a Reporter Lysis Buffer (Promega) using a protocol provided by the manufacturer, and 150 ~,1 of this lysate was used for the ~3-galactosidase assay using a (3-galactosidase assay kit (Promega).
Samples from each of triplicate culture dishes were evaluated for hGH and X3-galactosidase synthesis.
H1299 cells were similarly transfected with p53 expression vector or control vector. Two days after the transfection these cells were cotransfected with pPG-GH and PSV-[3-galactosidase control vector, and treated with 100 ~,M
pTpT. Twenty-four hours later, 250 ~.l of the medium and the cell lysate were harvested and processed as described above.
CAT assay.
The pCAT vector (Promega) was treated with benzo[a]pyrene-7,8-diol-9,10-epoxide (BP) as described (Athas et al. Cancer Res 1991) to produce less damaged and more damaged plasmids, previously shown to be instructive in studies examining different repair capacities in human cells. Based on the incorporation of 3H-BPDE into the DNA, the less damaged plasmid contained 25 adducts per 5 kb plasmid and the more damaged plasmid contained 50 adducts.
This non-replicating vector contains the chloramphenicol acetyltransferase gene under control of SV40 promoter and enhancer sequences. Human keratinocytes and p53-transfected H1299 cells were pre-treated with either 100 ~,M pTpT or an equal volume of diluent (DMEM) alone for 48 hours, then transfected with either BP-modified pCAT-control vector (0.5 ~,g/ml) or unmodified vector (0.5 ~g/ml) together with PSV-[i-galactosidase control vector (0.5 ~,g/ml). Cells were collected in a reporter lysis buffer (Promega) 24 hours after transfection.
CAT
enzyme activity was determined using the liquid scintillation counting protocol and components of the assay system kit (Promega). '4C-labeled chloramphenicol[50-60 mCi(1.85~2.22GBq)/rmnol] was purchased from New England Nuclear (Boston, MA). CAT activity was normalized with [i-galactosidase activity.
Western blot analysis.
Cells were treated with 100 ~,M pTpT or an equal volume of diluent alone for 48 hours. Total cellular proteins were collected in a buffer consisting of 0.25 M Tris HC1 (pH 7.5), 0.375 M NaCl, 2.5% sodium deoxycholate, 1%
Triton X-100, 25 mM MgCl2, 1 mM phenyhnethyl sulfonyl fluoride, and 0.1 mg ml aprotinin. Proteins (100 ~,g per sample) were separated by 7.5-15% SDS-PAGE and transferred to a nitrocellulose membrane (Schleicher & Schuell, I~eene, NH). After transfer, the gel was stained with Coomassie Blue to verify even loading as visualized by the residual high molecular weight proteins.
Membranes were blocked in 0.05% Tween-20/PBS with 5% milk, (Bio-Rad Laboratories, Hercules, CA). Antibody reactions were performed with the following antibodies: anti p53 (AB-6), anti PCNA (Ab-2) (Oncogene Science), and anti XPA (FL-273) (Santa Cruz Biotechnology). Sheep anti-mouse Ig linked to horseradish peroxidase (Amershariz, Arlington Heights, IL) (for p53 and PCNA) and goat anti-rabbit IgG (Bio-Rad)(for XPA) were used as the secondary antibodies. Binding was detected by the ECL detection lcit (Amersham).
To measure the repair of BP DNA adducts, non-replicating BP-damaged reporter plasmid system containing the bacterial chloramphenicol acetyltransferase (CAT) gene was used as described in Example 8. With first passage human keratinocytes, the transfection efficiency, as measured by the cotransfected [i-galactosidase expression vector, was 40-70%. Compared to diluent-treated cells, pTpT-treated human lceratinocytes showed an approximate doubling of CAT expression relative to paired cultures transfected with undamaged control CAT vector, when transfected with either the less BP-damaged (~ 25 adducts/plasmid) or the more BP-damaged (~50 adducts/
plasmid) vector.
To confirm the activation of p53 by pTpT in a second assay, a reporter plasmid expressing the human growth hormone (hGH) gene under the influence of a p53 inducible promoter was employed. Activation of p53 increases its binding to the consensus sequence in the plasmid, leading to transcription of the hGH coding sequence and ultimately to secretion of hGH into the medium.
pTpT-treated human keratinocytes showed a 45%+25% increase in hGH
secretion compared to diluent-treated cells. These data indicate that pTpT
activates p53 in normal human keratinocytes as well as in p53-transfected cells.
To confirm that pTpT enhances repair of BP-DNA adducts, at least in part, through p53 activation, p53-null H1299 cells were transfected with the p53 expression vector, and p53 protein expression was then confirmed by western blot analysis 48 hours after transfection. In p53+H1299 cells, repair was comparable to that observed in normal lceratinocytes; and the plasmid containing a low level of BP damage was repaired 80%+50% more efficiently in pTpT-pre-treated cells than in diluent pre-treated cells; and the plasmid containing a high level of BP damage was repaired more than three times as efficiently. In p53-H1299 cells, however, the repair capacity was the same as in both treatment groups. These data demonstrate that enhanced repair of BP-DNA adducts by pTpT involves p53.
pTpT activation of p53 in H1299 cells transiently transfected with the p53-responsive-hGH resulted in a 40% increase in hGH secretion compared to diluent-treated cells. These data further demonstrate that pTpT enhances p53 transcriptional activity through enhanced binding to its DNA consensus sequence.

Western blot analysis was used to examine the effect of pTpT treatment on the expression of selected genes known to be involved in DNA repair.
Normal human keratinocytes were treated with pTpT for 2 days before harvesting cellular protein. pTpT up-regulated the levels of p53, PCNA and the XPA protein 2 to 3-fold within 2 days of treatment.
EXAMPLE 10: Immunosuppression and Inhibition of Contact Hypersensitivity in a Murine Model.
C57B16 mice were subjected to the following treatment prior to sensitization with the allergen DNFB, by topical administration to abdominal skin; no pretreatment, WB irradiation (200 J/mz/dx4d), pTpT, pApA, or vehicle alone (30 ~,l of 100 ~M BID x Sd). Mice pretreated with WB or pTpT showed markedly suppressed ear swelling responses to DNFB challenge (0.6+0.2 and 0.9+0.3) compared to untreated or vehicle treated animals (4.3+0.6 and 3.3+0.2), whereas pApA-treated mice exhibited intermediate responses (2.5+0.6).
The immunomodulatory effect of pTpT was tested ifz vitro using human keratinocytes. Duplicate cultures of primary human keratinocytes were treated with pTpT, diluent or UVB irradiation (200 J/m2) or sham irradiation. Cells were collected at various times after treatment and analyzed for IL-10 protein by ELISA and for IL-10 mRNA byRT-PCR. An increase in lL,-10 mRNA was detected after 6 hours in irradiated cells and after 48 hours in pTpT treated cells.
An increase in IL-10 protein of 18 pglml was detected 24 hours after irradiation and 15+2 pg/ml 72 hours after treatment with pTpT. Previous work has demonstrated functional inhibition of the allogenic mixed lymphocyte reaction (MLR) assay by IL,-10. In the allogenic MLR, T cell activation is measured as lymphocyte proliferation, me~a,sured by 3H-thymidine incorporation. IL-10 activity of culture medium from the 72 hour pTpT sample was measured by addition of the > 10 kD components (containing the 18 kD IL-10 protein).
Reduction in T cell proliferation by 80+5% was demonstrated, compared to 8%+3 inhibition from diluent-treated control cultures. Thus, like UVB

irradiation, pTpT induces IL-10 in human lceratinocytes which is lilcely to cooperate with TNFa to inhibit contact hypersensitivity in pTpT treated slcin.
In another experiment, TNFa gene activation was measured by utilizing mice carrying a CAT reporter transgene bearing the entire TNFa promoter and 3'-untranslated region. Transgenic mice were subj ected to the following treatment prior to slcin assay for CAT expression: LJVB irradiation (200-700 J/mz), intracutaneous injection of pTpT (100 ~,M); lipopolysaccharide (LPS 1 ~,g/ml) as positive control, or vehicle alone. CAT activity was detected in skin treated with UVB, LPS, or pTpT (but not with vehicle alone).
EXAMPLE 11: Oligonucleotide Dependent W-Mimetic Activity:
Melanogenesis and p21/WafllCipl Expression The induction of melanogenesis in Cloudman S91 mouse melanoma cells by a five-nucleotide oligomer CATAC (SEQ m NO: 6) and a nine-nucleotide oligomer, GAGTATGAG (SEQ ID NO: 1) was examined. Duplicates of Cloudman S91 murine melanoma cells were incubated with either 100 ~M oligo or an equal volume of diluent (HZO) for 5 days. The cells were then collected, counted, and an equal number of cells were pelleted for melanin analysis. In three experiments, the pigment content after incubation with the 9-mer, 5-mer and pTpT increased 418%+267%, 61%~60% and 155%~60% of control levels, respectively. The 9-mer, but not the 5-mer, also stimulated melanogenesis in human melanocytes, producing a 51-62% increase after one week in culture.
Variations of this oligonucleotide were evaluated: a scrambled 9-mer ' (TAGGAGGAT; SEQ m NO: 2) and two truncated versions, a 7-mer (AGTATGA; SEQ m NO: 3) and second 5-mer (GTATG; SEQ m NO: 4).
Both 9-mers were equally active, inducing a 800% increase in melanin content.
The truncated versions (SEQ m NOs: 3 and 4) were also active, inducing 640%
and 670% increases, respectively. As with pTpT, SEQ ID NO: 1 (9-mer) oligonucleotide, but not SEQ ID NO: 6 (5-mer) induced the expression of the p2llWaf llCip 1 gene within 48 hours in a squamous cell carcinoma line, increasing the level of this mRNA 200-300%, compared to a 100-150% increase from pTpT.
Together, these data show that the UV-mimetic activity of pTpT can be duplicated quite dramatically by other oligonucleotides.

Melanogenesis Cultures of Cloudman S91 murine melanoma cells were treated for 5 days with SEQ m NO: 1, SEQ m NO: 6, pTpT as a positive control, or an equal volume of diluent as a negative control. Spectrophotometric analysis of S91 cell pellets after oligonucleotide treatment showed the melanin content of pTpT
treated cells to be 255 +/-60% that of control cells (Figure 12). SEQ m NO: 6 produced a slight increase in melanin, to 165+l-77% of control levels. SEQ m NO: 1 stimulated melanin content to an average of 600 +/-260% of control levels.
Oligonucleotides 5'pGTTAGGGTTAG3' (SEQ m NO: 5), 5'pCTAACCCTAAC3' (SEQ m NO: 9), or 5'pGATCGATCGAT3' (SEQ m NO
10), each comprising a 5' phosphate were added to cultures of Cloudman S91 melanoma cells as described in Example 11.
pTpT, shown previouslyto stimulate pigmentation in these cells, was used as a reference treatment, and diluent alone was used as a negative control.
After five days of treatment with the oligonucleotides, the cells were collected, counted, and an equal number of cells were pelleted for melanin analysis. The data shown in Figure 17 demonstrate that 10 wM pTpT increased melmin content to 3 times that of control diluent-treated cells. SEQ m NO: 5, representing the telomere over-hang sequence, also at 10 ~,M, increased the melanin level to 10 times that of control cells. SEQ m NO: 9 (telomere over-hang complement) and SEQ m NO: 10 (unrelated sequence) did not produce significant change in pigment content at concentration up to 10 ~.M. A truncated version of SEQ m NO: 5, comprising TTAGGG (SEQ m NO: 11) was also highly melanogeiuc, while the reverse complimentary sequence CCCTAA (SEQ m NO: 12) was less active (Figure 18), where both oligonucleotides contained a 5' phosphate.
The compounds of the present invention were tested for skin penetration and ira vivo melanogenic activity. Mice were treated (on their ears) with fluorescently-labeled pTpT or SEQ m NO: 1 (comprising a 5' phosphate) in propylene glycol for 4 hours, then ear skin was sectioned and examined by confocal microscopy. Treatment with either oligonucleotide resulted in brightly stained epidermis and hair follicles. Thus pTpT and SEQ m N0: 1 comparably penetrate the skin barner.
In another experiment, mice were treated once daily with either 100 p,M
pTpT or SEQ m NO: 1 containing a 5' phosphate in propylene glycol on one ear, or vehicle alone on the other ear. After 15 days, when the ears were sectioned and stained with Fontana Masson to detect melanin compared to vehicle controls, there was a 70% increase in pigmentation in pTpT-treated ears and a 250% increase with SEQ m N0: 1. Thus, both compounds comprising as few as 2 and as many as 9 nucleotides are effective at producing the ifz vitro UV-mimetic effects iya vivo.
p53 Activation and Cell Proliferation pTpT was previously found to inhibit cell cycle progression, at least in part through activation of p53 and subsequent upregulation of the cyclin dependent kinase inhibitor p21. Cultures of the human keratinocyte line SCC12F were treated with pTpT, SEQ B7 N0: 1, SEQ m NO: 6 or diluent alone as a negative control, collected and counted 48 hours later and processed for northern blot analysis of p21 mRNA expression. SEQ m NO: 1 was found to increase the level of p21 mRNA to almost 3-fold that of diluent control levels while pTpT-treated cells showed p21 mRNA levels twice that of control cells (Figure 13). Cells treated with SEQ a7 NO: 6 showed a 10-20% increase in p21 mRNA level. In these paired dishes, SEQ m NO: 1 also reduced cell number by approximately 50% after 2 days, while pTpT and SEQ m NO: 6 caused 40% and 25% reductions, respectively (Figure 14). Thus, the sequences of the present invention activate p53 and inhibit cell proliferation similar to the effect of pTpT.
Effect of size and sequence S91 cells were cultured in the presence of diluent alone, pTpT p9mer (SEQ m NO: 1), p7mer (AGTATGA; SEQ m NO: 7) or p5mer #2 (SEQ m NO: 4). After 5 days, the cells were collected, counted and an equal number of cells were pelleted for melanin analysis (Figure 15). pTpT produced a moderate increase in melanin content and SEQ m NO: 1, a larger increase. In addition, SEQ m NOS: 4 and 7 also strongly stimulated melanogenesis. Both SEQ m NOS: 4 and 7 stimulated a 7-8 fold increase in melanin. Because one p5mer was much more effective at inducing melanin production (compare results for SEQ
m NO: 4 and 6), these data suggest that oligonucleotide sequence plays a role in determining its melanogenic activity.
A p20mer was synthesized (pGCATGCATGCATTACGTACG; SEQ m NO: 8), with 3 repeats of the 4-base sequence GOAT, followed by two repeats , TACG, an oligonucleotide with an internal pTpT that resembles the 27-29 base fragment excised during excision repair of thymine dimers in eulcaryotic cells.
This oligonucleotide stimulated pigmentation to twice the level of control cells (Figure 16).
Effect of 5' phosphorylation S91 cells were cultured for 5 days in the presence of the thymidine dinucleotides or SEQ m NO: 1 with or without a 5' phosphate or diluent alone as a negative control. Removal of the 5' phosphate significantly reduced the melanogenic activity of pTpT by 80% and of the 9mer by 60% (p<0.04 and p<0.03, respectively, two-tailed Student's T-test, Figure 16. These data are consistent with an intracellular site of action of these oligonucleotides and with the reported requirement of a 5' phosphate for efficient cellular uptake.

5' phosphorylation increases oligonucleotide uptake.
Fluorescein phosphoramidite (FAM) labeled oligonucleotides were added to cultures of S91 cells for 4 hours and the cells were then prepared for confocal microscopy. Nuclei, identified by staining with propidium iodide, appeared red and FAM-labeled oligonucleotides appeared green. Co-localization of red and green signals was assigned a yellow color by the computer. Oligonucleotides with a 5' phosphate showed greater cellular uptake than those lacking this moiety. Confocal microscopy failed to detect uptalce of TpT and fluorescence-activated cell sorting (FAGS) analysis of these cells and gave a profile similar to that seen with untreated cells. pTpT-treated cells showed strong green fluorescence in the cytoplasm, but only a small amount of nuclear localization.
FACE analysis showed a shift in the peak fluorescence intensity, compared to TpT-treated cells, indicating more intensely stained cells. Similarly, the presence of the phosphate at the 5' end of SEQ ID NO: 1 greatly enhanced its uptake into the S91 cells. SEQ ID NO: 1 without 5' phosphorylation showed only moderate uptake and was localized predominantly in the cytoplasm, with faint nuclear staining in only some cells, whereas SEQ ID NO: 1 with 5' phosphorylation showed intense staining that strongly localized to the nucleus.
FACS analysis of SEQ ID NO: 1 without 5' phosphorylation showed a broad range of staining intensities with essentially two populations of cells, consistent with the confocal images. The phosphorylated SEQ ID NO: 1 containing cells also showed a range of staining intensities, but with more cells showing higher fluorescent intensity. Cells treated with phosphorylated SEQ ID NO: 8 showed a pattern of fluorescence very similar to that seen with phosphorylated SEQ m NO: l, both by confocal microscopy and FAGS analysis, indicating that its lower activity in the melanogenesis assay cannot be ascribed to poor uptake. These data show that uptake of these oligonucleotides by S91 cells is greatly facilitated by the presence of 5' phosphate and that melanogenic activity, while consistent with a nuclear site of action, is not solely dependent on nuclear localization.
Also, although the total intracellular fluorescence did not increase appreciably with increasing oligonucleotide length among the DNAs tested, the larger oligonucleotides more readily accumulated in the cell nucleus. There was no change in the profile of oligonucleotide uptake after 6 and 24 hours.
EXAMPLE 13: Oligonucleotides Can Induce Apoptosis Oligonucleotides homologous to the telomere overhang repeat sequence (TTAGGG; SEQ ID N0:11), sequence (llmer-1: pGTTAGGGTTAG; SEQ ID
NO: 5), complementary to this sequence (1 lmer-2: pCTAACCCTAAC; SEQ ID
NO: 9) and mrelated to the telomere sequence (llmer-3: pGATCGATCGAT;
SEQ ID NO: 10) were added to cultures of Jurlcat cells, a line of human T
cells, one of the cell types reported to undergo apoptosis in response to telomere disruption. Within 48 hours, 50% of the cells treated with 40 ~,M of SEQ ID
NO: 5 had accumulated in the S phase, compared to 25-30% for control cells (p<0.0003, non-paired t-test; see Figures 19A-19D), and by 72 hours, 13% of these cells were apoptotic as determined by a sub-Go/Gl DNA content, compared to 2-3% of controls (p<0.007, non-paired t-test; see Figures 19E-19H). At 96 hours, 20+3% of the 1 lmer-1 treated cells were apoptotic compared with 3-5%
of controls (p<0.0001, non-paired t-test). To exclude preferential uptake of the l lmer-1 as an explanation of its singular effects, Jurkat cells were treated with oligonucleotides labeled on the 3' end with fluorescein phosphoramidite, then subjected to confocal microscopy and FACS analysis. The fluorescence intensity of the cells was the same after all treatments at 4 hours and 24 hours.
Western analysis showed an increase in p53 by 24 hours after addition of l lmer-l, but not l lmer-2 or -3, with a concomitant increase in the level of the E2F1 transcription factor, known to cooperate with p53 in induction of apoptosis and to induce a senescent phenotype in human fibroblasts in a p53-dependent manner as well as to regulate an S phase checkpoint.
EXAMPLE 14: The Effect of DNA Fragments on DNA Mutation Frequency In.
vavo.
Transgenic mice carrying multiple genomic copies of a LacZ reporter plasmid were used. One hundred wM pTpT in polypropyleneglycol was applied to one ear and vehicle alone.to the other ear, daily for four days. On the fifth day, both ears were exposed to 100 mJlcmz UVB light. This procedure was repeated weelcly for 3, 5 or 7 weeks (3 mice/group). One week after the final irradiation, LacZ plasmids were harvested from the ear epidermis. Using methods well known in the art, the plasmids were recovered from genomic DNA
by restriction enzyme digestion and specific binding to the LacI protein.
Mutant LacZ plasmids were positively selected by transfection into bacteria and growth on selective medium and the mutation frequency was determined. After 3, 5, and 7 weeks, pTpT-treated skin exhibited a 20-30% lower mutation frequency than diluent treated skin (200 vs 293, 155 vs 216, and 261 vs 322, respectively).
These data showed that pTpT-enhanced DNA repair reduces UV-induced mutations in vivo and suggest that topical application could be used to lower the mutation rate in carcinogen-exposed human skin.
EXAMPLE 15: DNA Fragments Protect Against Oxidative Damage Primary newborn fibroblasts were treated for 3 days with 10 ~.M pTpT or diluent as control and then treated with 5 x 10-5 or 5 x 10-4 M H202. Within hours of H202 exposure, cell yields of pTpT pre-treated cultures were 45+1%
and 139+5% higher, respectively, compared to diluent pre-treated control samples. 72 hours after exposure of the low H202 dose, only 9.6~2.4% of the diluent pre-treated cells survived. In contrast, pTpT pre-treatment increased cell survival by 2-9 fold at 5 x 10-4 M HZOZ and conferred complete protection at the low dose. mRNA levels of Cu/Zn superoxide dismutase, an enzyme that participates in the process of oxygen radical quenching, were increased by greater than 3 fold 48 and 72 hours after pTpT treatment and remained elevated at least 24 hours after pTpT withdrawal (when the experiment was terminated).
EXAMPLE 16: Age Related Decline in DNA Repair Capacity Is Reversed by Oligonucleotides Human dermal fibroblasts (fb), derived from newborn, young adult (25-35y), and older adult (65-90y) donors were pre-treated with 10 ~,M pTpT or SEQ

LD NO: 1 containing a 5' phosphate pr diluent as a control for 24 hours. The samples were then UV irradiated with 5, 10 and 30 rn/cm2. DNA and proteins were collected at time 0 and up to 24 hours post-UV. There were age-associated decreases in the constitutive and UV-induced protein levels of p53, p21, XPA, RPA ERCC/PF and PCNA. However, in all age groups, pre-treatment with oligonucleotides resulted in up-regulated constitutive and LTV-induced levels of these proteins by 200-400%, Furthermore, slot blot analysis specific for thymine dimers and (6-4) photoproducts showed a significant decrease with aging in the DNA repair states in the first 16 hours post-UV. Pre-treatment with oligonucleotides increased the removal of photoproducts by 30-60 percent.
EXAMPLE 17: Phosphorothioate Version of the Telomere Overhang Homolog l lmer-1 Does Not Induce Apoptosis Cultures of Jurkat human T cells were treated with either diluent, l lmer-1 (SEQ LD NO:S) or the phosphorothioate 1 lmer-1 (1 lmer-1-S) for 96 hours, then collected and processed for FAGS analysis. Two concentrations of the oligonucleotides were tested, 0.4 ~M (Figures 20A-20C) and 40 ~M (Figures 20D-20F). At the 0.4 wM concentration, neither of the oligonucleotides affected the expected exponentially growing cell cycle profile of the Jurkat cells. At ~,M, the 11-mer-1 induced extensive apoptosis in these cells, indicated by a sub-Go/GI peak, while the l lmer-1-S had no effect.
EXAMPLE 18: Phosphorothioate Version of 1 lmer-1 Blocks Induction of S-Phase Arrest by the Phosphate Backbone 1 lmer-1 Cultures of a keratinocyte cell line (SSC12F, 100,000 cells/38 cm2) were treated for 48 hours with only the 1 lmer-1 (SEQ ID NO:S) or with the l lmer-1 in the presence of increasing concentrations of the 1 lmer-1-S. As shown previously in Example 13, the l lmer-1 induced an S-phase arrest as demonstrated by FAGS (Becton-Dickinson FacScan). Forty-three precent of the cells were in the S phase, compared to 26% of the control, diluent-treated cells.
However, when increasing concentrations of the phosphorothioate llmer-1 were also added to these cultures, fewer cells becaye arrested (Figures 21A-21G).
Complete inhibition of this arrest was seen with a ratio of l lmer-1: l lmer-1-S of 2:1. The llmer-1-S by itself did not induce the S-phase arrest.
EXAMPLE 19: Phosphorothioate Forms of the Telomere Oligonucleotides Reduce Constitutive and UV-W duced Pigmentation and Do Not Stimulate Melanogenesis Cultures of S91 mouse melanoma cells (100,000 cells/38 cmz) were treated with 100 ~M pTpT or phosphorothioate pTpT (pTspT) (Figure 22) or 40 ~M l lmer-1 or the phosphorothioate l lmer-1 (1 lmer-1-S) (Figure 23) for 6 days and were then collected, counted and assayed for melanin content. While the pTpT and l lmer-1 (Figure 22 and Figure 23, respectively) stimulated melanogenesis in these cells, pTspT and llmer-1-S did not (Figure 22 and Figure 23, respectively). Furthermore, both pTspT (Figure 22) and llmer-1-S
(Figure 23) reduced the constitutive pigmentation in these cells.
EXAMPLE 20: Phosphorothioate pTspT Inhibits LTV-Induced Melanogenesis Duplicate cultures of S91 cells (100,000 cells/39 cmz) were either sham-irradiated or irradiated with 5 mJ/cm2 solar-simulated light from a 1 kW
xenon arc solar-simulator (XMN 1000-21, Optical Radiation, Azuza, CA) metered at 285 ~ 5 nm using a research radiometer (model IL1700A, International Light, Newburyport, MA). Two sham-irradiated plates were then supplemented with 100 ~.M pTspT and two irradiated cultures were similarly treated with pTspT. After one week, cells were collected, counted and analyzed for melanin content by dissolving the cell pellets in 1 N NaOH and measuring the optical density at 475 nm. UV irradiation resulted in an increase in melanin content in these cells of about 40%. However, the LTV-induced increase in melanin was blocked by the addition of pTspT (Figure 24). In addition, the constitutive pigmentation of these cells was reduced by the pTspT in the sham-irradiated cultures, in agreement with the data presented in Figures 22 and 23.

EXAMPLE 21: pTpT Induces SOD2 Protein Level in Fibroblasts.
Fibroblasts (newborn human) were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% calf serum (CS) and 100 ~M
pTpT or diluent as control. Total cellular proteins were harvested at different intervals after stimulation and processed for western blot analysis. The blot was reacted with anti SOD2 antibodies (The Binding Site, Inc. San Diego, CA).
While fresh medium supplementation transiently induced the 37 kDa SOD2 in diluent treated control cultures, in pTpT treated cultures SOD2 induction was sustained at least through 32 hours when the experiment was terminated.
Coomassie blue-stained residual bands on the gel confirmed uniform loading of the different lanes.
EXAMPLE 22: Pretreatment With pTpT Protects Against Oxidative Damage Cells from the well differentiated squamous carcinoma line SCC12F (gift of Dr. James Rheinwald, Harvard University) were treated with 100 ~,M pTpT or diluent as control for 3 days. Then cells were re-plated in medium lacking pTpT
and were sham- or UV-irradiated with 10 J/cmz UVA (Sellas Sunlight UVA
lamp). Cell yields were determined at different intervals after UV
irradiation.
Figure 25 represents yields of UVA treated cells that were either pretreated with pTpT or pretreated with diluent, each as a percent of its own sham irradiated control. pTpT pretreatment increased the yields of UVA irradiated cells.
EXAMPLE 23: pTpT Induces Apurinic Endonuclease-1 (APE-1) Protein in Fibroblasts Fibroblasts (human newborn) were maintained in 10% CS-supplemented DMEM containing 100 ~,M pTpT, 10 wM or 60 wM oligonucleotide pGTTAGGGTTAG (SEQ ID NO:S), or diluent as control. Total cellular proteins were harvested at different intervals after supplementation and processed for western blot analysis. Expression of APE-1, the rate-limiting enzyme in repair of 8-oxoguanine (the principal form of oxidative DNA damage) was studied. The blot was incubated with anti APE-1 antibodies (APE/Ref 1 monoclonal IgG2b, Novus Biologicals, Inc. Littleton, CO). Within 24 hours of supplementation with pTpT or the l lmer oligonucleotide having nucleotide sequence SEQ ID NO:S, the 37 lcDa APE-1 protein was induced in samples stimulated with the oligonucleotides as compared to diluent control. The induction was sustained for at least 48 hours, when the experiment was terminated. Coomassie blue-stained residual bands on the gel confirmed uniform loading of the different lanes.
EXAMPLE 24: pTpT Induces Repair of UVA Damage to pCMV-Luc Plasmid The plasmid pCMV-Luc is a non-replicating vector containing a reporter gene, luciferase, under the control of a strong constitutive cytomegalovirus promoter (gift of Dr. Hedayati, Johns Hopkins University). Singlet oxygen-induced DNA damage of the plasmid was generated by irradiating the plasmid with visible light for different time intervals (0 min, 10 min and 20 min), in the presence of the photosensitizer methylene blue. Fibroblasts (human newborn), pretreated for 48 hours with 100 wM pTpT or with diluent alone, were transfected with 0.5 ~g of pCMV-Luc using Lipofect AmineTM (Promega, Madison, WI). After two days, plasmid-encoded luciferase activity was determined in cell extracts (Promega luciferase assay). Pretreatment with pTpT
enhanced the repair of singlet oxygen-induced DNA damage after 10 mJ/cm2 and 20 mJ/cmz UVA irradiation by 179% and 228% respectively. Figure 26 represents luciferase activity as a percent of luciferase activity assayed in fibroblasts transfected with sham irradiated plasmid.
EXAMPLE 25: DNA Oligonucleotides Protect Murine Skin From Photodamage Patients with a mutated xeroderma pigmentosum group A (XPA) gene have a greater than 1000 fold increased risk of UV-induced skin cancer. XPA-~-mice mimic the human syndrome [Kraemer, K. et czl., pp. 256-261 In: MoleculaY
Biology ofAgihg (Bohr, V.A., et al., eds.) Munksgaard, Copenhagen, 1999]. We have previously shown that oligonucleotides, particularly thymidine dinucleotide (pTT) and pGAGTATGAG (SEQ ID NO:1) that are partial homologs of the telomere overhang tandem repeat TTAGGG (SEQ m NO:11), increase levels of proteins involved in nucleotide excision repair and enhance the DNA repair rate.
To determine the effect of oligonucleotides in vivo, XPA+~+ and XPA~~- mice were treated with 100 ~,M pTT, 40 ~,M pGAGTATGAG (SEQ ID NO:l), or diluent alone, daily for 4 days. A 30 ~,1 volume of oligonucleotides or diluent in 90% propylene glycol/10% DMSO was applied to a 3 cmz area of skin along the spinal ridge of the mice. The mice were UV irradiated on day 5 with a previously determined erythemogenic LTVB dose. The course of treatment -- 4 days of oligonucleotide (or diluent) applications, starting with oligonucleotide on day 1 of each week, with LTV irradiation on day 5 -- was repeated for a total of 4 weeks. Seventy-two hours after the last dose of LTV irradiation, the mouse skin was processed histologically to assess general morphology, proliferation (Ki-67) [Ohike N. and T Morohoshi, Pathollnt (2001), 51:770-777; Billgren, A.M. et al., Breast Caf2ce~~ Res Teat (2002), 71:161-170], cyclobutane pyrimidine dimers (CPDs) determined by specific antibody binding, and on-going DNA
repair [proliferating cell nuclear antigen (PCNA); see Savio, M. et al., Carciyaogeraesis (1998) 19:591-596; Rudolph, P. et al., Hum Patl2ol (1998) 29:1480-1487].
Diluent treated XPA~~+ and XPA-~- skin showed spongiosis, blistering, and dyskeratosis, whereas oligonucleotide-treated samples lacked these features.
After 72 hours, only XPA-~- diluent-treated samples contained CPDs: 15 ~ 5 (+) cells per 400x microscopic field vs: none in samples from mice treated with pTT
or SEQ ID NO:1 (p < 0.0001). No XPA+~+ samples contained CPDs. Ki67 (+) cells were more numerous in diluent-treated than in oligonucleotide-treated XPA-~- skin, consistent with a hyperproliferative "rebound" after LTVB damage:
14 ~ 4 vs. 5 ~ 2 (p < 0.005). However, PCNA (+) cells were more numerous in both XPA+'+and XPA-~- oligonucleotide-treated skin: 38 ~ 2 vs. 52 ~ 6 (p <
0.01) and 125 + 8 vs. 89 + 11 (p < 0.001), consistent with the role of PCNA in on-going DNA repair, and with previously reported up-regulation of PCNA by oligonucleotides in vitro.

These data demonstrate that topical application of telomere homolog oligonucleotides enhances the skin's ability to repair repeated UVB damage, in large part through increased DNA repair capacity. The photoprotective effects were observed in both repair-proficient and severely repair-deficient animals, suggesting a therapeutic role in cancer prevention.
EXAMPLE 27: Single-Stranded DNA Homologous to the 3' Overhang Telomere Sequence Mimics UV Effects on Mitochondria) Gene Expression Exposure of keratinocytes to UVB irradiation affects the expression of genes involved in cell cycle arrest, DNA repair and cytokine production.
Single-stranded oligonucleotides sharing sequence homology with the telomere 3' overhang, when introduced into cells, mimic these UV effects. To identify other genes that may be similarly modulated, cells of keratinocyte origin (SCC 12F;
a human epidermal squamous cell carcinoma line provided by Dr. James Rheinwald of Harvard University) were exposed to solar simulated irradiation (SSR) (20 mJ/cmz, measured at 285 ~ 5 nm), to an 11-base oligonucleotide (pGTTAGGGTTAG; SEQ m NO:S) homologous to the telomere overhang repeat sequence (T-oligo), or to a complementary sequence as control [pCTAACCCTAAC; (SEQ m N0:9) control oligonucleotide]. Similar to SSR, T-oligo substantially inhibited cellular proliferation and arrested >43% of cells in the S-phase of the cell cycle, compared to <23% of control cells. Using DNA
microarray chips containing >700 genes known to be modulated by aging and stress conditions, we identified 8 genes that were downregulated by >50% with SSR and T-oligo treatment, but not with sham irradiation or control oligo treatment. Specifically, the modulated genes encode subunits of the mitochondria) enzymes ATPase and cytochrome c oxidase, known to be transcriptionally downregulated by UV, and NADH dehydrogenase. These are mitochondria)-transcribed genes whose encoded proteins participate in cellular respiration. Semi-quantitative RT-PCR and northern blot analysis confirmed the above data and showed that the genes were comparably downregulated by SSR
and T-oligo treatment as early as 24 hours and 48 hours, respectively. Using microarray chips that provide a rapid means for analyzing expression of many genes simultaneously, it was demonstrated that mitochondrial-encoded gene products involved in respiration are similarly modulated by LTV irradiation and DNA homologous to the telomere 3' overhang. The data suggest that DNA
damage responses mimiclced by telomere homolog DNA include temporary reduction of energy consumption by cells.
EXAMPLE 28: Induction of S-phase Arrest and Apoptosis by Telomere Overhang Oligonucleotide Materials and Methods Applying to Examples 28-37 Oligonucleotides Three DNA oligonucleotides were designed initially: one homologous to the telomere overhang (llmer-1: pGTTAGGGTTAG; SEQ ID NO:S), one complementary to this sequence (1 lmer-2: pCTAACCCTAAC; SEQ ID N0:9) and one unrelated (llmer-3: pGATCGATCGAT; SEQ ID NO:10). For later experiments, additional 11 base oligonucleotides were designed: one a simple permutation of l lmer-1 (pGGGTTAGGGTT; SEQ ID N0:13), one with the same number of G residues but roughly 50% rather than 100% homology to the overhang (pTAGATGTGGTG; SEQ ID NO:14), and one with roughly 50%
overhang homology but also containing cytosine bases (pCGGGCTTATTG;
SEQ ID NO:15) (Midland Certified Reagent Company, Midland, Texas).
Cell sources and culture Human neonatal fibroblasts were established and cultured as previously described (Ellen M.S., et al., 1997, PYOC Natl Acad Sci ZISA 94: 12627-12632).
Fibroblasts from a Nijmegen breakage syndrome (NBS) patient (#GM07166) and an age-matched control (GM03399) were purchased from the NIGMS
Human Cell Repository, Coriell Institute for Medical Research, Camden, NJ) and cultured in DMEM/15% FBS. Saos-2 cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA). SCC12F cells were the kind gift of Dr. James Rheinwald, Harvard University.

Cell cycle analysis The established line of human T lymphocytes, termed Jurlcat cells (180,000 cells /ml) were plated in RPMI medium 1640 supplemented with 3% FBS (both from GIBCO/BRL, Gaithersburg, MD). Duplicate cultures were treated with a final concentration of 40 ~M oligonucleotide or an equal volume of diluent (water) as a control. Cells were collected up to 96 hours after treatment, stained with propidium iodide and analyzed by FAGS. using a Becton-Dickinson FacsScan and CellQuest software.
Caspase activity Duplicate cultures of Jurlcat cells were cultured and treated with oligonucleotides as described above. At 48, 72 and 96 hours of treatment, cells were collected by centrifugation, washed and then the pellet lysed by repeated cycles of freeze-thawing. The lysate was then clarified by centrifugation and the supernatant used for protein analysis and Caspase-3 activity assay using the components and instructions of the Colorimetric CaspACE Assay System from Promega (Madison, Wisconsin). The assay uses the substrate Ac-DEVD-pNA and colorimetrically measures release of free pNA. Caspase activity is expressed as pmol of pNA produced/hour/~g protein.
TUNEL analysis for detection of apoptotic cells Cells were plated at a density of 180,000 cells/ml in RPMI 1640 medium supplemented with 3% FBS. Cells were treated with 40 ~M of either of the oligonucleotides for 72 hours. Cells were then collected, washed and fixed for minutes in 4% paraformaldehyde and then permeabilized with 0.1% Triton X-100 for 2 minutes. The TUNEL (terminal deoxynucleotidyltransferase mediated dUTP-biotin nick-end labeling) assay was carried out with components and instructions of the In Situ Cell Death Detection Kit using fluorescein-labeled dUTP from Boehringer Mannheim. TUNEL-positive cells were detected by FACS analysis and confocal microscopy.

_72_ Cellular uptalce of oligonucleotides For confocal analysis, Jurlcat cells were plated on Permanox chamber slides (Lab-Telc, Naperville, IL,) and treated for 4 hours with fluorescein phosphoramidite (FAM)-labeled oligonucleotides (Research Genetics, Inc., Huntsville, AL). Adherent cells were fixed in 4% paraformaldehyde in PBS, stained with propidium iodide (PI) to identify nuclei, mounted with Slowfade reagent (Molecular Probes, Eugene, OR), covered with a coverslip and stored at 4°C in the dark. Uptake of the oligonucleotides was assessed in 5-10 ~.m sections with a Carl Zeiss LSM 510 confocal laser microscope. Computer images assign green to the FAM-oligonucleotides, red to PI, and yellow to co-localization of the signals. FACS analysis was performed on Jurkat cells identically treated with the FAM-oligonucleotides for 4 hours, then fixed in 0.5 ml of 4%
paraformaldehyde overnight at 4°C. Fluorescence was measured on the FL-charnel.
Western blot analysis and antibodies Western blot analysis was performed as previously described [Eller, M.S., et al., (1996), Proc Natl Aead Sci ZISA 93, 1087-1092]. Antibody Ab-6 (DO-1 clone, Oncogene Research Products, Cambridge, MA) detected p53; antibody KH95 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) detected E2F1; antibody Ab-4 (Oncogene Research products) detected p73. Phospho-p53 (serine 15) was detected using the #9284 polyclonal antibody and phospho-p95/Nbsl (serine 343) was detected by a phospho-p95/Nbsl-specific polyclonal antibody, both antibodies from Cell Signaling Technology (Beverly, MA). p95/Nbsl antibody (#NB-100-143, Novus Biologicals, Littleton, CO) and an actin antibody (I-19, Santa Cruz Biotechnology, Santa Cruz; CA) were also used.
Modification of p95/Nbsl Jurkat and SCC12F cells were cultured and treated with the oligonucleotides as described above. After the indicated times, total protein was collected and analyzed by 8% PAGE and western blot using a polyclonal antibody to p95/Nbs 1 (#NB-100-143, Novus Biologicals, Littleton, CO). Immunoprecipitation and treatment with a serine/threonine phosphatase was performed as described by Lim et al. [Lim, D.S. et al., Nature 404:613-617, 2000].
Transient transfection and expression of TRF2Drr The Ad TRF2DN expression vector was the kind gift of Dr. Titia deLange (Rockefeller University). The control vector, pCMV LUC was the gift of Dr. M.
Hedayati, Johns Hopkins University. Cells were plated at a density of 3.5 x cells/cm2. One day later, the cultures were transfected with 1 ~,g plasmid DNA/35 mm culture dish, using the Fugene 6 Transfection Reagent (Roche Molecular Biochemicals, Indianapolis, IN), following the protocol supplied by the manufacturer. Cells were collected at the indicated times after transfection and p95/Nbsl was examined by western blot as described.
Telomere length Normal human fibroblasts were cultured for 5 days in the presence of either diluent or 40 ~M 1 lmer-1, -2 or -3. The cells were then collected and the genomic DNA isolated using the DNeasy Tissue Kit (Qiagen, Valencia, CA).
Telomere length was determined essentially as described by van Steensel and de Lange (van Steensel, B. and T. de Lange. 1997, Nature 385: 740-743) using the Telo TAGGG Telomere Length Assay from Roche Molecular Biochemicals (Indianapolis, Indiana) and the protocol supplied by the manufacturer. The mean telomere length (MTL) was calculated by densitometry and using the method of Harley et al. (Harley, C.B., et al., 1990, Nature 345: 458-460).
3' Overhang Assay Normal human fibroblasts were cultured in the presence of 40 ~,M l lmer-1 for up to 7 days. Cells were collected before treatment (time 0) and at 3, 5 and 7 days of treatment. The genomic DNA was isolated using the DNeasy kit.
Detection of the 3' overhang was carried out as described by van Steensel, Smogorzewska and de Lange (van Steensel, B., et al., 1998, Cell 92: 401-413).

A probe ([TTAGGG]ø) was hybridized to the genomic DNA to control for hybridization to telomeric double-stranded DNA. The test primer ([CCCTAA]4) was used to detect the overhang. , Oligonucleotides homologous to the 3' overhang sequence (llmer-1:
pGTTAGGGTTAG; SEQ ID NO:S), complementary to this sequence (1 lmer-2:
pCTAACCCTAAC; SEQ ID N0:9) and unrelated to the telomere sequence (1 lmer-3: pGATCGATCGAT; SEQ m NO:10) were synthesized. The three 11-mer oligonucleotides were added to cultures of Jurkat cells, an established line of human T lymphocytes, a cell type reported to undergo apoptosis in response to telomere disruption [Karlseder, J., et al., (1999) Science 283, 1321-1325] and DNA damaging ionizing radiation (IR) [Vigorito, E., et al, (1999), Hematol Cell They 41, 153-161]. Duplicate cultures of Jurlcat cells were treated with a final concentration of 40 ~,M l lmer-1, -2 or -3, oligonucleotides, or an equal volume of diluent (water) as a control. Cells were collected up to 96 hours after treatment, stained with propidium iodide and analyzed by FAGS. See also Example 13 and Figures 19A-19H.
Apoptosis was also detected by the DNA end-labeling TLJNEL assay.
Jurkat cells were treated with 40 ~,M oligonucleotides for 72 hours. Cells were then collected and the TUNEL assay was carried out. Only Jurkat cells treated with the overhang homolog SEQ m NO:S displayed an increase in fluorescence due to end-label incorporation as seen by FACS analysis and confocal microscopy. The activation of caspase-3, another marker of apoptosis, was also examined. Duplicate cultures of Jurkat cells were cultured and treated with oligonucleotides as described above for 48, 72 and 96 hours amd caspase-3 activity was measured. The activity of caspase-3 was 50% higher after 48 hours of treatment with the telomere overhang homolog, compared to controls, and 3-4 fold higher in these cells at 72 and 96 hours (Figure 27). Thus, the oligonucleotide homologous to the telomere 3' overhang specifically induces an S phase arrest and apoptosis in these cells.

EXAMPLE 29: Demonstration of a Role for p73 in Apoptosis The p53 transcription factor and tumor suppression protein was specifically implicated in the apoptosis following telomere loop disruption [Karlseder, J., et al., (1999), Scieface 283, 1321-1325]. However, although a role for p53 in apoptosis of murine embryonic fibroblasts after telomere loop disruption was demonstrated experimentally, the implied role for p53 in apoptosis of cells with presumptively compromised p53 in the same studies was not addressed [Karlseder, J., et al., (1999), Sciehee 283, 1321-1325]. Because the p53 homolog p73 has also been shown to mediate apoptosis in several cell types [Lissy, N.A., et al., (2000), Natuf~e 407: 642-644, Irwin, M., et al., (1997), Nature 407: 645-648], and because the telomere homolog DNA induced both proteins, experiments were designed to explore the contribution of this protein to oligonucleotide-induced cell death.
Because HTLV-1 infection has been shown to impair the p73 pathway [Lemasson, I. and Nyborg, J.K. (2001, JBiol Che~z 276, 15720-15727, Kaida, A., et al., (2000), OfZCOgehe 19, 827-830] as well as that of p53 [Akagi, T., et al., (1997), FEBS Lett 406, 263-266], a human melanoma line MM-AN known to express wild type p53 was selected for study. Matched cultures of human melanoma cell line MN-AN (obtained from H.R. Beyers, Boston University) were grown in DMEM with 5% FBS and treated with 40 ~.M l lmer-l, l lmer-2 or diluent alone for 24-48 hours and harvested at intervals for FACS analysis and western blotting.
Like the Jurkat cells, the MM-AN cells first entered an S-phase arrest, and by 72 hours a substantial portion of the cells were undergoing apoptosis, as indicated by their sub-Go/Gl DNA content. Proteins were analyzed by denaturing polyacrylarnide gel electrophoresis (SDS-PAGE), using 10% polyacrylamide, and western blot analysis was done with an E2F1-specific monoclonal antibody (KH95 Santa Cruz Biotechnology, Santa Cruz, CA), and p73 analyzed using a polyclonal antibody (AB-4 from Oncogene Research Products, San Diego, CA).
Western blot analysis revealed the same pattern of induction for E2F1 and p73 as observed for Jurkat cells, consistent with a causal role for these proteins in the observed apoptosis. In order to determine the contribution of p73 to cell death, MM-AN melanoma cells were stably transfected with a dominant negative p73 (p73DN) (Irwin et al., Nature 402:645-648) construct or empty vector as a control (p73DN-containing vector a gift from Dr. William Kaelin, Dana Farber Institute, Boston) and paired cultures were supplemented with 40 ~M l lmer-1 or diluent alone, then harvested at 72 hours for FAGS analysis. Cells expressing p73Drr underwent apoptosis at half the rate of control cells, confirming a substantial, but not exclusive role for the p73 protein in the process. Furthermore, by western blot, the differentiation markers Mart-1, tyrosinase, TRP-1 and gp-100 were upregulated 48-72 hours after T-oligo treatment of MM-AN melanoma cells.
The above-mentioned differentiation markers are expressed in normal human melanocytes. It appears that the loss of expression of these antigens is a mechanism that a subset of human melanomas uses to escape immunosurveillance defenses. Several peptides from these antigens are targets of tumor infiltrating cytotoxic lymphocytes. Encouraging results have been obtained in vivo with different vaccination strategies using antigenic peptides from the above antigens.
EXAMPLE 30: Effect of the T-oligo on Tumorigenicity of MM-AN Cells in SCID Mice To study the effect of T-oligo (pGTTAGGGTTAG; SEQ ID NO:S) on the tumorigenicity of MM-AN cells, cells were grown in DMEM with 5% fetal bovine serum (FBS) and treated with 40 ~M of T-oligo, complement, or diluent alone for 48 hours. The cells were then harvested with trypsin/EDTA and the percent viability determined by trypan blue exclusion. All cell populations at the time of injection had a viability of greater than 90%. The tumorigenicity of these melanoma cells was determined by inj ecting 1 x 10~ viable cells in balanced salt solution subcutaneously over the right scapular region of SCID mice (3 groups of 5 mice) to produce tumors. The animals were sacrificed by COZ inhalation 18 days after treatment and the tumor size evaluated with calipers. Tumors were processed for hematoxylin and eosin staining and the melanocytic origin of the tumors was confirmed by immunohistochemical staining of the lesion using HMB-45 monoclonal antibody, which recognizes gp-100, a conventional marker for human melanoma.
Tumors were palpable at the site injected with cells pretreated with diluent by day 10 and day 12 in the other groups. On day 18, animals were sacrificed and all tumors removed and measured. Mean tumor volume in the diluent-treated group was 322 ~ 45 mm3 vs. 56 ~ 13 mm3 in the T-oligo treated group (p = 0.04). The animals treated with the control oligonucleotide had a tumor volume of 238 ~ 50 mm3, not statistically less than the diluent control (Figure 28). Hematoxylin and eosin staining revealed large tumors present within the subcutis composed of large epitheloid cells with abundmt pigmented cytoplasm and large irregular nuclei demonstrating hyperchromasia and large nucleoli. The tumors were identified as melanoma by positive immunohistochemical staining with HMB-45, confirming their melanocytic origin.
To study the effect of T-oligo on the metastatic potential of melanoma cells, cultures of MM-AN cells were treated for 48 hours with T-oligo, complement or diluent alone and harvested as described above. 1 x 10~ cells in balanced salt solution were injected into the lateral tail vein of 5-week old SCm mice (3 groups of 5 mice). After 40 days, mice were sacrificed by COZ
inhalation, an autopsy performed and organs examined macroscopically for visible metastatic lesions.
All five animals treated with diluent alone or control oligonucleotide developed multiple metastases in comparison to one of five animals in the T
oligo-treated group. Metastases were seen in the adrenal gland, bone, brain, brown fat, kidney, liver, lung, pancreas and additional sites of the control animals. The average number of visible tumors per animal was 0.8 in the T-oligo pretreated group compared to 15.8 (p = 0.003) and 9.6 (p = 0.05) in the diluent and control oligo pretreated groups, respectively. The average volume of the macroscopic metastastic tumors was also found to be significantly reduced in _78_ the T-oligo treated animals by about 85% and 80% compared to the diluent treated and complement treated animals, respectively (Figure 29).

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Example 31: Activity Depends on Homology to Telomere Overhang Sequence To further confirm the specificity of these responses to the telomere overhang sequence, three variations were tested. Lilce 1 lmer-1, its permutation pGGGTTAGGGTT (SEQ m N0:13) is an exact homolog of the overhang and hence, predicted to be equally active. A second oligonucleotide, pTAGATGTGGTG (SEQ m NO:14) is equally as G-rich (5 of 11 bases) as 1 lmer-1 and has 55% homology to the overhang, similar to the 9-mer oligonucleotide, pGAGTATGAG (SEQ m NO:1), recently shown to induce p53 and pigmentation, and to enhance DNA repair capacity (see Example 12). A
third oligonucleotide, pCGGGCTTATTG (SEQ m NO:15), also has 55%
homology to the overhang, but differs from the other oligonucleotides in that it contains 18% cytosine bases that are not present in the overhang sequence.
These oligonucleotides were added individually to cultures of Jurkat cells at a final concentration of 10 ~.M. Cells were collected after 72 hours and processed for FAGS analysis. Each experimental condition was done in triplicate. The data shown in Figures 30A-30H are from one representative experiment of two. By 72 hours, 2% of the diluent-treated cells and cells treated with cytosine-containing oligonucleotide were apoptotic by FACE analysis. However, 13% of the homolog-treated and 10% of the partial homolog-treated cells were apoptotic.
After 96 hours, 28% of the hoinolo~-treated cells were apoptotic, compared to 16% of the partial homolog-treated cells, and 2 - 4% of cells in cultures treated with the cytosine-containing oligonucleotide or diluent alone. In separate paired cultures, the two complete telomere overhang homologs [pGTTAGGGTTAG
(SEQ m NO:S) and pGGGTTAGGGTT (SEQ m N0:13)] were shown to be equal in activity. The data suggest that oligonucleotide activity is a function of telomere homology rather than, for example, G content primarily. Furthermore, the presence of cytosine in the oligonucleotide greatly diminishes its activity independent of degree homology. Although pTAGATGTGGTG (SEQ m N0:14) and pCGGGCTTATTG (SEQ m NO:15) share comparable homology with the telomere overhang, the latter oligonucleotide failed to induce apoptosis and only induced a moderate S-phase arrest. This is consistent with work described in Example 12 comparing 2-20 base oligonucleotides in their ability to induce other LTV-mimetic DNA damage responses.
To determine if pTT, representing 33% of the 6 base telomere tandem repeat sequence, induces an S-phase arrest in Jurkat cells in the same way as the full telomere repeat sequence, we compared the effects of pTT and l lmer-1 on Jurkat cells. In order to determine relative efficacy of pTT and l lmer-1, cultures of Jurkat cells were treated with diluent, 20 ~,M or 5 ~,M pTT or llmer-1 for hours and collected for FACS analysis. Each experimental condition was performed in triplicate. The percentage of cells in each phase of the cell cycle, indicated as an average and standard deviation, was determined from the triplicate results. One representative experiment of three is shown in Figures 31A-31E. At a concentration of 20 ~M, the cell cycle profiles of pTT- and l lmer-1-treated Jurkat cells were nearly identical, with 53 ~ 1% and 58 ~ 1%, respectively, of the treated cells in the S-phase at 72 hours. However, at 5 ~M, 61% of the 1 lmer-1 treated cells had accumulated in the S-phase within 72 hours, compared to 45% of the pTT-treated cells and 36% of the diluent-treated cells. Although Jurkat cells treated with a higher 1 lmer-1 dose (40 ~,M, Figure 19F) also showed a comparable percentage of cells in S phase at 72 hours (38%), these cultures additionally contained 13% apoptotic (<Go) cells. No apoptosis was detected in Jurkat cultures treated with 20 ~,M pTT even at 96 hours.
Thus, pTT and the telomere overhang homolog have similar effects on the cell cycle in Jurlcat cells, although higher concentrations of the incomplete sequence are needed. These data, together with those in Figure 30, as well as other data for other oligonucleotides 2-20 bases in length (Example 12) further suggest that the ability of these oligonucleotides to induce DNA damage responses is directly related to their degree of homology to the telomere repeat sequence, although many sequences with only partial homology also have useful effects.

_87_ EXAMPLE 32: Induction of S-phase Arrest, E2F1, and p53 Protein Levels, and Phosphorylation of p53 in Normal Human Fibroblasts Preconfluent cultures of normal neonatal human fibroblasts were treated with the oligonucleotides (40 ~,M) or diluent alone and collected 24 hours later for FAGS analysis (Figures 32A-32D) or western blot. The averages and standard deviations presented in Figures 32A-32D were determined from triplicate samples. One representative experiment of three is presented.
Normal neonatal human fibroblasts respond within 24 hours to the telomere overhang homolog oligonucleotide, l lmer-1, by activation of an S-phase checkpoint, but no apoptotic cells were detected up to 72 hours after treatment. Control 11-base oligonucleotides complementary (llmer-2) or unrelated (llmer-3) to the telomere overhang had no effect on the cells. The selective effect of the telomere homolog oligonucleotide cannot be attributed to selective uptake, as comparable uptake has been shown among same length oligonucleotides regardless of sequence.
Activation of p53 as indicated by phosphorylation on serine-15 was determined by western blot analysis, using the phospho-p53 (ser-15) specific antibody 1668. The blot was then stripped and re-probed with the pantropic p53 DO-1. Lanes of the gel contained protein from fibroblasts treated with diluent alone, 1 lmer-1 or 1 lmer-2; collected at 24, 48 or 72 hours. As a positive control, fibroblasts from another donor were either sham- or X-irradiated (10 Gy) and collected after 3 hours. Densitometric analysis showed a 170-250 %
increase in E2F1 in l lmer-1 treated fibroblasts compared to diluent or l lmer-2 treated controls at 24 hours. p53 increased by 60-300% at 48 hours and 240-380% at 72 hours after 1 lmer-1 treatment compared to diluent or 1 lmer-2 treated controls.
All values were normalized to (3-actin and are based on two independent experiments. Western analysis of the treated fibroblasts showed that p53 and E2F1 are selectively induced by the telomere overhang homolog.
An increase in E2F1 was seen as early as 4 hours after addition of the 11-mer-1, peaked 12-24 hours after, and subsequently returned to control levels.
The E2F1 transcription factor is known to cooperate with p53 in induction of apoptosis (I~owalilc, T.F., et al., 1,995,.) T~ir~ol 69: 2491-2500, Kowalilc, T.F., et al., 1998, Cell Growth Differen.tiatiorz 9: 113-118) and to induce a senescent phenotype in human fibroblasts in a p53-dependent manner (Dimri, G.P., et al., 2000, Mol Cell Biol 20: 273-285). Furthermore, E2F1 is induced by DNA
damage from IR in a manner dependent on the ATM kinase (Lin, W.C., et al., 2001, Genes and Dev. 1 S: 1833-1844). To determine whether llmer-1, like pTT
(Eller, M.S., et al., 1997, P>~oc Natl Acad Sci USA 94: 12627-12632, Maeda, T., et al., 1999, Mutat Res 433: 137-145), activates as well as induces p53, we also examined p53 phosphorylation, a marker of transcriptional activation of p53 after various forms of DNA damage (Lambert, P.F., et al., 1998, JBiol Claern 273: 33048-33053, Dumaz, N. and D.W. Meelc. 1999. EMBO J 18: 7002-7010, Tibbetts, R.S., et al., 1999, Gerzes Dev 13: 152-157, Caspari, T., 2000, Curr~
Biol 10: 31 S-317, Unger, T., et al., 1999, Oncogene 18: 3205-3212). There was a striking and selective increase in p53 protein phosphorylated at serine 15 in response to the l liner, first detected at 4 hours and sustained through at least 48 hours.
EXAMPLE 33: p53 Phosphorylation and E2F1 Induction Are Dependent on ATM
Because the serine 15 site on p53 is known to be phosphorylated in an ATM-dependent mamzer after exposure to ionizing radiation (IR), we wished to examine the role of ATM in E2F1 induction and p53 serine 15 phosphorylation in response to the l liner-1. Fibroblasts from a patient with ataxia telangiectasia (AT) known to lack functional ATM protein, and age-matched normal control fibroblasts (N) were treated with either diluent, 40 ~,M l liner-1, sham irradiation or IR (10 Gy). Cells were collected 3 hours (sham or IR) or 48 hours (diluent, 1 liner-1) for western blot analysis to detect serine 15-phosphorylated p53 and total p53. In normal fibroblasts, the l liner-1 induced a 10-15 fold increase in the level p53 phosphorylation on serine 15 compared to diluent-treated controls.
In contrast, treatment of AT fibroblasts with the l liner-1 resulted in less than a 50% increase. The 1 liner-1 minimally induced the level of total p53 in these normal fibroblasts from a young adult donor, ,in contrast to the induction of p53 in newborn fibroblasts, consistent with the previously described reduced response to UV-induced DNA damage for adult versus newborn cells. As expected, AT fibroblasts showed only minimal phosphorylation of p53 serine 15 in response to IR. The l lmer-1 also, induced E2F1 levels in normal control fibroblasts, but not in AT fibroblasts. These data demonstrate a requirement for the ATM kinase in these responses to telomere overhang DNA and are consistent with ATM-dependent p53 induction by TRF2DN (Karlseder, J. et al., 1999, Science 283: 1321-1325).
EXAMPLE 34: Cell Cycle Arrest Is Not Dependent On p53 In order to determine whether these oligonucleotides similarly affect other human cell types and to investigate the role of p53, we treated a squamous cell carcinoma line (SCC12F) (Rheinwald, J.G., et al., 1983, Human caz~cizzogetzesis. pp. 86-96. Academic P>"ess (New York)) that over-expresses a presumptively mutated p53 (Ellen M.S. and B.A. Gilchrest. 2000, Pigment Cell Res. 13: 94-97) and a p53-null osteosarcoma cell line (Saos-2) (Wang, L.H., et al., 1998, Azzticancer Res 18: 321-325) with either diluent alone, l lmer-1, l lmer-2 or 1 lmer-3. Cultures were analyzed by FACS after 48 hours (Figures 33A-33H). As with the Jurkat cells and normal fibroblasts, the telomere overhang homolog selectively induced an S-phase arrest in both of these cell types within 48 hours. Although the percentage of cells in the S-phase in 1 lmer-1-treated SCC12F and Saos-2 cells was virtually identical (53 ~ 6% and 52 ~
1%, respectively), the Go/Gl arid GZ/M content of the two cell types were somewhat different. This may reflect differential uptake of the oligonucleotide leading to dose-dependent differences, and/or different pathways acting in these cells to control the cell cycle. No apoptotic response to the oligonucleotides was detected in either cell type within 72 hours. These data demonstrate that the l lmer-1 affects cells of both epithelial and mesenchymal origin and that the S
phase arrest in response to l lmer-1 is not dependent on p53. However, because many cell types with wild type p53 are relatively resistant to apoptosis after DNA damage (Sionov, R.V. and Y. Haupt. 1999. Oncogene 18: 6145-6157), the data do not address the role of p53 in the apoptotic response to 1 lmer-1, which notably occurs in Jurlcat cells that express a presumptively compromised p53 (Alcagi, T., et al., 1997, FEBS 406: 263-266).
EXAMPLE 35: Cells Lacking Functional p95/Nbsl Do Not Activate the S-phase Checkpoint In Response to the Telomere Overhang Homolog Because of the demonstrated role of p95/Nbsl in the S-phase arrest in response to IR and its known association with telomeric DNA, we next examined its role in the S-phase arrest seen in response to the telomere overhang homolog. Fibroblasts were obtained from an NBS (Nijmegen breakage syndrome) patient, in which p95/Nbsl was below the level of detection.
Preconfluent cultures or normal (control) fibroblasts and NBS fibroblasts were treated with 40 ~,M of the oligonucleotides and collected after 48 hours for FACS analysis (Figures 34A-34H). The averages and standard deviations were calculated from triplicate cultures of each condition. FACS profiles from one representative experiment of three are shown. As expected, l lmer-1-treated normal cells exhibit an S phase growth arrest within 24 hours, while the same cells treated with diluent alone, l lmer-2 or l lmer-3 are unaffected.
Unrelated oligonucleotide-treated and diluent-treated NBS cells display an FAGS profile similar to that of the normal cells arrested in the S phase and, compared to the diluent-treated NBS cells, their proliferation is minimally affected by the l lmer-1. These data suggest that p95/Nbsl has a previously unidentified role in normal progression of the S phase and that DNA synthesis is protracted in the absence of functional p95/Nbsl. Also of interest, the 8%
increase in the number of S-phase cells in llmer-1-treated NBS cultures compared to diluent-treated cells, although not statistically sigtuftcant (p<0.08), suggests that factors other than p95/Nbsl may contribute to the arrest following DNA damage or llmer-1 treatment. For example, unscheduled E2F1 activity during the S-phase has been shown to lead to activation of an S-phase checkpoint (Krelc, W., et al., 1995, Cell 83: 1149-1158).

Phosphorylation of p95/Nbsl by ATM, causally related to activation of the S-phase checkpoint after DNA damage, can be detected by PAGE as a subtle slowing of the protein migration in the gel (Lim, D.S., et al., 2000, Nature 404:
613-617, Zhao, S., et al., 2000, Nature 405:473-477, Wu, X., et al., 2000, Nature 405: 477-482). Western blot analysis detects such a shift in p95/Nbsl migration in protein harvested from Jurkat cells 48, 72 and 96 hours after addition of the llmer-1 but not l liner-2,or 1 liner-3. This change in apparent molecular weight of p95/Nbsl, presumably from covalent modification of the protein, coincides with the S-phase arrest as determined by FACS analysis.
This shift in p95/Nbsl migration also occurs in these cells after IR, as previously reported and ascribed to protein phosphorylation (Lim, D.S., et al., 2000, Natuf°e 404: 613-617, Zhao, S., et al., 2000, Nature 405: 473-477, Wu, X., et al., 2000, Nature 405: 477-482). SCC12F cells respond identically to addition of the l liner-1, again in a time frame in agreement with their activation of the S-phase checkpoint. The shift in SCC12F cells is more apparent with immunoprecipitated p95/Nbsl from l liner-1- treated and irradiated cells and is eliminated by treatment of the immunoprecipitated protein with a serine/threonine phosphatase as was reported for p95/Nbsl phosphorylated in response to IR (Lim, D.S., et al., 2000, Natuf°e 404: 613-617). Probing a western blot of proteins from diluent-and oligonucleotide-treated fibroblasts with a phospho-p95/Nbsl-specific antibody further demonstrates that phosphorylation of p95/Nbsl is induced by the telomere overhang homolog oligonucleotide l liner-1.
EXAMPLE 36: Telomere Disruption by TRF2DN Leads to Modification of p95/Nbsl Karlseder et al. (Karlseder, J., 1999, Science 283: 1321-1325) previously demonstrated that ectopic expression of TRF2DN, which disrupts the telomere loop and exposes the 3' overhang, induces an increase in p53 and apoptosis in certain cell types. In order to see if p95/Nbsl modification is also induced by telomere disruption, neonatal human fibroblasts were transiently transfected with the TRF2DN expression vector or a (control) vector without the TRFDN insert.

_87_ Cells were collected up to 40 hours after transfection and p95/Nbsl was analyzed by Western blot using a phospho-p95-specific antibody. By 24 hours post transfection, a distinct shift in p951Nbs1 mobility is detected, similar to that seen after exposure to IR (10 Gy). This shift to a higher molecular weight is still apparent after 40 hours. These data demonstrate that both telomere disruption and treatment with a homolog of the telomere 3' overhang induce modification of p95/Nbsl and hence, support our hypothesis that exposure of the 3' overhang is the primary signal for the DNA damage responses observed after various manipulations of the telomere.
EXAMPLE 37: Telomere Overhang Oligonucleotide Does Not Decrease Telomere Length or Alter the 3' Overhang To eliminate the possibility that the 1 lmer-1 oligonucleotide acts indirectly by disrupting the telomere loop structure, leading to critical telomere shortening and/or degradation of the 3' overhang as reported after TRF2Drr transfection (Karlseder, J., 1999, Sczefzce 283: 1321-1325), we analyzed mean telomere length after treatment of normal human fibroblasts with the oligonucleotides for 5 days, longer than necessary to induce p53 and the S
phase checkpoint. Normal human fibroblasts were treated with either diluent alone, ~,M l lmer-1, 1 lmer-2 or 1 lmer-3 for 5 days. The fibroblasts were harvested and the genomic DNA was isolated. Telomere length analysis was performed for diluent-treated cells, l lmier-1'-treated cells, l lmer-2-treated cells, l lmer-3-treated cells, and late passage fibroblasts (population doubling > 50), and compared with high molecular weight and low molecular weight telomere markers. The MTL values (in kilobases) for each experimental condition, calculated as described by Harley, et al. (Harley, C.B. et al., Nature 345:458-460, 1990), were as follows: 10.59 (diluent), 12.25 (llmer-1), 10.45 (l lmer-2), 10.22 (1 lmer-3), 8.86 (senescent), 10.19 (high molecular weight standard), 4.04 (low molecular weight standard). The 3' overhang assay was carried out on newborn fibroblasts treated for 3, 5, or 7 days with 40 ~,M l lmer-1.

_88-None of the oligonucleotides reduced mean telomere length (MTL).
Indeed, l lmer-1-treated cells showed a modest increase in MTL of approximately 20% during the 5-day experiment. Furthermore, treatment of fibroblasts with the l lmer-1 oligonucleotide for up to 7 days did not result in degradation of the telomere 3' overhang as was observed following telomere disruption by TRF2DN (Karlseder, J., 1999, Science 283:1321-1325). These data strongly suggest that the l lmer-1 does not affect telomere integrity, but likely mimics the signal created by this process. Similarly, the 1 lmer-1 effects cannot be attributed to inhibition of telomerase, for example by acting as a pseudosubstrate and hence preventing telomere elongation, because normal human fibroblasts, cells in which the effects are readily observed, do not express the catalytic subunit TERT (Greider, C.W. 1996, Annu Rev BioclzenZ 65: 337-365). Furthermore, telomere erosion due to telomerase inhibition would be expected to exert effects only after an extended period of time, more than 50 population doublings in previous reports (Naka, K., et al., 1999, Biochem Biophys Res Comm 255: 753-758, Marusic, L., et al., 1997, Mol Cell Biol 17:
6394-6401, Hande, M.P., et al., 1999, J Cell Bio 144: 589-601), far greater than the 24-48 hours reported here, and is in any case not observed.
EXAMPLE 38: Oligonucleotide Treatment of in Situ Melanoma in SLID
Mice by Various Delivery Methods (Hypothetical) MM-AN cells will be grown in DMEM with 5% FBS, the cells will then be harvested with trypsin/EDTA, and the percent viability will be determined by trypan blue exclusion. Only cell populations with 90% or greater viability will be used for the experiments. The tumorigenicity of these melanoma cells will be determined by subcutaneously injecting 1 x10 viable cells in balanced salt solution into the hind limb of SLID mice to produce subcutaneous tumors. For these studies, 3 groups of mice (5 mice/group) will be used. Group 1 will be given IP inj ections of 200 ~,g T-oligo five times weekly, starting 24 hours after injection of cells group 2 will be given IP injections with 200 ~g of the control oligonucleotide five times weekly and group 3 will be injected with diluent.

Oligonucleotides delivered IP have been found to be effective in reducing the size of subcutaneous melanoma in mice (Massod, R., et al., Blood 96:1904-1913, 2001). The size of the tumors will be monitored by twice weekly li examination of the mice and measurement of the tumors with calipers. The mice will be killed 4 wee~s after injection and the tumors will be processed for hematoxylin and eosin staining.
MM-AN melanoma cells will be treated and harvested as described above. Instead of delivering the oligonucleotide IP it will be delivered by an Alzet pump. 500 ~,g/per day of T-oligo or control oligo or diluent will be infused for 4 weeks. The size of the tumor will be evaluated as described above.
Alzet pumps have been found effective in treating subcutaneous melanoma tumors in mice (Hijiya, N., et al. Proc. Natl. Acad. Sci. vUSA 91:4499-4503, 1994).
MM-AN melanoma cells will be treated and harvested as described above. Group 1 will be given IV injections of 200 ~g T-oligo three times weelcly, group 2 will be given IV injections with 200 wg of control oligonucleotide three times weekly, and group 3 will be injected with diluent.
Mice will be killed 4 weeks after injection and the size of the tumors will be evaluated as described above.
EXAMPLE 39: Oligonucleotide Effect on Melanoma Metastasis and Tumorigenicity (Hypothetical) The pilot experiment protocol which results in maximum tumor regression will be selected. MM-AN melanoma cells will be treated and harvested as described above. 1x10 cells in balanced salt solution will be inj ected into the lateral tail vein of 4 week old SLID mice. There will be 3 groups of 25 mice for each of the above treatment groups. Group 1 will be inj ected with T-oligo; group 2 will be inj ected with the control oligonucleotide and group 3 will be injected with diluent. All treatments will begin 24 hours after injecting the cells. After 6 weeks, the mice will be killed by COZ
inhalation, an autopsy will be performed and organs will be examined macroscopically for visible lesions. Particular emphasis will be given to liver, pancreas and brain because these organs have been shown to be the primary sites of metastasis of MM-AN cells. For examination and sectioning, organs will be rinsed in PBS and fixed in 10% formalin for 48 hours. Organs will be examined under a dissecting microscope and the number of tumor nodules counted. The histological characteristics of tumors will be examined with hematoxylin and eosin-stained sections of tissue fixed in formalin and embedded in paraffin.
MM-AN melanoma cells will be treated and harvested as described above. The pilot experiment protocol which resulted in maximum tumor regression will be selected. The tumorigenicity of these melanoma cells will be determined by intradermally injecting 1x10 viable cells in balanced salt solution into the hind limb region of SCID mice to produce subcutaneous tumors. The size of the tumor will be monitored by twice weekly examination of the mice and measurement of tumors with calipers. The mice will be killed 4 weeks after injection and the tumors will be removed, measured and processed for hematoxylin and eosin staining. Three groups of mice will be used. Group 1 will be treated with T-oligo, group 2 will be treated with control oligonucleotide, and group 3 will be treated with diluent. Apoptosis will be determined in tumors by TUNEL in the groups of mice discussed above. The expression of differentiation markers gp-100, TRP-1 and Mart-1 will also be studied in tumors from the three groups of mice studied above.
EXAMPLE 40: Telomere Homolog Oligonucleotides Less Than 6 Nucleotides in Length Inhibit Proliferation of a Human Osteosarcoma Cell Line Human Saos-2 cells were plated in DME with 10% fetal bovine serum in p35 culture dishes, and dosed with 40 ~.M Smer (TTAGG), 3mer (TTA) 1 liner-1 (GTTAGGGTTAG; SEQ ID NO:S) or diluent alone as control, and cultured for an additional 36 hours after addition of oligonucleotides or diluent. Cells were then collected by trypsinization and counted by Coulter Counter. The Figure 36 graph represents averages of cell population doublings after additions, with standard error of mean, for duplicate cultures.

EXAMPLE 41: T-Oligo Inhibits Breast Cancer Cell Proliferation By Inducing Apoptosis and Senescence Inhibition of breast cancer proliferation by exposure to T-oligo.
Breast cancer cells were provided once with 40 ~M T-oligo (pGTTAGGGTTAG; SEQ ID NO:S), control oligonucleotide (pCTAACCCTAAC; SEQ ID N0:9) or diluent alone. Over 4 days, a single T-oligo treatment significantly reduced cell yields of MCF-7 (p<0.006) (Figure 37) and BT-20 (p<0.006) (Figure 38) cells as compared to control oligo or diluent alone.
MCF-7 cells were supplemented with T-oligo (40 ~,M) on day 1 after plating and then on day 3 were provided either with fresh medium and diluent alone or with fresh medium containing T-oligo. Duplicate cultures were continuously supplemented with medium and diluent alone and reached confluence at day 7. Even after T-oligo removal, the treated MCF-7 cells failed to resume growth for at least 14 days, when the experiment was terminated (Figure 39).
Exposure to T-oligo induces apoptosis and senescence.
MCF-7 (Figure 40) and BT-20 (Figure 41) cells were maintained in the presence of T-oligo, control oligo or diluent alone as above. In both cell lines, TLJNEL analysis showed a shift in the fluorescent peak, indicating DNA strand breaks, in T-oligo treated cultures. T-oligo treated MCF-7 cells display a prominent increase in senescence-associated ~3-galactosidase (S.A. ~3-gal) activity (Figure 42), a characteristic marker well known to identify aged cells and non-proliferative cells, both in vivo and in vitro (Dimri, G.P., et al., Mol Cell Biol.
20:273-285, 2000).
T-oligo induces p53 level and activity.
MCF-7 cells were maintained with T-oligo as above. Total cellular proteins were processed for western blotting. The blot was initially reacted with an antibody to phospho-p53 (serine-15), the form of the protein believed to indicate activation. After stripping, the blot was reacted with antibody recognizing total p53, and then with antibody against p21, a p53 transcriptionally regulated gene product. Within 48 hours, T-oligo induced p53 and p53 activity as determined by p53 phosphorylation and p21 induction. Similar results were obtained with BT-20 cells.
T-oligo is comparable to cisplatin and ICI 182,780 in inhibiting breast cancer cell proliferation.
MCF-7 and BT-20 cells were maintained as above. Cells were supplemented with T-oligo (40 ~M), with cisplatin (10 wM) as described (Rudin, C.M., et al., Cancer Res. 63:312-318, 2003 and Deng, X., et al. Bioclaem Biophys Res Commun. 296:792-798, 2002), with the antiestrogen ICI 182,780 (10-~ M, only MCF-7 cells) as described (Varma, H., et al., Cancer Res.
62:3985-3991, 2002; and Sun, J., et al., Endocrinology, 143:941-947, 2002) or with diluent alone. The T-oligo effect on cell proliferation is comparable to that of cisplatin, a chemotherapeutic agent commonly used to treat ovarian and breast cancer (Burstein, H.J., et al., Semin. Oncol. 28:344-358, 2001; Jakesz R., et al., Eur. J. Cancer 38:327-332, 2002; Otto, A.M., et al., J Cayacer Res Clin Oyacol.
122:603-612, 1996; and Sauter, C., et al., Oncology 43:46-49, 1986). The T-oligo effect on cell proliferation is also comparable to the effect of anti-estrogens used to control the growth of estrogen receptor positive cells like MCF-7 (Otto, A.M., et al., J Cancer Res Clin Oncol. 122:603-612, 1996). See Figure 43 and Figure 44.
EXAMPLE 42: T-Oligo Inhibits Growth of Human Pancreatic Adenocarcinoma Cells in Vitro HPAC cells, a human pancreatic adenocarcinoma epithelial cell line derived from a woman with moderate to well differentiated pancreatic adenocarcinoma of ductal origin, were purchased from American Type Culture Collection (Manassas, Virginia; ATCC Accession No. CRL-2119). The cells were plated in 35 mm dishes and were maintained in a 1:1 mixture of Dulbecco's Modified Eagle's Medium (DMEM) and Ham's fl2 medium containing 1.2 g/L sodium bicarbonate supplemented with 15 mM HEPES, 0.002 mg/ml insulin, 0.005 mg/ml transferrin, 40 ng/ml hydrocortisone, 10 ng/ml epidermal growth factor and 5% FBS. Twenty-four hours after plating, triplicate dishes were supplemented with 40 ~,M pGTTAGGGTTAG (T-oligo; SEQ ID
NO:S), 40 ~M pCTAACCCTAAC (control oligonucleotide; SEQ ID N0:9) or diluent alone. Cell yields were determined daily. T-oligo substantially inhibited HPAC cell growth, as seen by the results plotted in Figure 45.
EXAMPLE 43: T-Oligo Inhibits Growth of Breast Cancer Cells and Ovarian Cancer Cells Two DNA oligonucleotides were designed for the experiment and purchased from the Midland Certified Reagent Company, Midland Texas: one homologous to the telomere overhang repeat (T-oligo: pGTTAGGGTTAG;
SEQ ID NO: 5), and the other complementary to this sequence (pCTAACCCTAAC; SEQ ID N0:9) to be used as a control.
These oligonucleotides were stored as aliquots at -20°C until dilution in culture media immediately before cell treatments.
Cancer Cell Lines Human ovarian cancer cell line OVCARS (obtained from Dr. Tayyaba Hasan, Harvard Medical School) and breast cancer line MCF-7 (obtained from the ATCC Human Cell Repository) were used in our study. These cells were maintained in RPMI 1640 mediumsupplemented with 10% calf serum (CS) and cultured at 37°C in a humidified atmosphere containing 5% CO2, then passed into plates for experiments and cultured for 24 hours. After 24 hours, the cells were subj ected to treatment.

Culture of Cells RPMI 1640 medium and trypsin-EDTA (10x; 0.5% trypsin, 5.3 mM
EDTA) were obtained from Life Technologies, Roclcville, MD, and calf serum (CS) was obtained from Whittaker Bioproducts, Walkersville, MD.
The cancer cells were maintained as monolayers in RMPI 1640 medium supplemented with 10% calf serum (CS). All cells were cultured at 37°C
in 5%
COZ and 100% humidity.
The medium was first removed with a glass pipette connected to a vacuum flask. Phosphate buffered saline (PBS) was added for 10 minutes and then removed. The dish was then washed twice with EDTA, and was subsequently trypsinized by adding 1 ml of 0.5% trypsin and incubating until cells detached. One ml of 10% CS was added to stop the action of trypsin and to act as a source for nutrients. The solution was then removed, while flushing suspended cells into test tubes. This suspension was centrifuged for 5 minutes at 3,500 RPM and the supernatant subsequently removed. The cell pellet was then resuspended in 2 ml of fresh medium. A cell count was performed, and the cells distributed among 30 culture plates. Medium containing 10% CS was added to the new plates, which were then left to incubate for 24 hours.
Treatment of Cells Twenty four hours after plating, cells were treated with 40 ~M T-oligo, the control complement oligo, or an equal volume of diluent, added to 2 ml of medium containing 10% CS. The cells were then left to incubate for periods of 3, 4 and 5 days.
Experimental Design As shown in Tables 3 and 4, two separate sets of experiments were carried out, one for tracking cell growth, and the other to stain for ~i-galactosidase activity, which is a marker for senescence.

Measurement of Cell Growth Growth inhibition of OVCARS and MCF-7 cells was determined by use of a Coulter cell counter. Cells were collected 3, 4 and 5 days following treatment, pelleted, and resuspended in 1 ml of medium. Fifty ~1 of this cell suspension were added to 10 ml of isotonic salt solution and the cells in this solution were counted using the cell counter. This was done twice for each sample and the averages and standard deviation were calculated from these values.
Senescence - Associated ~i-galactosidase Histochemical Staining Another set of cells was stained to detect senescence-associated [3-galactosidase (S.A. ~3-gal) activity. Senescent cells express ~-galactosidase which cleaves the .X-gal substrate (5-bromo-4-chloro-3-indoyl ~3-D-galactopyranoside) to a product with a blue color.
The cells were washed twice with PBS solution and fixed with a solution prepared from 2.7 ml 2% formaldehyde, and 4 ml 0.2% glutaraldehye in 43 ml PBS for five minutes. The stain was freshly prepared each day, using 1 ml of 1 mg/ml X-gal in dimethylformaldehyde, 4 ml of 0.2 M citric acid /sodium phosphate buffer at pH 6.0, 1 ml of 100 mM potassium(aI) hexacyanoferrate solution, 1 ml of 100 mM potassium (IV) hexacyanoferrate solution, 600 ~l of 5 M sodium chloride, 40 ~1 of 1 M magnesium chloride and 13.4 ml of distilled water.
Two ml of stain were added to each plate. The plates were subsequently sealed with parafilm to prevent evaporation of the stain, and incubated for up to 3 days at 37°C. Fresh stain solution was added daily. For analysis, the cells were washed twice with PBS and the percentage of positively stained cells was determined.
Cell Count Data From the analysis of data obtained from the OVCARS cell counts, there was a reduction of ovarian cancer cell numbers for cells treated with T-oligo as compared to the control plates which were treated with diluent, demonstrating that the T-oligo induced a replicative arrest, suggestive of senescence (Table and Figure 46).
There was also a slight reduction in cell number in cultures treated with the control complement oligonucleotide. The reason for this growth inhibition is not clear, but it likely results from the sensitivity of some cell types to the presence of DNA in the culture medium.
From the analysis of the data obtained from the MCF-7 cells, it is seen that there is also a reduction of breast cancer cell numbers treated with T-oligo as compared to the control plates which were treated with diluent. Again there was a slight inhibition of cell growth with the control complement oligonucleotide (Table 6 and Figure 47).
S.A. ~3-galactosidase Stain Results Cells expressing ~3-galactosidase, a senescence marker, are typically much larger in size and multinucleated, both of which are morphological features indicative of senescence. Normal human fibroblasts stain positive for S.A. ~3-gal after T-oligo treatment.
W the OVCARS cells, it was seen that cells treated with diluent and the complement oligo did not stain blue' from producing ~-galactosidase, nor did they express any senescent phenotype. For the cells treated with T-oligo, only one cell stained blue. However, when viewed under l Ox magnification, it was observed that these cells were larger in size as compared to cells given control treatments and many of these cells were multinucleated as well.
In the MCF-7 cells, the percentage of cells that stained blue for the control (diluent-treated) plate was 5.975%, while that for the plate treated with T-oligo was 66.75%, and the percentage for the plate treated with the complement oligo was 6.675%. Cells in the T-oligo treated plate were also observed to be multinucleated and larger, while cells in the other two plates did not express this senescent-indicative morphology.

In summary, the T-oligo inhibits cell growth and induces a larger, flat, multinucleated morphology in both the ovarian and breast cancer cells studied.
Furthermore, the T-oligo induces the expression of S.A. ~i-gal in the MCF-7 breast cancer cells.
EXAMPLE 44: Effect of T-Oligo on Melanoma Cells Oligonucleotides Experiments were designed to test the effects of 3 oligonucleotides with 5' phosphate groups and phosphodiester linkages: one homologous to the 3' . telomere overhang repeat sequence (T-oligo: pGTTAGGGTTAG; SEQ m NO:S), one complementary to this sequence (pCTAACCCTAAC; SEQ m N0:9) and one unrelated to the telomere sequence (pGATCGATCGAT; SEQ m N0:10) (Midland Certified Reagent Company, Midland, Texas).
Cell culture and Cell cycle analysis.
MM-AN, MU, PM-WK, MM-RU, MM-MC and RPM-EP melanoma cells were grown in Minimum Essential Medium Eagle (MEM) with 10% FBS
(both from GibcoBRL, Gaithersberg, MD). Normal newborn human melanocytes were established and cultured as controls as previously described (Gilchrest, B.A., et al., JTnvest Def°fnatol 83:370-376, 1984).
Duplicate melanoma cell cultures were grown in MEM with 5% FBS and treated with a final concentration of 40 ~M of the 11-mer oligonucleotides homologous, complementary, or unrelated to the telomere 3' overhang sequence, or with an equal volume of diluent (water) as a control, for 96 hrs. Cells were collected, stained with propidium iodide and analyzed by a Becton Dickinson FACS Scan and CellQuest software.
TUNEL analysis.
To detect apoptosis by DNA end labeling, cells were grown and treated with the oligonucleotides as described above. Seventy-two hours after treatment, cells were fixed in 4% paraformaldehyde for 30 minutes and permeabilized with 0.1 % Triton X-100 for 2 minutes. The TUNEL assay was carried out using the cell death detection kit from Boehringer Mam~heim (Indianapolis, IN) using fluorescein-labeled dUTP. TUNEL positive cells were detected by FACS.
Western blot analysis and antibodies.
Melanoma cells were cultured in MEM with 5% FBS and treated with 40 ~.M T-oligo, complementary oligonucleotide, unrelated oligonucleotide, or an equal volume of diluent (water) for various times. Proteins were analyzed by denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis as described previously (Ellen M. S., et al., P~oc Natl Acad Sci USA
93:1087-1092, 1996) using an anti-tyrosinase monoclonal antibody (clone T311, Nova-castra, New Castle upon Tyne, UK); anti-TRP-1 monoclonal antibody (clone Ta99, Signet, Dedham, MA); anti-gp 100 monoclonal antibody (MO 634, Dako corporation, Carpinteria, CA) and anti-MART-1/Melan-A monoclonal antibody (clone M2-7C10, Signet, Dedham, MA); anti-livin/ML-IAP goat polyclonal antibody (AB798 Novus Biologicals Inc, Littleton, CO).
Animals.
Severe combined immunodeficient (SLID) mice were purchased from Fox-Chase Cancer Center, Philadelphia, PA and hosted in the pathogen-free animal facility at Boston University Medical Center.
Effect of T-oligo on melanoma metastasis.
At 40 ~,M, T-oligo (pGTTAGGGTTAG; SEQ ID NO:S) induced apoptosis of 30%-70% of cells from 6 human melanoma cell lines (MM-AN, MU, PM-WK, MM-RU, MM-MC and RPM-EP) within 72-96 hours as determined by FACS analysis and TUNEL staining, while 11-base oligonucleotides with complementary or unrelated sequences had no effect.
To study the effect of the T-oligo on the metastatic potential of MM-AN
cells, these cells were treated with 40 ~,M T-oligo, complementary oligonucleotide or diluent for 48 hrs in MEM with 5% FBS. The cells were then harvested with trypsin/EDTA and the percent viability was determined by trypan blue exclusion staining. Only cell populations with 90% or greater viability were used for this investigation. 1x10 cells in balanced salt solution were injected into the lateral tail vein of 5 week old SCm mice. There were 3 groups of 5 mice. Group 1 was injected with cells treated with the T-oligo, group 2 was injected with cells treated with the complementary control oligo, and group 3 was injected with diluent-treated cells. After 40 days, the mice were euthanized by COz inhalation, autopsies were performed, and organs were examined macroscopically for visible lesions. The lesions were also measured with calipers. Particular emphasis was given to lung, pancreas, brain, brown fat, kidney, and bone because these organs have been shown to be the primary sites of metastases of MM-AN cells (Byers, H. R., et al., Melanoma Res 3:247-253, 1993). For examination and sectioning, organs were rinsed in PBS and fixed in 10% formalin for 48 hrs. Organs were examined under a dissecting microscope and the number of tumor nodules counted. For histological examination, tumors were fixed in formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin. The melanocytic origin of the metastases was confirmed by the presence of melanin pigment.
Treatment of SCm mice for studying the effect of T-oligo on tumorigenicity.
MM-AN melanoma cells were cultured in MEM with 5% FBS and harvested with trypsin/EDTA. The tumorigenicity of these melanoma cells was determined by intradermally injecting 2x10 viable cells in balanced salt solution into the flank region of SCm mice to produce subcutaneous tumors. Three groups of 6 mice per group were studied. Twice daily intratumoral injections of oligonucleotide began 72 hrs after the melanoma cells were injected. Group 1 was given T-oligo (210 ~,g=60 nmoles in 150 ~,1 of PBS), group 2 was given the complementary control oligo; group 3 was injected with diluent alone. The mice were euthanized after 22 days, 24 hours after the last treatment. The tumors were measured with calipers, fixed in 10% formalin, and stained with hematoxylin and eosin. Immunohistochemical staining of tumors was done using gp-100 and TRP-1 monoclonal antibodies. For technical reasons, expression of tyrosinase and MART-1 could not be assessed in the tumors.
Apoptosis was determined by TUNEL analysis of tumor cross-sections using the ApopTag Kit from Intergen (Purchase, NY).
T-oligo induces apoptosis in melanoma cells.
MM-AN melanoma cells were treated with diluent, 40 ~M T-oligo, 40 ~,M complementary oligonucleotide or 40 ~,M unrelated oligonucleotide. The cells were collected after 96 hrs, stained with propidium iodide and analyzed by FACS. Data are presented on a logarithmic scale which allows better visualization of the apoptotic population (Figure 48). Following treatment with T-oligo, 65% of the cells were apoptotic as indicated by the sub G°/Gi population of cells. MM-AN cells were treated as described above and subjected to TLTNEL
analysis. Fluorescence indicates labeling of fragmented DNA, a maxl~er of apoptosis, selectively in the T-oligo treated cells (Figure 49). Normal human melanocytes were treated with diluent or the oligonucleotides as described above and were collected at 24 and 96 hours post-treatment for FACS analysis. Data are presented on a linear scale to display the S-phase arrest (Figure 50). MM-AN cells treated similarly and presented on a logarithmic scale in Figure 48 are also shown on the same FAGS profile here in white for comparison.
At 24 and 48 hours after treatment with the T-oligo, MM-AN cells were blocl~ed in S phase, as has been previously reported for other cell types (Ellen M.S., et al., Exp Cell Res 276:185-193, 2002; Eller, M.S., et al., Faseb J
17:152-162, 2003). At 72 hours the S-phase arrest persisted, but 22% of the MM-AN cells appeared in the sub Go/Gl region of the FAGS profile indicating apoptosis, and by 96 hours the percent of apoptotic cells increased to 65%
(Figure 48). Within 72 hours, TUNEL analysis demonstrated a distinct increase in fluorescence intensity of cells treated with the T-oligo, confirming widespread apoptosis (Figure 49). Neither the control oligonucleotides nor diluent alone affected the cells. MM-AN cells do not express detectable levels of p53, but as previously reported (Ellen M.S., et al., Exp Cell Res 276:185-193, 2002), T-oligo-induced apoptosis is preceded by an increase in the E2F1 and p73 transcription factors, and inhibited by 50% in MM-AN cells ectopically expressing a dominant-negative form of p73, suggesting that the apoptosis is mediated at least in part by the interaction of E2F1 and the p53 homolog p73.
In S contrast, the T-oligo did not induce apoptosis in normal human melanocytes within 96 hours, although it did induce an S-phase arrest most apparent at 24 hours (Figure 50), as reported previously for the 2-base thymidine dinucleotide (Pedeux, R., et al., Jlhvest Dernaatol 111:472-477, 1998) in these cells and for the 11-base T-oligo in other primary cell types and established cell lines (Eller, M. S., et al., Exp Cell Res 276:185-193, 2002; Eller, M. S., et al., Faseb J
17:152-162, 2003). To determine if the MM-AN cell response was unique, five other melanoma cell lines were examined. Of these, four underwent apoptosis at 96 hours after treatment with the T-oligo, with apoptosis rates ranging from 30%
to 60% as determined by FACS analysis.
The protein livin, also called ML-IAP or inhibitor of apoptosis protein, is a member of the protein family that is commonly up-regulated in melanoma cells and largely responsible for resistance to chemotherapy-induced cell death in at least some cell lines (Kasof, G.M. et al., J. Biol. Cherya. 276:3238-3246, 2001;
Vucic, D. et al., Cu~~. Biol 10:1359-1366, 2000; Li, J. et al., Efzclocs°is2ology 142:370-380, 2001; Sasaki; H. et al., Ca~iceY Res. 60:5659-5666, 2000).
T-oligo reduces expression of livin/ML-IAP.
MM-AN cells were treated with 40 ~,M T-oligo, complementary oligonucleotide, unrelated oligonucleotide, or an equal volume of diluent alone, for 24, 48 or 72 hours. Cells were harvested and western blotting was performed using an antibody for livin/ML-IAP. MM-AN cells expressed a high constitutive level of livin/ML-IAP, and treatment with the T-oligo selectively and dramatically reduced the expression within 72 hours. Therefore, in this cell type the apoptotic response to T-oligo appears likely to be modulated not only by the induction of the pro-apoptotic proteins E2F1 and p73, as reported previously (Ellen M.S., et al., Exp. Cell. Res. 276:185-193, 2002), but also by down-regulation of livin/ML-IAP.
Progression of melanoma is often associated with the loss of melanocyte-specific antigens, presumptively allowing the tumor cells to escape immunosurveillance and defeating attempts at immunotherapy (Slingluff, C. L., Jr. et al., Cancer Inamunol. Immunother 48:661-672, 2000). Because melanocyte differentiation is associated with slower cell proliferation, as observed after treatment with T-oligo (Eller, M.S. et al., Proc. Natl. Acad. Sci. USA 93:1087-1092, 1996; Hadshiew, LM., et al., J. Der~matol. Sci. 25:127-138, 2001;
Pedeux, R. et al., J. Invest. Dermatol. 111:472-477, 1998), and because oligonucleotides sharing at least 50% nucleotide sequence identity with the telomere overhang repeat enhance differentiation (melanization) of human melanocytes and S91 marine melanoma cells (Ellen M.S. et al., Natz~~-e 372:413-414, 1994; Eller, M.S. et al., PPOG. Natl. Acad. Sci. USA 93:1087-1092, 1996; Pedeux, R. et al., J.
Invest. Dermatol. 111:472-477, 1998), we examined the expression of melanocyte differentiation antigens that have been targeted for immunotherapy.
T-oligo induces markers of melanocyte differentiation.
Cells were treated as described above and analyzed for protein expression of TRP-1, gp-100, tyrosinase, and melan-A/MART-1.
T-oligo induced the melanocyte-associated differentiation markers tyrosinase-related protein 1 (TRP-1 or gp 75)' and glycoprotein 100 (gp 100), two antigens whose expression is frequently lost in more aggressive, poorly differentiated melanomas (Slingluff, C.L., Jr. et al., Cancer Immunol.
InZmunother 48:661-672, 2000). T-oligo also induced tyrosinase, the rate-limiting enzyme in melanogenesis, previously shown to be induced in normal human melanocytes by other oligonucleotides with a sequence sharing at least 50% nucleotide sequence identity with the telomere overhang repeat (Eller, M.S. et al., PPOG. Natl. Acad. Sci. USA 93:1087-1092, 1996) and in human and marine melanoma cells (Ellen M.S. et al., Nature 372:413-414, 1994; Eller, M.S. et al., Proc. Natl. Acad. Sci. USA 93:1087-1092, 1996; Pedeux, R. et al., J.

Invest. De~rnatol. 111:472-477, 1998) and MART-1, a protein expressed in melanocytes and melanomas which is a recognized target for a subclass of lymphocytes in melanoma immunotherapy (Rivoltini, L. et al., J. Immunol.
154:2257-2265, 1995).
The above data suggested that.oligonucleotides sharing at least 50%
nucleotide sequence identity with the telomere overhang repeat may reduce MM-AN tumorigenesis and metastasis in vivo. In order to explore this possibility, MM-AN cells were treated for 48 hours with 40 ~,M T-oligo, complementary oligonucleotide or diluent alone and then collected, washed and placed in oligonucleotide-free medium. Cells treated for 48 hours with the oligonucleotides or diluent alone were then injected into the tail vein of SCm mice (5 mice per group), using lOG viable cells per injection, with viability determined by trypan blue exclusion. Forty days after the inj ections, control mice receiving the complementary oligonucleotide had lost approximately 12%
of initial body weight, showed limited movement, and appeared severely ill; T-oligo-treated animals had increased their weight by 27% and appeared healthy.
Forty days after injection, the mice were euthanized, autopsies were performed, and the average number and size of macroscopic metastases in the brain, liver, pancreas and bone were determined by visual inspection of the organs. In contrast to animals injected with control cells, animals injected with T-oligo treated cells were virtually tumor-free, with no detectable metastases in four out of five mice. T-oligo treatment of MM-AN cells before injection also reduced the average volume of the metastases by 80-85%, compared to control groups (Figure 51), and the average number of metastatic tumors was reduced by 90-95% (Figure 52). Although pretreatment of malignant cells is not equivalent to treating metastases already established in vivo, these data demonstrate that relatively brief exposure to the T-oligo can have a long-lasting effect on these cells that cannot be reversed by the tissue environment. Because oligonucleotides of this size with physiologic phosphodiester linkage have a half life in culture of approximately 4-6 hours (Wright, W.E. et al., EMBO J.

15:1734-1741, 1996), the effective exposure time of MM-AN cells to T-oligo in these experiments is probably far less than 48 hours.
In order to determine whether T-oligo can affect the growth of pre-existing tumors, 2xl0~MM-AN cells were injected subcutaneously into the flank of SCID mice (6 mice/group). Beginning 72 hours later, when small tumor nodules were first apparent, the sites were inj ected twice daily with 60 nmoles of T-oligo (150 ~l of a 0.4 mM solution), complementary oligonucleotide as control, or an equal volume of diluent alone. After 22 days, the animals were euthanized and the tumor size was determined. There was no significant difference in tumor size between diluent and control oligo-treated mice, but T-oligo treatment reduced tumor volume (p<0.05) by 84% and 88% respectively, as determined by measurement of the tumors with calipers. Control mice developed large tumors, but mice treated with T-oligo developed tumors that were very small (3 mice) or undetectable (3 mice). As also determined in tissue cross-sections obtained 24 hours after the last injections, T-oligo induced tumor cell apoptosis as seen by TUNEL anlysis of tumors on day 22, 24 hours after the last injection. T-oligo treatment also greatly enhanced expression of the differentiation associated proteins gp100 and TRP-1 in the small residual tumor nodules. The tissue surrounding the injected tumor nodules appeared normal in all animals, as seen in immunostained sections.
The T-oligo effects persisted in vivo for at least 40 days after an initial 48-hour exposure of MM-AN cells in vitro; and were observed to a comparable degree in MM-AN cell tumors established in vivo prior to T-oligo exposure and then subj ected to twice daily local inj ections.

EXAMPLE 45: Effect of Oligonucleotide Sequence on Induction of Apoptosis in Melanoma Cells Table 7 Apoptotic Cells SEQ ID 72 Hours 96 Hours NO:

Diluent 2.8 + 0.3 2.3 0.4 TTAGAGTATGAGTTA 16 8.5 + 0.6 22 + 1 TTAGAGTATGAG 17 13.6 + 1.3 33.1 2.8 GAGTATGAGTTA 18 12.2 + 2.5 22.8 1.5 GTTAGGGTTAG 5 18.5 + 1 I 43.4 + 1 Oligonucleotides (all with 5' phosphate groups) with sequences sharing at least 50% nucleotide sequence identity with the telomere overhang repeat were synthesized. Cultures of the human melanoma line MM-AN were treated with these oligonucleotides at a concentration of 40 ~,M for 72 and 96 hours and the percent of apoptotic cells was determined by FACS analysis. All of the oligonucleotides induced apoptosis (Table 7).
EXAMPLE 46: Hydrolysis of the T-oligo is Necessary for Activity Oligonucleotides of SEQ ID NO:S were synthesized. Oligonucleotide 1 was synthesized entirely with a phosphorothioate backbone. Oligonucleotide 2 had two phosphorothioate linkages on each end, with the other linkages in the middle being phosphodiester linkages. Oligonucleotide 3 had two phosphorothioate linkages on the 5' end (5' end bloclced), with the rest of the linkages being phosphodiester linkages. Oligonucleotide 4 had two phosphorothioate linkages on the 3' end (3' end blocked), with the rest of the linkages being phosphodiester linkages. See Figure 53.

These oligonucleotides were added to cultures of normal neonatal fibroblasts. After 48 hours, cells were collected to be analyzed for p53 serine 15 phosphorylation and p95/Nbsl phosphorylation by western blot. Other cultures were left in the presence of the oligonucleotides for one week and then the cells were stained for senescence-associated [3-galactosidase activity (S.A. [3-Gal). [3-galactosidase positive cells were scored and presented as a percent of total cells (Figure 54).
Oligonucleotides with a nuclease-accessible 3' terminus are the most effective at stimulating "early" responses such as p53 and p95/Nbsl phosphorylation. However, oligonucleotides with a nuclease-accessible 5' terminus can also induce the senescent phenotype after one week, but not the phosphorylation reactions at 48 hours, suggesting that 3' -~ 5' nuclease susceptibility is preferable for activity in inducing senescence.
EXAMPLE 47: T-oligo Induces Differentiation BT-20 cells, human breast cancer estrogen receptor negative cells, purchased from American Type Culture Collection (ATCC Accession No. HTB-19) were maintained in minimum essential medium (Eagle) with 2 mM L-glutamine and Earle's BSS adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids, and 1.0 mM sodium pyruvate, 90%; fetal bovine serum, 10%. Twenty-four hours after plating, dishes were supplemented with 40 ~.M T-oligo pGTTAGGGTTAG (SEQ ID NO:S), 40 ~.M pCTAACCCTAAC
(SEQ ID NO:9) control oligonucletotide, or diluent alone. Total cellular proteins were harvested 7 days later and were processed for western blot analysis. The blot was reacted with anti-estrogen receptor a antibody. Forty ~,M
pGTTAGGGTTAG (SEQ ll~ NO:S) substantially induced the level of estrogen receptor a as compared to the level of the receptor in diluent treated or pCTAACCCTAAC (SEQ ID N0:9) treated cells. This experiment shows that treatment with oligonucleotides that share at least 50% nucleotide sequence identity with the telomere overhang repeat sequence induces the differentiation of breast cancer cells and may render them susceptible to treatment with anti-estrogens.
EXAMPLE 48: T-oligo Induces Senescence-Associated (3-Galactosidase (SA-(3-Gal) Activity in Human Fibrosarcoma Cells Human fibrosarcoma cells were treated for 4 days with diluent alone, 40 ~,M T-oligo (pGTTAGGGTTAG; SEQ ID NO:S), or the complementary oligonucleotide pCTAACCCTAAC (SEQ ID N0:9), then stained with X-gal to assay for SA-~3-Gal activity as described by Dimri et al. (Pf~oc. Natl. Acad.
Sci.
USA 92:9363-9367, 1995).
In another experiment, parallel cultures were treated for 4 days with diluent alone, 40 ~M T-oligo (pGTTAGGGTTAG; ~SEQ ID NO:S), or the complementary oligonucleotide pCTAACCCTAAC (SEQ ID N0:9). The cells were washed once with PBS and refed with complete medium for 24 hours or 48 hours, then stained with X-gal to assay for SA-(3-Gal activity.
T-oligo Treated Fibrosarcoma Cells Lose Clonogenic Capacity Human fibrosarcoma cells were treated with diluent, 40 ~,M T-oligo or complementary oligonucleotide for one week and were then trypsinized and counted. Three hundred cells were seeded into 60 mm culture dishes in triplicate, and then incubated in 4 ml of complete medium for 2 weeks; medium was changed twice per week. Subsequently, the medium was removed, and the cells were fixed for 5 min in 4 ml of methanol. The methanol was removed, the culture dishes were rinsed briefly with water, the cell colonies were stained for 10 min in 4 ml of 4% (w/v) methylene blue solution in PBS, washed once again with water, and then comited. The yield of colonies, as a percentage of cells plated, was ~ 100% for the diluent-treated cells, ~ 6% for the cells treated with T
oligo, and ~91% for the cells treated with complementary oligonucleotide.

T-Oligo Activates Both the p53 and pRb Pathways Human fibrosarcoma cells were treated with diluent, 40 ~,M T-oligo or complementary oligonucleotide for four days and were then collected for protein analysis by western blot using 30 ~,g total protein per condition, and probed for total p53, phospho-p53 (serine 15) and phospho-pRb (serine 780). For p53 detection, the blot was first probed for phospho-p53 (serine-15) and then stripped and re-probed with the pantropic p53 antibody DO-1.
Protein from all three protein preparations was bound by p53 antibody DO-1. Protein from the diluent-treated cells and from the complementary oligonucleotide-treated cells was bound by antibody to pRb phosphorylated at serine 780. Protein from only the T-oligo-treated cells was bound by antibody to p53 phosphorylated at serine 15.
EXAMPLE 49: Pretreatment With pTT Delays Tumor Formation and Decreases the Number of UVB-Induced Tumors in XPC+~ Mice SKH1 hairless wild type (WT) and XPC+~- knockout (KO) mice (8 animals per treatment group) were pretreated topically with thymidine dinucleotide (pTT, 100 ~,M), or diluent (propylene glycol 75%/DMSO 25%) alone once a day for 5 days, then were LTVB irradiated for 3 weeks once a day for 5 days (WT mice with 30 mJ/cm2 and KO with 5 mJ/cm2 solar-simulated UV
metered at 285 ~ 5 nm).
Mice received 5 consecutive cycles (150 days) of one weelc daily topical pTT (or diluent alone) followed by 3 weeks of daily UV irradiation. One week of pretreatment followed by three weeks of LTVB were designated as one cycle.
Initial UVB dose 25 mJ/cm2 was increased 1.5 times with subsequent cycle. One hundred sixty days after the initial UVB irradiation, the mice were sacrificed.
Tumor prevalence and multiplicity were evaluated on a weekly basis starting on week 5 after the initial UVB irradiation.
By week 6, 25% of diluent-treated mice developed tumors on UVB
irradiated areas, whereas all of the pTT-treated mice were tumor free up to 12 weeks post-UVB. By 12 weeks, already 75% of diluent-treated mice had tumors.

One hundred sixty days after initial IJVB irradiation, when all mice were sacrificed, all of the diluent-treated mice had developed one or more tumors, whereas 33% of the pTT-treated mice continued to be free of tumors. See Figure 55.
Compared to diluent treatment, pTT treatment reduced by one-half the average number of tumors in WT and XPC+~- mice 160 days after the initial UVB
irradiation, 7.25 ~ 0.87 vs. 3 ~ 0.94 (diluent vs pTT, respectively, p<0.006).
A
statistically significant difference in the average number of tumors/animal in pTT-treated vs diluent-treated mice was also detectable between weelcs 12 to (p<0.05), despite the small number of experimental animals. See Figure 56.
All tumors in the irradiated mouse skin measuring at least 1 mmz on the surface were excised at the time of euthanasia, formalin fixed, embedded, vertically sectioned and stained with hematoxylin and eosin. Sections of each tumor were then examined under light microscopy (40x) by a dermatopathologist in a blinded manner, without knowing in which treatment group (pTT or vehicle control) the tumors had arisen. Conventional pathologic criteria for diagnosing normal, premalignant and malignant lesions were employed and included degree of nuclear atypia, failure of cells to differentiate, and invasion of cells across the basement membrane into the dermis. As expected, premalignant actinic keratoses were more numerous than invasive squamous cell carcinomas, but both types of lesions were more than twice as numerous in diluent-treated than in pTT-treated animals. See Figure 57.
EXAMPLE 50: UV-Induced Tumors Regress After Intratumoral Injections of pTT
Hairless XPC homozygous knockout (ISO) mice were UVB irradiated 5 days a week for 12 weeks. The first UV-induced tumors started to appear after weeks, and by 160 days after the initial UVB irradiation, the majority of tumors were more than 2-4 mm2 in surface appearance. Four animals were separated into two groups of 2 animals in each. Three tumors per animal of the same size and appearance were selected for diluent and pTT intratumoral injections.

Tumors were inj ected with 15-20 ~,l of pTT (40 ~,M) or diluent three times a weelc for 2 weeks. Tumors were measured 11 and 19 days after the first intratumoral inj ection.
A total of six tumors per treatment condition were evaluated for change in growth pattern after intratumoral injections of diluent or pTT. Already by day 1 l, pTT injections led to a statistically significant decrease in LTV-induced tumor size. By day 19 some of the tumors completely disappeared (p<0.003). No inhibition of malignant growth was detected in diluent-injected tumors. By day 19, there was about a 25% increase in the size of tumors in this treatment group.
See Figure 58.
EXAMPLE 51: pTT Treatment Reduces LTV-Induced Mutations in the Skin of Wildtype and XPC Knockout Mice after Single Dose And Chronic UV
Irradiation Hairless wild type (WT) and homozygous XPC knockout (KO) mice also transgenic for the LacZ/pUR288 mutation indicator gene (2-5 animals per treatment group) were pretreated with thymidine dinucleotide (pTT, 100 ~M), or diluent alone, once a day for 10 days. The mice were UVB irradiated once (WT
mice with 25 mJ/cmz and KO mice with 5 mJ/cmz solar-simulated LTV metered at 285 + 5 nm). After 14 days, animals were sacrificed. Epidermal DNA was isolated and processed for analysis of mutations arising in the LacZ gene.
Baseline mutation frequencies were similar in mice of both genotypes. UV
irradiation led to approximately 10 fold increases in LacZ gene mutation frequency in the epidermis of W irradiated mice pretreated with diluent (WT
and KO, respectively). Mutation analysis of pTT treated WT mouse slcin showed a decrease (p<0.05) of 31 + 5% in mutation frequency. Compared to the diluent-pretreated control KO mice, the pTT-pretreated KO mice showed a 26%
lower mutation frequency, although this was not statistically significant.
Mice received 5 consecutive cycles (150 days) of one week daily topical treatment with pTT or diluent alone, followed by 3 weeks of daily UV
irradiation. After 160 days, mice were evaluated for mutation frequency. After chronic W irradiation, the mutation frequency in mouse skin was 10 times higher than after a single dose of UV radiation. Compared to diluent treatment, pTT treatment decreased mutation frequency by 28% and 31% in irradiated WT
and KO mouse skin (p<0.08 and p<0.04, respectively). Earlier data with cultured mouse fibroblasts transgenic for Lac2 showed that T-oligo pretreatment decreases IJV-induced mutation frequency.
EXAMPLE 52: Oligonucleotides Homologous to the Telomeric 3' Overhang Stimulate p21-Dependent Adaptive Responses to Oxidative Damage.
Fibroblast exposure to 40 ~,M T-oligo pGTTAGGGTTAG (SEQ ID
N0:5) activates p53, as determined by p53 phosphorylation on serine 15, by 24 and 48 hrs, with a 3-fold higher response than that produced by pTT (100 ~,M) (western blot). A dichlorofluorescein diacetate assay revealed increases in reactive oxygen species within 36 hrs of treatment with pGTTAGGGTTAG
(SEQ m N0:5) and pTT compared to treatment with control oligonucleotides pCTAACCCTAAC (SEQ m N0:9) and pCC or diluent alone (p<0.002). The specific NADPH oxidase inihibitor diphenyliodonium chloride blocked reactive oxygen species production, and T-oligo failed to induce reactive oxygen species in p21-null mouse embryonic fibroblasts as compared with wild-type control cells. Within 48 and 72 hrs~ pGTTAGGGTTAG (SEQ m N0:5), but not pTT, modestly induced senescence-associated-[3-galactosidase staining, consistent with the ability of prolonged T-oligo treatment (>_7 days) to induce fibroblast senescence. In fibroblasts, the effects produced appear to depend on degree of homology with the telomeric 3' overhang as well as on length of treatment:
only prolonged exposures lead to senescence, while exposures of <_72 hrs induce reactive oxygen species levels in a p53- and p21-dependent manner and induce the level of superoxide dismutase (SOD) enzymes.
Normal newborn fibroblasts were treated with pTT or wth diluent as control for up to 4 days, and were harvested for RNA isolation. Northern blot analysis showed that SOD2 mRNA is induced by pTT.

Normal newborn fibroblasts were treated with pTT or with diluent as control for up to 32 hours, and were harvested to extract proteins for Western blot analysis. The results showed that SOD2 protein is induced by pTT.
Normal newborn fibroblasts were treated with pTT or with diluent as control for up to 4 days, and were harvested for RNA isolation. Northern blot analysis showed that SOD1 mRNA is induced by pTT.
Normal newborn fibroblasts were treated with pTT or with diluent as control for up to 32 hours, and were harvested to extract proteins for Western blot analysis. The results showed that SOD 1 protein is induced by pTT.
Newborn fibroblasts were treated for up to 72 hours with 40 ~M T-oligo pGTTAGGGTTAG (SEQ ID NO:S) or with diluent as control, and were harvested for RNA isolation and northern blot analysis. Two known transcripts of SOD2 both were induced during T-oligo treatment.
Normal newborn fibroblasts were treated for 3 days with pTT or with diluent as control, re-seeded to equal densities, and exposed to 50 ~M
hydrogen peroxide solution in DME or DME alone as control. Cells were then harvested at 8, 24, 48 and 72 hours for cell yield determination by Coulter counter.
Pretreatment with pTT was seen to stimulate resistance to hydrogen peroxide.
Newborn fibroblasts were treated for two days with pTT or with diluent as control, irradiated with 10 J/cm2 UVA. Cells were replated at 200 cells/100 nun dish, and grown for 15 days. Cell colonies were fixed, stained with methylene blue, and counted. Pretreatment with pTT increased cell viability following oxidative stress created by UVA irradiation, as measured by colony-forming efficiency.
EXAMPLE 53: T-oligo Increases DNA Repair Capacity and Accelerates Removal of DNA Photoproducts in Fibroblasts Two different DNA repair assays were used to assess DNA repair in cultured primary neonatal human fibroblasts two days after pretreatment with T-oligo (40 ~,M), two different control oligonucleotides (40 ~,M), or diluent control.

The first assay was a host cell reactivation assay using the plasmid pCMVluc. In this assay, the plasmid was irradiated with WB in vitro, and subsequently transfected into human fibroblasts, using lipofectamine. The UVB-induced DNA damage (DNA photoproducts; mainly thymine dimers and 6,4-photoproducts) must be repaired by cellular DNA repair enzymes before the plasmid-encoded luciferase can be expressed. Therefore, luciferase activity in cell extracts two days after transfection reflects the efficiency of the host cells to repair DNA photoproducts.
The host cell reactivation (which reflects DNA repair capacity) is shown as luciferase activity, relative to the luciferase activity in parallel samples transfected with unirradiated control plasmids. Shown in Figure 59 are results of one representative experiment with triplicate samples; mean ~ standard deviation. A repeat experiment with cells from a different donor produced similar results. T-oligo increased DNA repair capacity in the fibroblasts.
The second assay measured the amount of DNA photoproducts in samples of fibroblasts assayed at various times after irradiation. Cells were irradiated with 2 J/mz UVB and lysed either immediately afterwards, or 2, 4, or 6 hours later. Alkaline elution of cellular DNA was performed in the presence of T4 endonuclease V, which cleaves DNA at sites with pyrimidine dimers. For quantitative evaluation of the number of T4 eridonuclease V sites (= number of pyrimidine dimers), a standard sample with a known number of DNA lesions was used for calibration. Shown in Figure 60 are results of 3 to 5 independent samples of fibroblasts; mean ~ standard deviation. T-oligo accelerated the removal of DNA photoproducts in the fibroblasts.
EQUIVALENTS
While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

-114- What is claimed is:

1. A method for treating a hyperproliferative disorder in a mammal, said method comprising administering to the human an effective amount of a composition comprising one or more oligonucleotides which share at least 50% nucleotide sequence identity with (TTAGGG)n.

2. The method of Claim 1, wherein the mammal is a human.

3. The method of Claim 1, wherein the composition comprises one or more oligonucleotides selected from the group consisting of:
a) oligonucleotides 2-200 nucleotides long;
b) oligonucleotides 2-20 nucleotides long;
c) oligonucleotides 5-11 nucleotides long; and d) oligonucleotides 2-5 nucleotides long.

4. The method of Claim 1 wherein the oligonucleotide(s) contain one or more non-phosphodiester linkages but are enzymatically cleavable to release the 3' terminal nucleotide.

5. The method of Claim 1 wherein the composition comprises an oligonucleotide which is pGAGTATGAG (SEQ 117 NO:1), pGTTAGGGTTAG (SEQ ID NO:5), pGGGTTAGGGTT (SEQ ID
NO:13), pTAGATGTGGTG (SEQ ID NO:14), or pTT.

6. The method of Claim 1 wherein the composition comprises an oligonucleotide which is pTTAGAGTATGAGTTA (SEQ ID NO:16), pTTAGAGTATGAG (SEQ ID NO:17) or pGAGTATGAGTTA (SEQ
ID NO:18)

7. A method for inhibiting the growth of cancer cells in a human, comprising administering to the human an effective amount of a composition comprising one or more oligonucleotides which share at least 50% nucleotide sequence identity with the human telomere overhang repeat.

8. The method of Claim 7, wherein the composition comprises an oligonucleotide comprising nucleotide sequence SEQ ID NO:1, SEQ ID
NO:5, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17 or SEQ ID NO:18.

9. The method of Claim 7 wherein the cancer cells are selected from the group consisting of: lymphoma cells, osteosarcoma cells, melanoma cells, leukemia cells, cervical cancer cells and squamous cell carcinoma cells.

10. The method of Claim 7 wherein the cancer cells are selected from the group consisting of: breast cancer cells, ovarian cancer cells, pancreatic cancer cells and fibrosarcoma cells.

11. A method for promoting differentiation of malignant cells in a mammal, said method comprising administering to the mammal an effective amount of a composition comprising one or more oligonucleotides which share at least 50% nucleotide sequence identity with (TTAGGG)n.

12. A method for enhancing the expression of one or more surface antigens indicative of differentiation of cancer cells in a human, said method comprising administering to the human an effective amount of a composition comprising one or more oligonucleotides which share at least 50% nucleotide sequence identity with (TTAGGG)n.

13. The method of Claim 12 wherein the cancer cells are melanoma cells.

14. The method of Claim 12 wherein the antigen is MART-1, tyrosinase, TRP-1 or gp-100.

15. The method of Claim 12 wherein the composition comprises pTT.

16. The method of Claim 12 wherein the composition comprises an oligonucleotide comprising SEQ ID NO:5.

17. The method of Claim 12 wherein the cancer cells are breast cancer cells.

18. A method for inducing apoptosis in cancer cells in a human, said method comprising administering to the human an effective amount of a composition comprising one or more oligonucleotides which share at least 50% nucleotide sequence identity with the human telomere overhang repeat sequence.

19. The method of Claim 18 wherein the oligonucleotide(s) contain one or more non-phosphodiester linkages but are enzymatically cleavable to release the 3' terminal nucleotide.

20. The method of Claim 18 wherein the cancer cells are melanoma cells, ovarian cancer cells, or breast cancer cells.

21. The method of Claim 18 wherein the oligonucleotide(s) comprise SEQ
ID NO:5, SEQ ID NO:16, SEQ ID NO:17 or SEQ ID NO:18.

22. A method for inhibiting the growth of cancer cells in a mammal, said method being independent of the presence or activity of telomerase in said cells, said method comprising administering to the human an effective amount of a composition comprising one or more oligonucleotides which share at least 50% nucleotide sequence identity with (TTAGGG)n.

23. A method for inhibiting the growth of cancer cells in a mammal, said method not requiring the presence or activity of p53 normal function in said cells, said method comprising administering to the human an effective amount of a composition comprising one or more oligonucleotides which share at least 50% nucleotide sequence identity with (TTAGGG)n.

24. A method for inhibiting the growth of cancer cells in a mammal, said method resulting in S-phase arrest in said cells, said method comprising administering to the human an effective amount of a composition comprising one or more oligonucleotides which share at least 50%
nucleotide sequence identity with (TTAGGG)n.

25. The method of Claim 24 wherein the composition comprises one or more oligonucleotides less than 6 nucleotides long.

26. A method for preventing spongiosis, blistering or dyskeratosis in the skin of a mammal, following exposure to ultraviolet light, said method comprising applying to the skin an effective amount of a composition comprising one or more oligonucleotides which share at least 50%
nucleotide sequence identity with (TTAGGG)n.

27. The method of Claim 26 wherein the composition comprises pGAGTATGAG (SEQ ID NO:1).

28. The method of Claim 26 wherein the composition comprises pTT.

29. A method for reducing the occurrence of skin cancer in a human, said method comprising applying to the skin an effective amount of a composition comprising one or more oligonucleotides which share at least 50% nucleotide sequence identity with the human telomere overhang repeat sequence.

30. The method of Claim 29 wherein the composition comprises pGAGTATGAG (SEQ ID NO:1).

31. The method of Claim 29 wherein the composition comprises pTT.

32. A method for reducing the occurrence of skin cancer in a human with xeroderma pigmentosum or other genetic predisposition to skin cancer, said method comprising administering to the skin an effective amount of a composition comprising one or more oligonucleotides which share at least 50% nucleotide sequence identity with the human telomere overhang repeat.

33. The method of Claim 32 wherein the composition comprises pGAGTATGAG (SEQ ID NO:1).

34. The method of Claim 32 wherein the composition comprises pTT.

35. A method for enhancing repair of ultraviolet irradiation-induced damage to skin in a human, said method comprising applying to the skin an effective amount of a composition comprising one or more oligonucleotides which share at least 50% nucleotide sequence identity with the human telomere overhang repeat sequence.

36. The method of Claim 35 wherein the composition comprises pGAGTATGAG (SEQ ID NO:1).

37. The method of Claim 35 wherein the composition comprises pTT.

38. A method for reducing oxidative damage in a mammal, said method comprising administering to the mammal an effective amount of a composition comprising one or more oligonucleotides which share at least 50% nucleotide sequence identity with (TTAGGG)n.

39. The method of Claim 38 wherein the composition is administered to the shin.

40. The method of Claim 38 wherein the composition comprises pGTTAGGGTTAG (SEQ ID NO:5).

41. The method of Claim 38 wherein the composition comprises pTT.

42. A method for treating melanoma in a mammal, comprising administering to the mammal an effective amount of a composition comprising one or more oligonucleotides that share at least 50% nucleotide sequence identity with (TTAGGG)n.

43. The method of Claim 42 wherein the mammal is a human.

44. The method of Claim 42 wherein the composition comprises pGTTAGGGTTAG (SEQ ID NO:5).

45. The method of Claim 42 wherein the composition comprises pTT.

46. A method for reducing proliferation of lceratinocytes in the skin of a human, said method comprising administering to the skin an effective amount of a composition comprising one or more oligonucleotides that share at least 50% nucleotide sequence identity with the human telomere overhang repeat.

47. The method of Claim 46, wherein the human has seborrheic keratosis, actinic keratosis, Bowen's disease, squamous cell carcinoma, or basal cell carcinoma.

48. The method of Claim 46, wherein the composition comprises pGTTAGGGTTAG (SEQ ID NO:5).

49. The method of Claim 46 wherein the composition comprises pTT.

50. A composition comprising an oligonucleotide, wherein the oligonucleotide is pGAGTATGAG (SEQ ID NO:1), pCATAC (SEQ ID
NO:6), pGTTAGGGTTAG (SEQ ID NO:5), pGGGTTAGGGTT (SEQ
ID NO:13), or pTAGATGTGGTG (SEQ ID NO:14).

51. A composition comprising an oligonucleotide, wherein the oligonucleotide is pTTAGAGTATGAGTTA (SEQ ID NO:16), pTTAGAGTATGAG (SEQ ID NO:17) or pGAGTATGAGTTA (SEQ
ID NO:18).

52. A method of inhibiting proliferation of epithelial cells in a mammal, comprising administering to the epithelial cells an effective amount of a composition comprising at least one oligonucleotide, wherein the oligonucleotide comprises a base sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID
NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 12, and a contiguous portion of any of the foregoing sequences.

53. The method of Claim 52, wherein the oligonucleotide is single-stranded.

54. The method of Claim 52, wherein the oligonucleotide comprises a 5' phosphate.

55. The method of Claim 52, wherein the composition comprises oligonucleotide at a concentration of about 1 µM to about 500 µM.

56. The method of Claim 52, wherein said oligonucleotide is ultraviolet-irradiated.

57. The method of Claim 52, wherein the composition comprises oligonucleotide in liposomes.

58. The method of Claim 52, wherein the composition comprises propylene glycol.

59. The method of Claim 52, wherein the composition is administered orally.

60. The method of Claim 52, wherein the composition is administered by aerosol.

61. The method of Claim 52, wherein the mammal is a human.

62. The method of Claim 52, wherein the epithelial cells are carcinoma cells.

63. A method of preventing or reducing DNA damage in cells in a mammal, wherein said DNA damage is caused by radiation or DNA-damaging chemicals, comprising contacting said cells with an effective amount of a composition comprising at least one oligonucleotide, wherein the oligonucleotide comprises a base sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID
NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ
ID NO:11, SEQ ID NO:12, and a contiguous portion of any of the foregoing sequences.

64. The method of Claim 63, wherein the cells are epithelial cells.

65. The method of Claim 63, wherein said oligonucleotide is single-stranded.

66. The method of Claim 63, wherein the oligonucleotide comprises a 5' phosphate.

67. The method of Claim 63, wherein the composition comprises the oligonucleotide at a concentration of about 1 µM to about 500 µM.

68. The method of Claim 63, wherein the composition comprises a physiologically acceptable carrier.

69. A composition comprising at least one oligonucleotide, wherein the oligonucleotide comprises a base sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID
NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ
ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID
NO:16, SEQ ID NO:17 and SEQ ID NO:18.

70. The composition of Claim 69, wherein the oligonucleotide comprises a 5' phosphate.