CA2263888C - Methods and compositions for the detection of cervical cancer - Google Patents

Abstract

The invention provides a wide range of methods and compositions for detecting and treating cervical cancer in an individual.
Specifically, the invention provides target cervical cancer-associated proteins, which permit a rapid detection, preferably before metastases occur, of cervical cancer. The target cervical cancer-associated protein, may be detected, for example, by reacting the sample with a labeled binding moiety, for example, a labeled antibody capable of binding specifically to the protein. The invention also provides kits useful in the detection of cervical cancer in an individual. In addition, the invention provides methods utilizing the cervical cancer-associated proteins either as targets for treating cervical cancer or as indicators for monitoring of the efficacy of such a treatment.

Description

METHODS AND COMPOSITIONS FOR THE
DETECTION OF CER YICAL CANCER
Field of the Invention.
The present invention relates generally to methods and compositions for the detection of cervical cancer. More specifically, the present invention relates to cervical cancer-associated proteins which act as cellular markers useful (i) in detecting cervical cancer, and (ii) as molecular s targets for cervical cancer therapy.
Background of the Invention.
Cancer of the uterine cervix is one of the most common malignancies in women and remains a significant public health problem throughout the world. In the united States alone, invasive cervical cancer accounts for approximately 19% of all gynecological cancers (Miller et al.
to (1993) in "Surveillance Epidemiology, and End Results Program cancer Statistics Review: 1973-1990 ", NIH Pub. No. 93-2789, Bethesda, MD: National Cancer Institute). In 1996, it is estimated that there will be 14,700 newly diagnosed cases and 4900 deaths attributed to this disease (American Cancer Society, Cancer Facts & Figures 1996, Atlanta, GA:
American Cancer Society, 1996). In many developing countries, where mass screening programs are not widely 15 available, the clinical problem is more serious. Worldwide, the number of new cases is estimated to be 471,000 with a 4 year survival rate of 40% (Munoz et al. ( 1989) "Epidemiology of Cervical Cancer" in "Human Papillomavirus ", New York, Oxford Press, pp 9-39; and National Institutes of Health, Consensus Development Conference Statement on Cervical Cancer, April 1-3, 1996).
The precursor to cervical cancer is dysplasia, also known in the art as cervical 2o intraepithelial neoplasia (CIN) or squamous intraepithelial lesions (SIL,) (Brinton et al. ( 1992) "Epidemiology of Cervical Cancer: Overview" in "The Epidemiology of Cervical Cancer and Human Papillomavirus", Lyon, France: International Agency for Research on Cancer; and Tabbara et al. (1992) "The Bethesda classification for sqzramous intraepithelial lesions:
histologic, cytologic and viral correlates", Obstet. Gynecol. 79: 338-346}.
While it is not 25 understood how normal cells become transformed, the concept of a continuous spectrum of histopathological change from normal, stratified epithelium through CIN to invasive cancer has

-2-been widely accepted for many years (see, , f'or example, IvLitchell et al. ( 1994) "The natural history of cervical intraepithelial neoplasia: an argument of intermediate endpoint biomarkers", Cancer Epidmiol. Biomark. Prev. 3: 619-626). A large body of epidemiological and molecular biological evidence has been gathered that establishes human papillomavirus (HPV) infection as a causative factor in cervical cancer (Munoz et aL (1992) in "The Epidemiology of Human Papillomavirus and Cervical Cancer ", IRAC publication no. 119, Lyon France:
Int. Agency for Research on Cancer, pp 251-261 ). HPV is found in 85% or more of squamous cell invasive lesions, which represent the most common histologic type seen in cervical carcinoma (Cox et al.
(1995) Baillierre's Clin. Obstet Gynaecol. 91-37). Additional cofactors include, for example, oncogenes activated by point mutations, and chromosomal translocations of deletions (Spandidos et al. (1989) J. Pathol. 157: 1-10).
Cytological examination of Papanicolaou-stained cervical smears (also referred to as Pap smears) currently is the method of choice for detecting cervical cancer.
Despite the historical success of this test, concerns have arisen regarding its ability to predict reliably the behavior of same preinvasive lesions (Ostor et a1 (1993) Int. J. Gynecol. Pathol. 12: 186-192; and Genest et a1 (1993) Human Pathol. 24: 730-736). The identification of a cervical cancer-associated tumor marker for reliably detecting early onset of cervical cancer and/or providing early prognostic information will greatly aid the management of cervical cancer.
All eukaryotic cells have a nucleus containing DNA, or chromatin, which is organized by an internal protein scaffolding known as the nuclear matrix (NNI). The nuclear matrix was first described in 1974 by Berezney et al. (Berezney et al. (1974) Biochem. Biophys.
Res. Commun., 60: 1410-1417). Penman et al. describe a method for selectively extracting insoluble interior nuclear matrix proteins and their associated nucleic acids from cells and determining the particular cell type by analyzing the proteins by two-dimensional gel electrophoresis (see for example, U.S.
Pat. Nos. 4,882,268, issued 11/21/89, and 4,885,236, issued 12/5!89.
The nuclear matrix is believed to be involved in a wide variety of nuclear functions fundamental to the control of gene expression. For a general review see, for example, Fey et al.
(1991) Crit. Rev. Euk. Gene Express. 1: 127-143. Tissue-specific nuclear matrix proteins have been identified in the rat, mouse and human. Fey et al. (1986) Proc. Natl.
Acad Sci. USA 85:
121-125; Stuurman et al. (1990) J. Biol. Chem. 265: 5460-5465; and Getzenberg et al. (1990)

-3-Mol. Endocrinol. 4: 1336-1342. Changes in the presence or absence of specific nuclear matrix proteins have been associated with cellular transformation and differentiation (Bidwell et al.
( 1993) Proc. Natl. Acad. Sci. USA 90: 3162-3166; Brancolini et al. ( 1991 ) Proc. Natl. Acad.
Sci. USA 88: 6936-6940; and Greenfield et al. (1991) Proc. Natl. Acad. Sci.
USA 88: 11217-11221).
Several recent studies using similar methodology have identified tumor-specific nuclear matrix proteins in cancers of the prostate (Partin et al. (1993) Cancer Res.
53: 744-746), breast (Khanuja et al. (1993) Cancer Res. 53: 3394-3398), colon cancer (Keesee et al.
{1994) Proc.
Nail. Acaa! Sci. USA 91: 1913-1916), bone (Bidwell et al. (1994) CartcerRes.
54: 28-32), to bladder (Getzenberg et al. ( 1996) Cancer Res. 56: 690-694) and the larynx (Donat et al. ( 1996) Otolaryngol. Head Neck Surg. 114: 387-393). Molecular characterization of the specific nuclear matrix proteins, however, remains poorly defined, due to the low abundance of these proteins in the cell and their generally insoluble character.
There is, however, a need in the art for specific, reliable markers that are expressed differentially in normal and cancerous cervical tissue and that may be useful in the detecting cervical cancer or in the prediction of its onset. Accordingly, it is an object of this invention to provide cervical cancer-associated molecules which are useful as markers for the early and/or rapid detection of cervical cancers in an individual. It is another object of this invention to provide methods for detecting cervical cancers in an individual. It is another object of the 2o invention to provide methods and compositions for treating cervical cancers in an individual and for monitoring the efficacy of such a treatment in the individual.

-4-Summary of the Invention.
The invention provides a variety of methods and compositions for detecting and/or prognosing cervical. cancer in a tissue or body fluid sample of an individual.
The invention is based, in part, upon the discovery of cervical cancer-associated proteins which are present at detectable levels in cervical cancer cells, but which are not detectable in normal cervical cells, as determined by two-dimensional gel electrophoresis.
In one aspect, the invention provides a method for detecting cervical cancer in a human. The method comprises the step of detecting the presence of a cervical cancer-associated protein in a tissue or body fluid sample of the human thereby to indicate the presence of a cervical cancer or a 1o precursor of a cervical cancer. The cervical cancer-associated protein is characterized as having a molecular weight of from about 44,900 Daltons to about 69,400 Daitons, as determined by standard polyacrylamide gel electrophoresis techniques and an isoelectric point of from about 5.1 to about 6.6 as determined by standard isoelectric focusing techniques. In addition, the cervical cancer-associated protein is further characterized as being a non-chromatin protein which is 15 detectable at a higher level in a human cervical cancer cell than in a normal human cervical cell, as determined by two-dimensional gel electrophoresis. It is contemplated, however, that the accuracy and/or reliability of the method may be further enhanced by detecting the presence of a plurality of cervical cancer-associated proteins in the preselected tissue or body fluid sample.
As used herein, the term "cervical cancer" is understood to mean any cancer or cancerous 20 lesion associated with cervical tissue or cervical cells and, in addition, includes precusors to cervical cancer, for example, dysplasia (also known in the art as a cervical intraepithelial neoplasia or a squamous intraepithelial lesion).
As used herein, the term "cervical cancer-associated" molecules refers to molecules originating from and isolatable from a cervical cancer cell or cells, and substantially neither 25 originating from nor isolatable from a normal cervical cancer cell or cells. As used herein, the term "cervical cancer-associated protein" is understood to mean any protein which is detectable at a higher level in cervical cancer cells than in normal cervical cells, as determined by two-dimensional (2-D) gel electrophoresis. It is not necessary that the target molecule or target protein be unique to a cervical cancer cell; rather it is preferred that the target molecule or protein 3o has a signal to noise ration high enough to discriminate between samples originating from a

-5-cervical cancer tissue or body fluid and samples originating from normal cervical tissue or body fluid.
In a preferred embodiment, methods of the invention comprise the step of detecting one or more cervical cancer (CvC) associated proteins, referred to herein as CvC-1 through CvC-5, which can be purified or co-purified using nuclear matrix protein purification methodologies, well known and thoroughly documented in the art. See, for example, Fey et al.
(1986) Proc. Nat1 Acad. Sci, USA 85: 121-125. As used herein, the term "nuclear matrix protein"
is understood to mean any non-cytoskeletal, non-lamin, non-chromatin protein that (i) is isolated fibm mammalian cell nuclei, (ii) is resistant to solubilization from the nuclei in 0.25M
ammonium sulfate, (iii) remains in solution following dialysis into physiological buffer from 8M urea and (iv) is detectable on a silver stained two-dimensional electrophoresis gel.
Accordingly, one or more of the resultant cervical cancer-associated proteins may be fiu-ther defined as being a nuclear matrix protein.
In a preferred embodiment, methods of the invention may comprise the step of detecting the protein CvC-I, a protein having a molecular weight of about 69,400 Daltons, as determined by polyacrylamide gel electrophoresis, and a pI of about 5.8, as determined by isoelectric focusing techniques. Alternatively, the methods of the invention may comprise the step of detecting the protein CvC-2, a protein having a molecular weight of about 53,800 Daltons, as determined by polyacrylamide gel electrophoresis, and a pI of about 5.5, as determined by isoelectric focusing techniques. Alternatively, the methods of the invention may comprise the step of detecting the protein CvC~3, a protein having a molecular weight of about 47,900 Daltons, as determined by polyacrylamide gel electrophoresis, and a pI of about 5.6, as determined by isoelecuic focusing techniques. Alternatively, the methods of the invention may comprise the step of detecting the protein CvC-4, a protein having a molecular weight of about 46,000 Daltons, as determined by polyacrylamide gel electrophoresis, and a pI of about 5.1, as determined by isoelectric focusing techniques. Alternatively, the methods of the invention may comprise the step of detecting the protein CvC-5, a protein having a molecular weight of about 44,900 Daltons, as determined by polyacrylamide gel electrophoresis, and a pI of about 6.6, as determined by isoelectric focusing techniques.
In another preferred embodiment, the methods of the invention may comprise the step of detecting a cervical cancer-associated protein which comprises a continuous amino acid sequence r

-6-selected from the group consisting of: SEQ m NO.: 1; SEQ ID NO.: 2; SEQ ID
NO.: 3; SEQ m NO.: 4; SEQ ID NO.: 5; SEQ ID NO.: 6; SEQ 117 NO.: 7; SEQ ID NO.: 8; and SEQ
ID NO.: 9.
Alternatively, the method of the invention may comprise the step of detecting a cervical cancer-associated protein having the amino acid sequence set forth in SEQ 117 NO.:
10, commonly referred to in the art as IEF SSP 9502. See, for example, Honore et al. (1994) Gene 151: 291-296, In another preferred embodiment, the methods of the invention may comprise the step of detecting a cervical cancer-associated protein which comprises a continuous amino acid sequence selected from the group consisting of: SEQ )D NO.: 11; SEQ ID NO.: 12; SEQ ID
NO.: 13; SEQ
ID NO.: 14; SEQ ID NO.: 15; SEQ B7 NO.: 16; and SEQ ID NO.: 17. Alternatively, the method of the invention may comprise the step of detecting a cervical cancer-associated protein having the amino acid sequence set forth in SEQ ID NO.: 18, and commonly referred to in the art as Cytokeratin I7. See, for example, Troyanovsky et al. (1992) J. Biol. Biol. 59:
127-137, In another preferred embodiment, the methods of the invention may comprise the step of detecting a cervical cancer-associated protein which comprises a continuous amino acid sequence selected from the group consisting of: SEQ >D NO.: 19; SEQ ID NO.: 20; SEQ ID
NO.: 21; SEQ
>D NO.: 22; SEQ ID NO.: 23; SEQ ID NO.: 24; and SEQ ID NO.: 25. Alternatively, the method of the invention may comprise the step of detecting a cervical cancer-associated protein having the amino acid sequence set forth in SEQ m NO.: 26, commonly referred to in the art as TDP-43.
See, for example, Ou et al. (1995) J. Virol. 69: 3584-3596.
In another preferred embodiment, the methods of the invention may comprise the step of detecting a cervical cancer-associated protein which comprises a continuous amino acid sequence selected from the group consisting of: SEQ ID NO.: 27; SEQ ID NO.: 28; SEQ ID
NO.: 29; SEQ
ID NO.: 30; SEQ ID NO.: 31; SEQ ID NO.: 32; and SEQ ID NO.: 33. alternatively, the method of the invention may comprise the step of detecting a cervical cancer-associated protein having the amino acid sequence set forth in SEQ ID NO.: 34, commonly referred to in the art as Nup358.
See, for example, Wu et al. (1995) J. Biol. Chem. 270: 14209-14213, _7.
In another preferred embodiment, the methods of the invention may comprise the step of detecting a cervical cancer-associated protein which comprises a continuous amino acid sequence selected from the group consisting of: SEQ ID NO.: 35; SEQ ID NO.: 36; SEQ ID
NO.: 37; SEQ
)D NO.: 38; SEQ U~ NO.: 39; SEQ ID NO.: 40; SEQ m NO.: 41; SEQ >D NO.: 42; SEQ
U~
NO.: 43; SEQ ID NO.: 44; and SEQ ID NO.: 45. Alternatively, the method of the invention may comprise the step of detecting a cervical cancer-associated protein having the amino acid sequence set forth is SEQ )D NO.: 46, commonly referred to in the art as lamin A. See, for example, Fisher et al. (1986) Proc. Natl. Acad. Sci. USA. 83: 6450-6454, The methods of the invention may be performed on any relevant tissue or body fluid sample. For example, methods of the invention may be performed on cervical tissue, more preferably cervical biopsy tissue, and most preferably on Pap smears.
Alternatively, the methods of the invention may be performed on a human body fluid sample selected from the group consisting of blood; serum; plasma; fecal matter; urine; vaginal secretion;
spinal fluid; sativa;
ascitic fluid; peritoneal fluid; sputum; and breast exudate. It is contemplated, however, that the methods of the invention also may be useful in assays for metastasized cervical cancer cells in other tissue or body fluid samples.
Marker proteins associated with a cervical cancer in a tissue or body fluid sample may be detected using any of a number of assay methods available in the art. In one embodiment, for example, the marker cervical cancer-associated protein may be reacted with a labeled binding moiety capable of specifically binding to the marker protein thereby to produce a labeled complex of the binding moiety and the marker protein. The labeled complex thereafter may be detected, using conventional methodologies well known in the art. Detection of the presence of the labeled complex may provide an indication of the presence of the cervical cancer cells or pre-cancerous cells in the individual being tested. As used herein, the term "binding moiety" is understood to mean any binding partner capable of specifically binding to a cervical cancer-associated protein with a binding affinity greater than about I Os M''. As used herein the terms "specifically binding", "specifically bound" and "binds specifically" refer to a binding interaction with a binding affinity of greater than about 105 M-1. As used herein, the binding moiety is labeled with a detectable moiety, for example, a radioactive, fluoroscopic, spectroscopic, or enzymatic label, using techniques well known in the art.

.$_ It is appreciated that, binding moieties which interact and bind specifically with the target protein, may be designed using conventional methods well known in the art. In the invention, the binding moiety can be an antibody, for example, a monoclonal or a polyclonal antibody.
Monoclonal antibodies are preferred. It is contemplated, however, that other useful binding moieties useful in the practice of the instant invention may include, for example, biosynthetic antibody binding sites, also referred to in the art as BABS or sFv's, and antibody fragments, for example, Fv, Fab, Fab' and (Fab')z fragments. Procedures for preparing, testing, and labeling BABS and antibody fragments are well known in the art, and so are not discussed in detail herein.
In another embodiment, one or more marker proteins in a sample may be detected by first isolating the proteins from the sample, and then separating the proteins by two-dimensional gel electrophoresis to produce a characteristic two-dimensional gel electrophoresis pattern. The gel electrophoresis pattern then may be compared with a standard, for example, a standard gel pattern obtained from a data base of gel electrophoresis patterns. Thus, in another embodiment, the invention provides electrophoresis gel patterns or electropherograms of cervical cancer-associated proteins which are useful in detecting a cervical cancer in an individual.
The cervical cancer-associated proteins of the invention can be purified or co-purified from cervical cancer cells using nuclear matrix protein isolation procedures, such as those disclosed in U.S. Patent No. 4,885,236 and U.S. Patent No. 4,882,268.
Alternatively, the marker proteins, once identified and characterized may be isolated from the sample by any of a range of protein purification protocols well known to those skilled in the art, such as affinity chromatography, to yield isolated proteins. As used herein, the term "isolated" is understood to mean substantially free of undesired, contaminating proteinaceious material.
Furthermore, the skilled artisan may produce nucleic acid sequences encoding the entire isolated marker protein, or fragments thereof, using methods currently available in the art (see, for example, Maniatis et al., eds. (1989) "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor Press). For example, an isolated cervical cancer-associated protein may be sequenced using conventional peptide sequencing protocols, and then oligonucleotide hybridization probes designed for screening a cDNA library. The cDNA library then may be screened with the resultant oligonucleotide to isolate full or partial length cDNA sequences which encode the isolated protein.

Furthermore, the skilled artisan, using the methodologies described in U.S.
Patent Nos.
4,885,236 and 4,882,268 may isolate from a cell sample a nucleic acid molecule having a sequence capable of recognizing and being specifically bound by a cervical cancer-associated protein. In such a procedure, the soluble proteins are separated from the nucleus and cytoskeleton by extracting mammalian cells with a non-ionic detergent solution at physiological pH and ionic strength. The insoluble protein and nucleic acids then are digested with DNAase and then eluted with a buffered ammonium sulfate solution to yield a nucleic acid molecule capable of recognizing and being specifically bound by a cervical cancer-associated protein. Any remaining proteins then are separated from the target nucleic acid molecule.
~o Detection of the aforementioned nucleic acid molecules thus can serve as an indicator of the presence of cervical cancer and/or metastasized cervical cancer in an individual. Accordingly, in another aspect, the invention provides another method for detecting cervical cancer in a human. The method comprises the step of detecting the presence of a nucleic acid molecule in a tissue or body fluid sample thereby to indicate the 15 presence of a cervical carcinoma in the individual. The nucleic acid molecule is selected from the group consisting of (i) a nucleic acid molecule comprising a sequence capable of recognizing and being specifically bound by a cervical cancer-associated protein, and (ii) a nucleic acid molecule comprising a sequence encoding a cervical cancer-associated protein. As defined herein, the cervical cancer-associated protein is characterized as being 2o selected from the group consisting of (i) a protein having a molecular weight of about 69,400 Daltons and an isoelectric point of about 5.8; (ii) a protein having a molecular weight of about 53,800 Daltons and an isoelectric point of about 5.5; (iii) a protein having a molecular weight of about 47,900 Daltons and an isoelectric point of about 5.6; (iv) a protein having a molecular weight of about 46,000 Daltons, and an isoelectric point of 25 about 5.1; and (v) a protein having a molecular weight of about 44,900 Daltons and an isoelectric point of about 6.6, wherein in each example, the molecular weight is determined by standard polyacrylamide gel electrophoresis techniques and the isoelectric point is determined by standard isoelectric focusing techniques, and wherein the cervical cancer-associated protein is further characterized as being a non-chromatin protein which 30 is detectable at a higher level in a human cervical cancer cell than in a normal human cervical cell, as determined by two-dimensional gel electrophoresis.

A target nucleic acid molecule in a sample may be detected, for example, by Northern blot analysis by reacting the sample with a labeled hybridization probe, for example, a 32P labeled oligonucleotide probe, capable of hybridizing specifically with at least a portion of the nucleic acid molecule encoding the marker protein. Detection of a nucleic acid molecule either encoding a cervical cancer-associated protein or capable of being specifically bound by a cervical cancer-associated protein, thus can serve as an indicator of the presence of a cervical cancer in the individual being tested.
In another aspect, the invention provides a kit for detecting the presence of cervical cancer or for evaluating the efficacy of a therapeutic treatment of a cervical cancer. Such kits may 1o comprise, in combination, (i) a receptacle for receiving a human tissue or body fluid sample from the individual, (ii) a binding partner which binds specifically either to an epitope on a marker cervical cancer-associated protein or a nucleic acid sequence encoding at least a portion of the marker cervical cancer-associated protein, (iii) means for detecting the binding of the binding partner with either the cervical cancer-associated protein or the nucleic acid sequence encoding at 15 least a portion of the cervical cancer-associated protein, and (iv) a reference sample.
In one embodiment of the kit, the binding moiety binds specifically to a cervical cancer-associated protein selected from the group of proteins further defined as having: a molecular weight of about 69,400 Daltons and an isoelectric point of about 5.8; a molecular weight of about 53,800 Daltons and an isoelectric point of about 5.5; a molecular weight of about 47,900 Daltons 2o and an isoelectric point of about 5.6; a molecular weight of about 46,000 Daltons and an isoelectric point of about 5.1, or a molecular weight of about 44,900 Daltons and an isoelectric point of about 6.6, wherein the molecular weight is determined by conventional polyacrylamide gel electrophoresis methodologies, and the isoelectric point is determined by conventional isoelectric focusing methodologies.
25 In another embodiment of the kit, the reference sample may comprise a negative and/or positive control. The negative control being indicative of a normal cervical cell type and the positive control being indicative of cervical cancer.
In another aspect, the invention provides a method for treating cervical cancer. The method comprises administering to a patient with cervical cancer, a therapeutically-effective amount of a 3o compound, preferably an antibody, and most preferably a monoclonal antibody, which binds specifically to a target cervical cancer-associated protein thereby to inactivate the protein. The target protein being characterized as having a molecular weight of from about 44,900 Daltons to about 69,400 Daltons, as determined by standard polyacrylamide gel electrophoresis techniques and an isoelectric point of from about 5.1 to about 6.6, as determined by standard isoelectric focusing techniques, and wherein the target protein is further characterized as being a non-chromatin protein which is detectable at a higher level in a human cervical cancer cell than in a normal human cervical cell, as determined by two-dimensional gel electrophoresis. Similarly, it is contemplated that the compound may comprise a small molecule, for example, as small organic molecule, which inhibits or reduces the biological activity of the target cervical cancer-associated protein.
In another aspect, the invention provides another method for treating cervical cancer. The method comprises the step of administering to a patient diagnosed as having cervical cancer, a therapeutically-effective amount of a compound which reduces in vivo the expression of a target cervical cancer-associated protein thereby to reduce in vivo the expression of the target protein.
In a preferred embodiment, the compound is a nucleobase containing sequence, such as, an anti-sense nucleic acid sequence or anti-sense peptide nucleic acid (PNA) molecule, complementary to a nucleic acid sequence encoding at least a portion of the target protein.
After administration; the anti-sense nucleic acid sequence or anti-sense PNA molecule binds to the nucleic acid sequences encoding, at least in part, the target protein thereby to reduce in vivo expression of the target cervical cancer-associated protein.
In another aspect, the present invention provides a method of detecting cervical cancer in a human, comprising: detecting the presence of a nucleic acid molecule in a tissue or body fluid sample of the human thereby to indicate the presence of a cervical cancer in the human, wherein the nucleic acid molecule is selected from the group consisting of: (a) a nucleic acid molecule comprising a sequence capable of binding specifically to a cervical cancer-associated protein comprising an amino acid sequence selected from the group consisting of SEQ ID
NO: l, SEQ ID
N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ >D NO:S, SEQ >D N0:6, SEQ ID N0:7, SEQ ID
N0:8, SEQ )D N0:9, and SEQ )D NO:10; and (b) a nucleic acid molecule comprising a nucleotide sequence encoding a cervical-cancer associated protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID N0:2, SEQ
B7 N0:3, SEQ B~ N0:4, SEQ )D NO:S, SEQ ID N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ 1D N0:9, and SEQ )D NO:10.

- lla-In another aspect, the present invention provides use a therapeutically-effective amount of a compound which binds specifically to a cervical cancer-associated protein thereby to inactivate said protein, said protein comprising an amino acid sequence selected from the group consisting of SEQ m NO:1, SEQ )D N0:2, SEQ >D N0:3, SEQ )D N0:4, SEQ )D N0:5, SEQ
ID N0:6, SEQ )D N0:7, SEQ )D N0:8, SEQ )D N0:9, and SEQ )D NO:10, for treating cervical cancer.
In another aspect, the present invention provides use of a therapeutically-effective amount of a compound which reduces expression of a cervical cancer-associated protein thereby to reduce expression of said protein, said protein comprising an amino acid sequence selected from the group consisting of SEQ )D NO:1, SEQ )D N0:2, SEQ >D N0:3, SEQ ID
N0:4, SEQ
ID N0:5, SEQ >D N0:6, SEQ )D N0:7, SEQ >D N0:8, SEQ >D N0:9, and SEQ B7 NO:10, for treating cervical cancer.
In another aspect, the present invention provides a method of detecting cervical cancer in a human, comprising: detecting the presence of a nucleic acid molecule in a tissue or body fluid sample of the human thereby to indicate the presence of a cervical cancer in the human, wherein the nucleic acid molecule is selected from the group consisting of: (a) a nucleic acid molecule comprising a sequence capable of binding specifically to a cervical cancer-associated protein comprising an amino acid sequence selected from the group consisting of SEQ m N0:19, SEQ
ID N0:20, SEQ ID N0:21, SEQ )D N0:22, SEQ )D N0:23, SEQ )D N0:24, SEQ 1D
N0:25, and SEQ ID N0:26; and (b) a nucleic acid molecule comprising a nucleotide sequence encoding a cervical cancer-associated protein comprising an amino acid sequence selected from the group consisting of SEQ )D N0:19, SEQ )D N0:20, SEQ )D N0:21, SEQ )D N0:22, SEQ )D
N0:23, SEQ )D N0:24, SEQ )D N0:25, and SEQ )D N0:26.
In another aspect, the present invention provides use of a therapeutically-effective amount of a compound which binds specifically to a cervical cancer-associated protein thereby to inactivate said protein, said protein comprising an amino acid sequence selected from the group consisting of SEQ )D N0:19, SEQ ID N0:20, SEQ ID N0:21, SEQ )D N0:22, SEQ >D
N0:23, SEQ ID N0:24, SEQ ID N0:25, and SEQ )D N0:26, for treating cervical cancer.

llb to reduce the expression of said protein, said protein comprising an amino acid sequence selected from the group consisting of SEQ ID N0:19, SEQ >I7 N0:20, SEQ ID N0:21, SEQ ID
N0:22, SEQ ID N0:23, SEQ ID N0:24, SEQ ID N0:25, and SEQ ID N0:26, for treating cervical cancer.
In another aspect, the present invention provides a method for detecting cervical cancer in a human, the method comprising:
detecting, in a tissue or body fluid sample from the human, a ribonucleic acid molecule that encodes a protein comprising the amino acid sequence set forth in SEQ
ID NO:10, wherein the ribonucleic acid molecule, if present in the sample, is indicative of the presence of cervical cancer in the human.
In another aspect, the present invention provides a method for detecting cervical cancer in a human, the method comprising:
(a) contacting a tissue or body fluid sample from the human with a binding moiety selected from the group consisting of a nucleic acid and a peptide nucleic acid that binds to a target nucleic acid indicative of cervical cancer, if present in the tissue or body fluid sample, to produce a complex comprising the binding moiety and the target nucleic acid, wherein the binding moiety binds specifically to a nucleic acid encoding a protein having an amino acid sequence set forth in SEQ
ID NO:10; and (b) detecting the complex, which if present in the sample is indicative of cervical cancer in the human.
Thus, the invention provides a wide range of methods and compositions for detecting and treating cervical cancer in an individual. Specifically, the invention provides cervical cancer-associated proteins, which permit specific and early, preferably before metastases occur, detection of cervical cancer in an individual. In addition, the invention provides kits useful in the detection of cervical cancer in an individual. In addition, the invention provides methods utilizing the cervical cancer-asssociated proteins as targets and indicators, for treating cervical cancers and for monitoring of the efficacy of such a treatment. These and other numerous additional aspects and advantages of the invention will become apparent upon consideration of the following figures, detailed description, and claims which follow.

WO 98!09170 PCT/US97/14526 Brief Description of the Drawings.
Figure 1 a is a high resolution two-dimensional gel electrophoresis pattern of nuclear matrix proteins isolated from a cervical cancer tissue sample. Tumor-associated proteins encircled and marked with reference numbers 1-5 correspond to proteins CvC-1 to CvC-5, listed in Table 2.
Figure lb is a high resolution two-dimensional gel electrophoresis pattern of nuclear matrix proteins isolated from a normal cervical tissue sample. As a reference, the relative positions corresponding to the CvC-1 to CvC-5 proteins of Figure 1 a are encircled and marked with reference numbers 1-5.
to Figure 2a is a high resolution two-dimensional gel electrophoresis pattern of nuclear matrix proteins isolated from the cervical carcinoma-derived cell line C33A.
Tumor-associated proteins CvC-2 and CvC-5 are encircled and marked with reference numbers 2 and 5.
Figure 2b is a high resolution two-dimensional gel electrophoresis pattern of nuclear matrix proteins isolated from CaSki cells. Tumor-associated proteins CvC-l and CvC-3 are ~ 5 encircled and marked with reference numbers 1 and 3.
For each of the above figures, molecular weight standards are indicated on the ordinate axes (Mrx103) and isoelectric points are shown on the abscissae.

Detailed Description of the Invention.
The present invention provides methods and compositions for the detection and treatment of cervical cancer. _The invention is based, in part, upon the discovery of cervical cancer-associated proteins which generally are present at detectably higher levels in cancerous cervical cells than in normal cervical cells, as determined by two-dimensional gel electrophoresis.
The cervical cancer-associated proteins may act as marker proteins useful in the detection of cervical cancer or as target proteins for therapy of cervical cancer. For example, it is contemplated that, the marker proteins and binding moieties, for example, antibodies that bind to the marker proteins or nucleic acid probes which hybridize to nucleic acid sequences encoding the to marker proteins, may be used to detect the presence of cervical cancer in an individual.
Furthermore, it is contemplated that, the skilled artisan may produce novel therapeutics for treating cervical cancer which include, for example: antibodies which can be administered to an individual that bind to and reduce or eliminate the biological activity of the target protein in vivo;
nucleic acid or peptide nucleic acid sequences which hybridize with genes or gene transcripts I5 encoding the target proteins thereby to reduce expression of the target proteins in vivo; or small molecules, for example, organic molecules which interact with the target proteins or other cellular moieties, for example, receptors for the target proteins, thereby to reduce or eliminate biological activity of the target proteins.
Set forth below are methods for isolating cervical cancer-associated proteins, methods for 2o detecting cervical cancer using cervical cancer-associated proteins as markers, and methods for treating individuals afflicted with cervical cancer using cervical cancer-associated proteins as targets for cancer therapy.
1. Identircation and Purification of Cervical Cancer-associated Proteins.
Marker proteins of the invention, as disclosed herein are identified by (i) isolating proteins 25 from normal cervical tissue and from cervical cancer tissue using a nuclear matrix purification protocol, such as those described generally in U.S. Pat. Nos. 4,882,268 and 4,885,236, or Fey et al. ( 1986) .rupra (ii) fractionating the resulting nuclear matrix protein preparations by 2-D gel electrophoresis, (iii) visualizing the resulting protein patterns, for example, by silver staining, and (iv) identifying polypeptide spots on the resulting 2-D gel electropherograms which generally are 3o detectable in samples isolated from cervical cancer cells but not detectable in samples isolated from normal cervical cells.

Marker proteins associated with cervical cancer tissue were isolated as described herein using a modification of the method of Fey et al. (Fey et al. (1986) supra). Briefly, cervical cancer tissue is minced into small (1 mm3) pieces and homogenized with a Teflon pestle on ice and treated with a buffered solution containing 0.5% Triton-X-100, vanadyl riboside complex plus a protease inhibitor cocktail (phenylmethyl sulfonyl fluoride, aprotinin, and leupeptin) to remove lipids and soluble proteins. Tumor cells from cell lines can be harvested by trypsinization and treated in the same way as for homogenized tumor tissue. Stromal aggregates are removed by filtering the homogenate through a 250 micron nylon screen followed by a centrifugation step.
Soluble cytoskeletal proteins are removed by incubating the pellet in an extraction buffer 1o containing 250 mM (NH4)2504, 0.5% Triton-X-100, vanadyl riboside complex plus a protease inhibitor cocktail for 10 minutes on ice followed by centrifugation. Chromatin is removed by incubating the pellet in DNAase I in a buffered solution containing a protease inhibitor cocktail for 45 minutes at 25°C.
The remaining pellet fraction, containing the target proteins and intermediate filaments, is 15 solubilized in a disassembly buffer containing 8 M urea, protease inhibitor cocktail plus 1 % 2-mercaptoethanol. Insoluble contaminants, primarily carbohydrates and extracellular matrix, are removed by ultracentrifugation. Intermediate filaments are allowed to reassemble upon removal of urea by dialysis in assembly buffer containing protease inhibitor cocktail and removed by ultracentrifugation, leaving the target proteins in the supernatant fraction.
Protein concentration 2o can be determined by the Coomassie Plus Protein Assay Kit (Pierce Chemicals, Rockford, IL) using a bovine gamma globulin standard. Proteins are immediately precipitated in 80% ethanol and stored at -80°C until use.
It is contemplated that, after identification, the resulting cervical cancer-associated proteins may be isolated by preparing a nuclear matrix protein preparation, such as the one described 25 above, electrophoresing the resulting proteins on a 2-D gel, and after some means of visualization, isolating the protein of interest from the resulting 2-D gel. Alternatively, it is contemplated that the marker protein, once identified, can be isolated, using standard protein purification methodologies well known to those of ordinary skill in the art, such as affinity chromatography, to yield substantially pure marker proteins. As used herein, the term "substantially pure" is 3o understood to mean at least 80% pure as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

2. Detection of Cervical Cancer.
Once cervical cancer-associated proteins have been identified, they may be used as markers to determine whether an individual has cervical cancer and/or cervical dysplasia, and if so, suitable detection methods can be used to monitor the status of the disease.
Using the marker proteins, the skilled artisan can produce a variety of detection methods for detecting cervical cancer in a human. The methods, typically comprise the steps of detecting, by some means, the presence of one or more cervical cancer-associated proteins in a tissue or body fluid sample of the human. The accuracy and/or reliability of the method for detecting cervical cancer in a human may be further enhanced by detecting the presence of a plurality of cervical to cancer-associated proteins in a preselected tissue or body fluid sample.
The detection step may comprise one or more of the protocols described hereinbelow.
2.A. Protein Detection Methods.
The marker protein in a sample may be reacted with a binding moiety capable of specifically binding the marker protein. The binding moiety may comprise, for example, a member of a ligand-receptor pair, i.e., a pair of molecules capable of having a specific binding interaction. The binding moiety may comprise, for example, a member of a specific binding pair, such as antibody-antigen, enzyme-substrate, nucleic acid-nucleic acid, protein-nucleic acid, protein-protein, or other specific binding pair known in the art. Binding proteins may be designed which have enhanced affinity for a target protein. Optionally, the binding moiety may be linked with a 2o detectable label, such as an enzymatic, fluorescent, radioactive, phosphorescent or colored particle label. The labeled complex may be detected, e.g., visually or with the aid of a spectrophotometer or other detector.
The marker proteins also may be detected using gel electrophoresis techniques available in the art. In two-dimensional gel electrophoresis, the proteins are separated first in a pH gradient gel according to their isoelectric point. The resulting gel then is placed on a second polyacrylamide gel, and the proteins separated according to molecular weight (see, for example, O'Farrell ( 1975) J. Biol. Chem. 250: 4007-4021 ).
One or more marker proteins may be detected by first isolating proteins from a sample obtained from an individual suspected of having cervical cancer, and then separating the proteins 3o by two-dimensional gel electrophoresis to produce a characteristic two-dimensional gel electrophoresis pattern. The pattern then may be compared with a standard gel pattern produced by separating, under the same or similar conditions, proteins isolated from normal or cancer cells.
The standard may be stored or obtained in an electronic database of electrophoresis patterns. The presence of a cervical cancer-associated protein in the two-dimensional gel provides an indication of the presence of a cervical cancer in the sample being tested. The detection of two or more proteins in the two-dimensional gel electrophoresis pattern further enhances the accuracy of the assay. The presence of a plurality, e.g., two to five, cervical cancer-associated proteins on the two-dimensional gel provides a strong indication of the presence of a cervical cancer in the sample. The assay thus permits the early detection and treatment of cervical cancer.
to 2B. Immunoassay.
A marker cervical cancer-associated protein may also be detected using any of a wide range of immunoassay techniques available in the art. For example, the skilled artisan may employ the sandwich immunoassay format to detect cervical cancer in a body fluid sample.
Alternatively, the skilled artisan may use conventional immuno-histochemical procedures for detecting the presence 15 of the cervical cancer-associated protein in a tissue sample, for example, in a Pap smear, using one or more labeled binding proteins (See Example S, hereinbelow).
In a sandwich immunoassay, two antibodies capable of binding the marker protein generally are used, e.g., one immobilized onto a solid support, and one free in solution and labeled with detectable chemical compound. Examples of chenucal labels that may be used for the second 2o antibody include radioisotopes, fluorescent compounds, and enzymes or other molecules which generate colored or electrochemically active products when exposed to a reactant or enzyme substrate. When a sample containing the marker protein is placed in this system, the marker protein binds to both the immobilized antibody and the labeled antibody, to form a "sandwich"
immune complex on the support's surface. The complexed protein is detected by washing away 25 non-bound sample components and excess labeled antibody, and measuring the amount of labeled antibody complexed to protein on the support's surface.
Both the sandwich immunoassay and the tissue immunohistochemical procedure are highly specific and very sensitive, provided that labels with good limits of detection are used. A detailed review of immunological assay design, theory and protocols can be found in numerous texts in the 3o art, including "Practicallmmunology", Butt, W.R., ed., (1984) Marcel Dekker, New York and "Antibodies, A Laboratory Approach" Harlow et al. eds.(I988) Cold Spring Harbor Laboratory.

In general, immunoassay design considerations include preparation of antibodies (e.g., monoclonal or polyclonal antibodies) having sufficiently high binding specificity for the target protein to form a complex that can be distinguished reliably from products of nonspecific interactions. As used herein, the term "antibody" is understood to mean binding proteins, for example, antibodies or other proteins comprising an immunoglobulin variable region-like binding domain, having the appropriate binding affinities and specificities for the target protein. The higher the antibody binding specificity, the lower the target protein concentration that can be detected. A preferred binding specificity is such that the binding protein has a binding affinity for the target protein of greater than about 105 M-I, preferably greater than about 10~ M-1 to Antibodies to an isolated target cervical cancer-associated protein which are useful in assays for detecting a cervical cancer in an individual may be generated using standard immunological procedures well known and described in the art. See, for example, Practical Immunology, Butt, N.R., ed., Marcel Dekker, NY, 1984. Briefly, an isolated target protein is used to raise antibodies in a xenogeneic host, such as a mouse, goat or other suitable mammal.
15 The marker protein is combined with a suitable adjuvant capable of enhancing antibody production in the host, and injected into the host, for example, by intraperitoneal administration.
Any adjuvant suitable for stimulating the host's immune response may be used.
A commonly used adjuvant is Freund's complete adjuvant (an emulsion comprising killed and dried microbial cells and available from, for example, Calbiochem Corp., San Diego, or Gibco, Grand Island, NY).
2o Where multiple antigen injections are desired, the subsequent injections comprise the antigen in combination with an incomplete adjuvant (e.g., cell-free emulsion).
Polyclonal antibodies may be isolated from the antibody-producing host by extracting serum containing antibodies to the protein of interest. Monoclonal antibodies may be produced by isolating host cells that produce the desired antibody, fusing these cells with myeloma cells using 2s standard procedures known in the immunology art, and screening for hybrid cells (hybridomas) that react specifically with the target protein and have the desired binding affinity.
Antibody binding domains also may be produced biosynthetically and the amino acid sequence of the binding domain manipulated to enhance binding affinity with a preferred epitope on the target protein. Specific antibody methodologies are well understood and described in the 30 literature. A more detailed description of their preparation can be found, for example, in "Practicallmmunology" (1984) supra) In addition, genetically engineered biosynthetic antibody binding sites, also known in the art as BABS or sFv's, may be used in the practice of the instant invention.
Methods for making and using BABS comprising (i) non-covalently associated or disulfide bonded synthetic VH and VL
dimers, (ii) covalently linked VH-VL single chain binding sites, (iii) individual VH or VL domains, or (iv) single chain antibody binding sites are disclosed, for example, in U.
S. Patent Nos.: 5,091,513; 5,132,405; 4,704,692; and 4,946,778. Furthermore, BABS having requisite specificity for the cervical cancer-associated proteins can be derived by phage antibody cloning from combinatorial gene libraries (see, for example, Clackson et al. (1991) Nature 352:
624,628). Briefly, a library of phage each of which express on their coat surface, BABS having immunoglobulin variable regions encoded by variable region gene sequences derived from mice pre-immunized with isolated cervical cancer-associated proteins, or fragments thereof, are screened for binding activity against immobilized cervical cancer-associated protein. Phage which bind to the immobilized cervical cancer-associated proteins are harvested and the gene encoding the BABS sequenced. The resulting nucleic acid sequences encoding the BAr3S of interest then may be expressed in conventional expression systems to produce the BABS
protein.
The isolated cervical cancer-associated protein also may be used for the development of diagnostic and other tissue evaluating kits and assays to monitor the level of the proteins in a tissue or fluid sample. For example, the kit may include antibodies or other specific binding proteins which bind specifically with the cervical cancer-associated proteins and which permit the presence and/or concentration of the cervical cancer-associated proteins to be detected and/or quantitated in a tissue or fluid sample.
Suitable kits for detecting cervical cancer-associated proteins are contemplated to include, e.g., a receptacle or other means for capturing a sample to be evaluated, and means for detecting the presence and/or quantity in the sample of one or more of the cervical cancer-associated proteins described herein. As used herein, "means for detecting" in one embodiment includes one or more antibodies specific for these proteins and means for detecting the binding of the antibodies to these proteins by, e.g., a standard sandwich immunoassay as described herein.
Where the presence of a protein within a cell is to be detected, e.g., as from a tissue sample, the kit also may comprise means for disrupting the cell structure so as to expose intracellular proteins.

2. C. Nucleic Acid based Assays.
The presence of a cervical cancer in an individual also may be determined by detecting, in a tissue or body fluid sample, a nucleic acid molecule encoding a cervical cancer-associated protein.
Using methods well known to those of ordinary skill in the art, the cervical cancer-associated proteins of the invention may be sequenced, and then, based on the determined sequence, oligonucleotide probes designed for screening a cDNA library (see, for example, Maniatis et al.
(1989) supra).
A target nucleic acid molecule encoding a marker cervical cancer-associated protein may be detected using a labeled binding moiety, capable of specifically binding the target nucleic acid.
to The binding moiety may comprise, for example, a protein, a nucleic acid or a peptide nucleic acid.
Additionally, a target nucleic acid, such as an mRNA encoding a cervical cancer-associated protein, may be detected by conducting, for example, a Northern blot analysis using labeled oligonucleotides, e.g., nucleic acid fragments complementary to and capable of hybridizing specifically with at least a portion of a target nucleic acid. While any length oligonucleotide may 15 be utilized to hybridize an mRNA transcript, oligonucleotides typically within the range of 8-100 nucleotides, preferably within the range of 15-50 nucleotides, are envisioned to be most useful in standard hybridization assays.
The oligonucleotide selected for hybridizing to the target nucleic acid, whether synthesized chemically or by recombinant DNA methodologies, is isolated and purified using standard 2o techniques and then preferably labeled (e.g., with 35S or 32P) using standard labeling protocols.
A sample containing the target nucleic acid then is run on an electrophoresis gel, the dispersed nucleic acids transferred to a nitrocellulose filter and the labeled oligonucleotide exposed to the filter under suitable hybridizing conditions, e.g. 50% formamide, 5 X SSPE, 2 X Denhardt's solution, 0. I % SDS at 42oC, as described in Maniatis et al. ( 1989) supra.
Other useful 25 procedures known in the art include solution hybridization, and dot and slot RNA hybridization.
The amount of the target nucleic acid present in a sample optionally then is quantitated by measuring the radioactivity of hybridized fragments, using standard procedures known in the art.
In addition, oligonucleotides also may be used to identify other sequences encoding members of the target protein families. The methodology also may be used to identify genetic sequences 3o associated with the nucleic acid sequences encoding the proteins described herein, e.g., to identify non-coding sequences lying upstream or downstream of the protein coding sequence, and which may play a functional role in expression of these genes. Additionally, binding assays may be conducted to identify and detect proteins capable of a specific binding interaction with a nucleic acid encoding a cervical cancer-associated protein, which may be involved e.g., in gene regulation or gene expression of the protein. In a further embodiment, the assays described herein may be used to identify and detect nucleic acid molecules comprising a sequence capable of recognizing and being specifically bound by a cervical cancer-associated protein.
In addition, it is anticipated that using a combination of appropriate oligonucleotide primers, i.e., more than one primer, the skilled artisan may determine the Ievel of expression of a target gene in vivo by standard polymerase chain reaction (PCR) procedures, for example, by quantitative PCR. Conventional PCR based assays are discussed, for example, in Innes et al (1990) "PCR Protocols; A guide to methods and Applications", Academic Press and Innes ei al.
(1995) "PCR Strategies" Academic Press, San Diego, CA.
3. Identification of Proteins Which Interact In Vivo l~tl: Cervical Cancer-associated Proteins.
In addition, it is contemplated that the skilled artisan, using procedures Like those described hereinbelow, may identify other molecules which interact in vivo with the cervical cancer-associated proteins described herein. Such molecules also may provide possible targets for chemotherapy.
By way of example, cDNA encoding proteins or peptides capable of interacting with cervical cancer-associated proteins can be determined using a two-hybrid assay, as reported in Durfee et al. (1993) Genes & Develop. 7:555-559. The principal of the two hybrid system is that noncovalent interaction of two proteins triggers a process (transcription) in which these proteins normally play no direct role, because of their covalent linkage to domains that function in this process. For example, the two-hybrid assay, detectable expression of a reporter gene occurs when two fusion proteins, one comprising a DNA-binding domain and one comprising a transcription initiation domain, interact.
The skilled artisan can use a host cell that contains one or more reporter genes, such as yeast strain YI53, reported in Durfee et al. (1993) supra. This strain carries two chromosomally located reporter genes whose expression is regulated by Gal4. A first reporter gene, is the E. coli IacZ gene under the control of the Gala promoter. A second reporter gene is the selectable HISS

gene. Other useful reporter genes may include, for example, the luciferase gene, the LEU2 gene, and the GFP (Green Fluorescent Protein) gene.
Two sets of plasmids are used in the two hybrid system. One set of plasmids contain DNA encoding a Gal4 DNA-binding domain fused in frame to DNA encoding a cervical cancer-associated protein. The other set of plasmids contain DNA encoding a Gal4 activation domain fused to portions of a human cDNA library constructed from human lymphocytes.
Expression from the first set of plasmids result in a fusion protein comprising a Gal4 DNA-binding domain and a cervical cancer-associated protein. Expression from the second set of plasmids produce a transcription activation protein fused to an expression product from the lymphocyte cDNA
to library. When the two plasmids are transformed into a gal-deficient host cell, such as the yeast Y153 cells described above, interaction of the Gal DNA binding domain and transcription activation domain occurs only if the cervical cancer-associated protein fused to the DNA binding domain hinds to a protein expressed from the lymphocyte cDNA library fused to the transcription activating domain. As a result of the protein-protein interaction between the cervical cancer-15 associated protein and its in vivo binding partner detectable levels of reporter gene expression occur.
In addition to identifying molecules which interact in vivo with the cervical cancer-associated proteins, the skilled artisan may also screen for molecules, for example, small molecules which alter or inhibit specific interaction between a cervical cancer-associated protein 2o and its in vivo binding partner.
For example, host cell can be transfected with DNA encoding a suitable DNA
binding domain/cervical cancer-associated protein hybrid and a translation activation domain/putative cervical cancer-associated protein binding partner, as disclosed above. The host cell also contains a suitable reporter gene in operative association with a cis-acting transcription activation element 25 that is recognized by the transcription factor DNA binding domain. The level of reporter gene expressed in the system is assayed. Then, the host cell is exposed to a candidate molecule and the level of reporter gene expression is detected. A reduction in reporter gene expression is indicative of the candidate's ability to interfere with complex formation or stability with respect to the cervical cancer-associated protein and its in vivo binding partner. As a control, the candidate 3o molecule's ability to interfere with other, unrelated protein-protein complexes is also tested.
Molecules capable of specifically interfering with a cervical cancer-associated protein/binding partner interaction, but not other protein-protein interactions, are identified as candidates for production and further analysis. Once a potential candidate has been identified, its efl'lcacy in modulating cell cycling and cell replication can be assayed in a standard cell cycle model system.
Candidate molecules can be produced as described hereinbelow. For example, DNA
encoding the candidate molecules can be inserted, using conventional techniques well described in the art (see, for example, Maniatis (1989) supra) into any of a variety of expression vectors and transfected into an appropriate host cell to produce recombinant proteins, including both full length and truncated forms. Useful host cells include E. coli, Saccharomyces cerevisiae, Pichia pastoris, the insect/baculovirus cell system, myeloma cells, and various other mammalian cells.
1o The full length forms of such proteins are preferably expressed in mammalian cells, as disclosed herein. The nucleotide sequences also preferably include a sequence for targeting the translated sequence to the nucleus, using, for example, a sequence encoding the eight amino acid nucleus targeting sequence of the large T antigen, which is well characterized in the art. The vector can additionally include various sequences to promote correct expression of the recombinant protein, including transcription promoter and termination sequences, enhancer sequences, preferred ribosome binding site sequences, preferred mRNA leader sequences, preferred protein processing sequences, preferred signal sequences for protein secretion, and the like. The DNA sequence encoding the gene of interest can also be manipulated to remove potentially inhibiting sequences or to minimize unwanted secondary structure formation. As will be appreciated by the practitioner 2o in the art, the recombinant protein can also be expressed as a fusion protein.
After translation, the protein can be purified from the cells themselves or recovered from the culture medium. The DNA can also include sequences which aid in expression and/or purification of the recombinant protein. The DNA can be expressed directly or can be expressed as part of a fusion protein having a readily cleavable fusion junction.
The DNA may also be expressed in a suitable mammalian host. Useful hosts include fibroblast 3T3 cells, (e.g., NIH 3T3, from CRL 1658) COS (simian kidney ATCC, CRL-1650) or CHO (Chinese hamster ovary) cells (e.g., CHO-DXB 11, from Chasin ( 1980) Proc.
Nat'l. Aca~l Sci. USA 77 :4216-4222), mink-lung epithelial cells (MVILu), human foreskin fibroblast cells, human glioblastoma cells, and teratocarcinoma cells. Other useful eukaryotic cell systems include 3o yeast cells, the insect/baculovirus system or myeloma cells.

WO 98!09170 PCT/IJS97/14526 In order to express a candidate molecule, the DNA is subcloned into an insertion site of a suitable, commercially available vector along with suitable promoter/enhancer sequences and 3' termination sequences. Useful promoter/enhancer sequence combinations include the CMV
promoter (human cytomegalovirus (MIE) promoter) present, for example, on pCDMB, as well as the mammary tumor virus promoter (MMTV) boosted by the Rous sarcoma virus LTR
enhancer sequence (e.g., from Clontech, Inc., Palo Alto). A useful inducable promoter includes, for example, A Zn2+ induceable promoter, such as the Zn2+ metallothionein promoter (Wrana et al.
(1992) Cell 71: 1003-1014) Other induceable promoters are well known in the art and can be used with similar success. Expression also can be further enhanced using traps-activating 1o enhancer sequences. The plasmid also preferably contains an amplifiable marker, such as DHFR
under suitable promoter control, e.g., SV40 early promoter (ATCC #37148).
Transfection, cell culturing, gene amplification and protein expression conditions are standard conditions, well known in the art, such as are described, for example in Ausubel et al., ed.,(1989) "Current Protocols in Molecular Biology", John Wiley & Sons, NY. Briefly, transfected cells are cultured 15 in medium containing 5-10% dialyzed fetal calf serum (dFCS), and stably transfected high expression cell lines obtained by amplification and subcloning and evaluated by standard Western and Northern blot analysis. Southern blots also can be used to assess the state of integrated sequences and the extent of their copy number amplification.
The expressed candidate protein is then purified using standard procedures. A
currently 2o preferred methodology uses an affinity column, such as a ligand affinity column or an antibody af~mity column. The column is then washed, and the candidate molecules selectively eluted in a gradient of increasing ionic strength, changes in pH, or addition of mild detergent. It is appreciated that in addition to the candidate molecules which bind to the cervical cancer-associated proteins, the cervical cancer associated proteins themselves may likewise be produced 25 using such recombinant DNA technologies.
4. Cervical Cancer Therany and Methods for Monitoring Therany.
The skilled artisan, after identification of cervical cancer-associated proteins and proteins which interact with the cervical cancer-associated proteins, can develop a variety of therapies for treating cervical cancer. Because the marker proteins described herein are present at detectably 3o higher levels in cervical cancer cells relative to normal cervical cells, the skilled artisan may employ, for example, the marker proteins and/or nucleic acids encoding the marker proteins as target molecules for a cancer chemotherapy.
4.A. Anti-sense-based Therapeutics.
A particularly useful cancer therapeutic envisioned is an oligonucleotide or peptide nucleic acid sequence complementary and capable of hybridizing under physiological conditions to part, or all, of the gene encoding the marker protein or to part, or all, of the transcript encoding the marker protein thereby to reduce or inhibit transcription and/or translation of the marker protein gene. Alternatively, the same technologies may be applied to reduce or inhibit transcription and/or translation of the proteins which interact with the cervical cancer-associated proteins.
Anti-sense oligonucleotides have been used extensively to inhibit gene expression in normal and abnormal cells. See, for example, Stein et al. ( I988) Cancer Res.
48: 2659-2668, for a pertinent review of anti-sense theory and established protocols. In addition, the synthesis and use of peptide nucleic acids as anti-sense-based therapeutics are described in PCT publications PCT/EP92/01219 published November 26, 1992, PCT/US92/10921 published June 24, 1993, and PCT/IlS94/013523 published June 1, 1995. Accordingly, the anti-sense-based therapeutics may be used as part of chemotherapy, either alone or in combination with other therapies.
Anti-sense oligonucleotide and peptide nucleic acid sequences are capable of hybridizing to a gene and/or mRNA transcript and, therefore, may be used to inhibit transcription and/or translation of the protein described herein. It is appreciated, however, that oligoribonucleotide sequences generally are more susceptible to enzymatic attack by ribonucleases than are deoxyribonucleotide sequences. Hence, oligodeoxyribonucleotides are preferred over oligoribonucleotides for in vivo therapeutic use. It is appreciated that the peptide nucleic acid sequences, unlike regular nucleic acid sequences, are not susceptible to nuclease degradation and, therefore, are likely to have greater longevity in vivo. Furthermore, it is appreciated that peptide nucleic acid sequences bind complementary single stranded DNA and RNA strands more strongly than corresponding DNA sequences (see, for example, PCTIEP92/20702 published November 26, 1992). Accordingly, peptide nucleic acid sequences are preferred for in vivo therapeutic use.
Therapeutically useful anti-sense oligonucleotides or peptide nucleic acid sequences may be synthesized by any of the known chemical oligonucleotide and peptide nucleic acid synthesis methodologies well known and thoroughly described in the art. Alternatively, a complementary sequence to part or all of the natural mRNA sequence may be generated using standard recombinant DNA technologies.
Since the complete nucleotide sequence encoding the entire marker protein as well as additional 5' and 3' untranslated sequences are known for each of the marker proteins and/or can be determined readily using techniques well known in the art, anti-sense oligonucleotides or peptide nucleic acids which hybridize with any portion of the mRNA transcript or non-coding sequences may be prepared using conventional oligonucleotide and peptide nucleic acid synthesis methodologies.
Oligonucleotides complementary to, and which hybridizable with any portion of the mRNA transcripts encoding the marker proteins are, in principle, effective for inhibiting translation of the target proteins as described herein. For example, as described in U.S. Pat. No.
5,098,890, issued March 24, 1992, oligonucleotides complementary to mRNA at or near the translation initiation codon site may be used to inhibit translation.
Moreover, it has been suggested that sequences that are too distant in the 3' direction from the translation initiation site may be less effective in hydbridizing the mRNA transcripts because of potential ribosomal "read-through", a phenomenon whereby the ribosome is postulated to unravel the anti-sense/sense duplex to permit translation of the message.
A variety of sequence lengths of oligonucleotide or peptide nucleic acid may be used to hybridize to mRNA transcripts. However, very short sequences (e.g., sequences containing less than 8-15 nucleobases) may bind with less specificity. Moreover, for in vivo use, short oligonucleotide sequences may be particularly susceptible to enzymatic degradation. Peptide nucleic acids, as mentioned above, likely are resistant to nuclease degradation. Where oligonucleotide and peptide nucleic acid sequences are to be provided directly to the cells, very long sequences may be less effective at inhibition because of decreased uptake by the target cell.
Accordingly, where the oligonucleotide or peptide nucleic acid is to be provided directly to target cells, oligonucleotide and/or peptide nucleic acid sequences containing about 8-50 nucieobases, and more preferably 15-30 nucleobases, are envisioned to be most advantageous.
An alternative means for providing anti-sense oligonucleotide sequences to a target cell is gene therapy where, for example, a DNA sequence, preferably as part of a vector and associated with a promoter, is expressed constituitively inside the target cell. Recently, Oeller et al. (Oeller et al. (1992) Science 254: 437-539), described the in vivo inhibition of the ACC synthase enzyme using a constitutively expressible DNA sequence encoding an anti-sense sequence to the full length ACC synthase transcript.
Accordingly, where the anti-sense oligonucleotide sequences are provided to a target cell indirectly, for example, as part of an expressible gene sequence to be expressed within the cell, longer oligonucleotide sequences, including sequences complementary to substantially all the protein coding sequence, may be used to advantage.
Finally, therapeutically useful oligonucleotide sequences envisioned also include not only native oligomers composed of naturally occurring nucleotides, but also those comprising modified nucleotides to, for example, improve stability and lipid solubility and thereby enhance cellular to uptake. For example, it is known that enhanced lipid solubility andlor resistance to nuclease digestion results by substituting a methyl group or sulfur atom for a phosphate oxygen in the internucleotide phosphodiester linkage. Phosphorothioates ("S-oligonucleotides" wherein a phosphate oxygen is replaced by a sulfur atom}, in particular, are stable to nuclease cleavage, are soluble in lipids, and are preferred, particularly for direct oligonucleotide administration. S-15 oligonucleotides may be synthesized chemically using conventional synthesis methodologies well known and thoroughly described in the art.
Preferred synthetic internucleoside linkages include phosphorothioates, alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidate, and carboxymethyl esters.
Furthermore, one or more of 2o the 5'-3' phosphate group may be covalently joined to a low molecular weight (e.g., 15-500 Da}
organic group, including, for example, lower alkyl chains or aliphatic groups (e.g., methyl, ethyl, propyl, butyl), substituted alkyl and aliphatic groups (e.g., aminoethyl, aminopropyl, aminohydroxyethyl, aminohydroxypropyl), small saccharides or glycosyl groups.
Other low molecular weight organic modifications include additions to the internucleoside phosphate linkages 25 such as cholesteryl or diamine compounds with varying numbers of carbon residues between the amino groups and terminal ribose. Oligonucleotides with these linkages or with other modifications can be prepared using methods well known in the art (see, for example, U.S.
Pat. No. 5,149,798).
Suitable oligonucleotide and or peptide nucleic acid sequences which inhibit transcription and/or translation of the marker proteins can be identified using standard in vivo assays well 3o characterized in the art. Preferably, a range of doses is used to determine ei~ective concentrations for inhibition as well as specificity of hybridization. For example, in the cases of an oligonucleotide, a dose range of 0-100pg oligonucleotide/ml may be assayed.
Further, the oligonucleotides may be provided to the cells in a single transfection, or as part of a series of transfections. Anti-sense ei~icacy may be determined by assaying a change in cell proliferation over time following transfection, using standard cell counting methodology and/or by assaying for reduced expression of marker protein, e.g., by immunofluorescense.
Alternatively, the ability of cells to take up and use thymidine is another standard means of assaying for cell division and may be used here, e.g., using 3H thymidine. Effective anti-sense inhibition should inhibit cell division sui~iciently to reduce thymidine uptake, inhibit cell proliferation, and/or reduce detectable levels of marker proteins.
to It is anticipated that therapeutically effective oligonucleotide or peptide nucleic acid concentrations may vary according to the nature and extent of the neoplasm, the particular nucleobase sequence used, the relative sensitivity of the neoplasm to the oligonucleotide or peptide nucleic acid sequence, and other factors. Useful ranges for a given cell type and oligonucleotide and/or peptide nucleic acid may be determined by performing standard dose range is experiments. Dose range experiments also may be performed to assess toxicity levels for normal and malignant cells. It is contemplated that useful concentrations may range from about 1 to 100 ug/ml per 105 cells.
For in vivo use, the anti-sense oligonucleotide or peptide nucleic acid sequences may be combined with a pharmaceutical carrier, such as a suitable liquid vehicle or excipient, and 20 optionally an auxiliary additive or additives. Liquid vehicles and excipients are conventional and are available commercially. Illustrative thereof are distilled water, physiological saline, aqueous solutions of dextrose, and the like. For in vivo cancer therapies, the anti-sense sequences preferably can be provided directly to malignant cells, for example, by injection directly into the tumor. Alternatively, the oligonucleotide or peptide nucleic acid may be administered 25 systemically, provided that the anti-sense sequence is associated with means for directing the sequences to the target malignant cells.
In addition to administration with conventional carriers, the anti-sense oligonucleotide or peptide nucleic acid sequences may be administered by a variety of specialized oligonucleotide delivery techniques. For example, oligonucleotides may be encapsulated in liposomes, as 3o described in Mannino et al. (1988) BioTechnology 6: 682, and Felgner et al.
(1989) Bethesda Res. Lab. Focus 11:21. Lipids useful in producing liposomal formulations include, without WO 98!09170 PCT/US97/14526 limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art (see, for example, in U.S. Pat. No. 4,235,871; U.S. Pat. No. 4,501,728; U.S. Pat.
No. 4,837,028; and U.S. Pat. No. 4,737,323). The pharmaceutical composition of the invention may further include compounds such as cyclodextrins and the like which enhance delivery of oligonucleotides into cells. When the composition is not administered systemically but, rather, is injected at the site of the target cells, cationic detergents (e.g. Lipofectin) may be added to enhance uptake. In addition, reconstituted virus envelopes have been successfully used to deliver RNA and DNA to cells (see, for example, Arad et al. (1986) Biochem. Biophy. Acta. 859: 88-94).
to For therapeutic use in vivo, the anti-sense oligonucleotide and/or peptide nucleic acid sequences are administered to the individual in a therapeutically ei~ective amount, for example, an amount sufficient to reduce or inhibit target protein expression in malignant cells. The actual dosage administered may take into account whether the nature of the treatment is prophylactic or therapeutic in nature, the age, weight, health of the patient, the route of administration, the size 15 and nature of the malignancy, as well as other factors. The daily dosage may range from about 0.01 to 1,000 mg per day. Greater or lesser amounts of oligonucleotide or peptide nucleic acid sequences may be administered, as required. As will be appreciated by those skilled in the medical art, particularly the chemotherapeutic art, appropriate dose ranges for in vivo administration would be routine experimentation for a clinician. As a preliminary guideline, 2o effective concentrations for in vitro inhibition of the target molecule may be determined first.
4.B. Binding Protein-based Therapeutics.
As mentioned above, a cancer marker protein or a protein that interacts with the cancer marker protein may be used as a target for chemotherapy. For example, a binding protein designed to bind the marker protein essentially irreversibly can be provided to the malignant cells, 25 for example, by association with a ligand specific for the cell and known to be absorbed by the cell. Means for targeting molecules to particular cells and cell types are well described in the chemotherapeutic art.
Binding proteins maybe obtained and tested using technologies well known in the art. For example, the binding portions of antibodies maybe used to advantage. It is contemplated, 3o however, that intact antibodies or BABS, which preferably, have been humanized may be used in the practice of the invention. As used herein, the term "humanized" is understood to mean a process whereby the framework region sequences of a non-human immunoglobulin variable region are replaced by human variable region sequences. Accordingly, it is contemplated that such humanized binding proteins will elicit a weaker immune response than their unhumanized counterparts. Particularly useful are binding proteins identified with high affinity for the target protein, e.g., greater than about 109 M-'~ Alternatively, DNA encoding the binding protein may be provided to the target cell as part of an expressible gene to be expressed within the cell following the procedures used for gene therapy protocols well described in the art. See, for example, U.S.
Patent No. 4,497,796, and "Gene Transfer", Vijay R. Baichwal, ed., (1986). It is anticipated that, once bound by binding protein, the target protein the will be inactivated or its biological activity 1o reduced thereby inhibiting or retarding cell division.
As described above, suitable binding proteins for in vivo use, may be combined with a suitable pharmaceutical carrier, such as physiological saline or other useful carriers well characterized in the medical art. The pharmaceutical compositions may be provided directly to malignant cells, for example, by direct injection, or may be provided systemically, provided the 15 binding protein is associated with means for targeting the protein to target cells. Finally, suitable dose ranges and cell toxicity levels may be assessed using standard dose range experiments.
Therapeutically effective concentrations may range from about 0.01 to about 1,000 mg per day.
As described above, actual dosages administered may vary depending, for example, on the nature of the malignancy, the age, weight and health of the individual, as well as other factors.
20 4. C. Small Molecule-based Therapeutics.
After having isolating cervical cancer-associated nuclear matrix proteins, the skilled artisan can, using methodologies well known in the art, can screen smali molecule libraries (either peptide or non-peptide based libraries) to identify candidate molecules that reduce or inhibit the biological function of the cervical cancer-associated proteins. The small molecules preferably accomplish 25 this function by reducing the in vivo expression of the target molecule, or by interacting with the target molecule thereby to inhibit either the biological activity of the target molecule or an interaction between the target molecule and its in vivo binding partner.
It is contemplated that, once the candidate small molecules have been elucidated, skilled artisan may enhance the efficacy of the small molecule using rational drug design methodologies 3o well known in the art. Alternatively, the skilled artisan may use a variety of computer programs which assist the skilled artisan to develop quantitative structure activity relationships (QSAR) which further to assist the design of additional candidate molecules de novo.
Once identified, the small molecules may be produced in commercial quantities and subjected to the appropriate safety and efficacy studies.
It is contemplated that the screening assays may be automated thereby facilitating the screening of a large number of small molecules at the same time. Such automation procedures are within the level of skill in the art of drug screening and, therefore, are not discussed herein.
Candidate peptide based small molecules may be produced by expression of an appropriate nucleic acid sequence in a host cell or using synthetic organic chemistries.
Similarly, non-peptidyl-based small molecules may be produced using conventional synthetic organic chemistries well known in the art.
t1s described above, for in vivo use, the identified small molecules may be combined with a suitable pharmaceutical carrier, such as physiological saline or other useful carriers well characterized in the medical art. The pharmaceutical compositions may be provided directly to malignant cells, for example, by direct injection, or may be provided systemically, provided the binding protein is associated with means for targeting the protein to target cells. Finally, suitable dose ranges and cell toxicity levels may be assessed using standard dose range experiments. As described above, actual dosages administered may vary depending, for example, on the nature of the malignancy, the age, weight and health of the individual. as well as other factors.
4. D. ~4lerhods for :Ylonitorin~ the Status of Cervical Cancer in an Individual The progression of the cervical cancer or the therapeutic efficacy of chemotherapy may be measured using procedures well known in the art. For example, the efficacy of a particular chemotherapeutic agent can be determined by measuring the amount of a cervical cancer-associated protein released from cervical cancer cells undergoing cell death.
As reported in PCT
publication PCT/US92/Q9220, published May 13, 1993, soluble nuclear matrix proteins and fragments thereof are released by cells upon cell death. Such soluble nuclear matrix proteins can be quantitated in a body fluid and used to monitor the degree or rate of cell death in a tissue.
For example, the concentration of a body fluid-soluble nuclear matrix proteins or a fragment thereof released from cells is compared to standards from healthy, untreated tissue.
Fluid samples are collected at discrete intervals during treatment and compared to the standard. It is contemplated that changes in the level of a body fluid soluble cervical cancer-associated protein, will be indicative of the efficacy of treatment (that is, the rate of cancer cell death). It is contemplated that the release of body fluid soluble interior nuclear matrix proteins can be measured in blood, plasma, urine, sputum, vaginal secretion, and breast exudate.
Where the assay is used to monitor tissue viability or progression of cervical cancer, the step of detecting the presence and abundance of the marker protein or its transcript in samples of interest is repeated at intervals and these values then are compared, the changes in the detected concentrations reflecting changes in the status of the tissue. For example, an increase in the level of cervical cancer-associated proteins may correlate with progression of the cervical cancer.
to Where the assay is used to evaluate the efficacy of a therapy, the monitoring steps occur following administration of the therapeutic agent or procedure (e.g., following administration of a chemotherapeutic agent or following radiation treatment). Similarly, a decrease in the level of cervical cancer-associated proteins may correlate a regression of the cervical cancer.
Thus, cervical cancer may be identified by the presence of cervical cancer-associated proteins as taught herein. Once identified, the cervical cancer may be treated using compounds which reduce in vivo the expression and/or biological activity of the cervical cancer-associated proteins. Furthermore, the methods provided herein can be used to monitor the progression of the disease and/or treatment of the disease. The following non limiting examples provide details of the isolation and characterization of cervical cancer-associated proteins and methods for their 2o use in the detection of cervical cancer.
Example 1 Isolation of Cervical Cancer-Associated Nuclear Matrix Proteins From Cervical Cancer Tissue Samples and Cell Lines Cervical cancer-associated proteins were identified by comparing silver stained 2-D gel patterns of proteins isolated from normal and cancerous cervical cells.
Fresh cervical carcinoma tissue was obtained from patients undergoing hysterectomy for clinically localized (stage IB, II or III, International Federation of Gynecology and Obstetrics or FIGO classification) carcinomas of the cervix from the Instituto Nacional de Cancerologia in Mexico City, Mexico, in accordance with Scientific and Ethics Committee Review Board 3o approval. A small number of tumor tissues were obtained under Institutional Review Board approval from the Pittsburgh Cancer Center (Pittsburgh, PA). Normal cervical tissue was obtained under Institutional Review Board approval from patients undergoing hysterectomy for causes unrelated to abnormal cervical histopathology, via the Cooperative Human Tissue Network (Columbus, OH). Clinical staging and tumor histopathology for twenty patients who provided tissue samples for use in these experiments are shown in Table 1.
With the exception of one case of adenosquamous carcinoma, all of the tumors were squamous cell carcinomas. A
majority of these were of the large cell non-keratinizing type. All the patients had localized disease with clinical stages ranging from IB to IIIB (Table 1 ).
TABLE 1. Patient Aye, Clinical.Sta~inp and HistopatholoPV.
Case Number Patient Age FIGO Stage Histopathological Diagnosis to 1 37 IB LCNKSt 2 49 IB LCKS~

3 32 IB Squamous, mod. well diff.

5 63 III Adenosquamous

7 44 IIIB LCNKS

8 31 IB Squamous, poorly diff.

9 31 IB LCNKS

12 ~3 5 IB LCNKS

17 33 ~ IB LCNKS

FIGO Stage IB IIA IIB III IIIB
n 9 1 5 3 2 15 t, Large cell nonkeratinizing squamous cell carcinoma $, Large cell keratinizing squamous cell carcinoma *, Squamous cell carcinoma, moderately well differentiated ~, Squamous cell carcinoma, poorly differentiated Fresh tissue was obtained during surgery, placed into transport medium (RPMI

supplemented with gentamicin and 10% fetal calf serum (GIBCO)), packed in ice, and shipped to Matritech, Inc. by overnight carrier. In a small number of cases where immediate shipment could not be arranged, tissues specimens were snap-frozen in liquid nitrogen and sent on dry ice to Matritech, Inc. by overnight carrier. Minimum size of tissue specimens was 0.2 gram. Diagnosis was obtained from pathology reports that accompanied each specimen.
Nuclear matrix proteins were isolated from cervical cancer tissue using a modification of the method of Fey et al. ( 1986) supra. Fresh cervical cancer tissue specimens, ranging in size from about 0.2 g to about 1.0 g, were obtained from 20 different patients.
Tissue specimens to were minced into small (1 mm3) pieces and homogenized with a Teflon pestle on ice and treated with a buffered solution containing 0.5% Triton-X-100, vanadyl riboside complex (RNAase inhibitor, Five Prime-Three Prime, Inc.) plus a protease inhibitor cocktail containing phenylmethyl sulfonyl fluoride (Sigma Chemical Co.), aprotinin and leupeptin (Boehringer Mannheim), to remove lipids and soluble proteins.
Stromal aggregates were removed by filtering the homogenate through 250 micron Nitex nylon screen (Tetko, Inc.) followed by a centrifugation step (600 x g, 4°C, 5min). Soluble cytoskeletal proteins were removed by incubating the pellet in an extraction buffer containing 250 mM (NH4)ZS04, 0.5% Triton X-100, vanadyl riboside complex and protease inhibitor cocktail on ice for 10 minutes followed by centrifugation (600 x g, 4°C, 5min).
2o Chromatin was removed by incubating the pellet in DNAase ( 100 mg/mL, Boehringer-Mannheim) in a buffered solution containing protease inhibitor cocktail for 45 minutes at 25°C.
The remaining pellet fraction, which contained nuclear matrix proteins and intermediate filaments, was solubilized in disassembly buffer containing 8 M urea, protease inhibitor cocktail and 1 (voUvol) 2-mercaptoethanol. Insoluble contaminants, primarily carbohydrates and extracellular matrix were removed by ultracentrifugation (163,000 x g, 20°C, 1 hr).
Intermediate filaments were allowed to reassemble upon removal of urea by dialysis in an assembly buffer containing 150 mM KCI, 24 mM imidazole HCI, 5 mM MgCl2, 0.125 mM EGTA and 2 mM dithiothreitol (DTT) with protease inhibitors and were removed by ultracentrifugation (109,000 x g, I 5°C, 1.5 hr), leaving the nuclear matrix proteins in the supernatant fraction.
3o In addition, cervical cancer-associated proteins were isolated from CaSki, ME-180, C33A, HeLa (S3 subline), SiHa, C4-1, C4-11, and HT-3 cervical tumor cell lines. Each cell line was obtained from the American Type Culture Collection (ATCC) and maintained at 37°C in 5% COZ
in Dulbecco's Modified Eagles Medium supplemented with 10% fetal calf serum, gentamicin, fungizone and 0.12% SeraExtend (Irvine Scientific). For nuclear matrix extraction studies, cells were grown to approximately 80% confluence in 10 stage cell culture factories {Nunc), harvested by trypsinization, counted and extracted in the same manner as homogenized tumor tissue.
Protein concentration of nuclear matrix proteins was determined by the Coomassie Plus Protein Assay Kit (Pierce Chemical) using a bovine gamma globulin standard. Proteins were immediately precipitated in 80% ethanol and stored at -80°C until use.
The resulting nuclear matrix proteins were next characterized by high-resolution two-to dimensional gel electrophoresis according to the procedure of O'Farrell (1975) J. Biol. Chem.
250: 4007-4021 (1975), on an Investigator 2-D system (Oxford Glycosystems, Bedford, MA).
Nuclear matrix proteins were solubilized for isoelectric focusing (IEF) analysis in sample buffer containing 9 M urea, 65 mM 3-[(cholamidopropyl)dimethylamino]-1-propanesuifate (CHAPS), 2.2% ampholytes, and 140 mM dithiothreitol (DTT). Two hundred micrograms of nuclear matrix 15 proteins were loaded per gel.
One-dimensional isoelectric focusing was carried out for 18,000 volt-hours using 1 mm x 18 mm gel tubes. Following first dimension electrophoresis, gels were extruded from gel tubes, equilibrated for 2 minutes in a buffer containing 0.3 M Tris base, 0.075 M
Tris-HCI, 3.0% SDS, 50 mM DTT, and 0.01% bromophenol blue and placed on top of 1 mm 10% Tris-glycine-SDS
2o Duracryl (Oxford Glycosystems) high tensile strength polyacrylamide electrophoresis slab gels.
Second dimension slab gels were electrophoresed at 16 Watts per gel and 12 °C constant temperature for approximately S hours. Molecular weight standards consisted of bovine albumin (Mr 66,000), ovalbumin (Mf 45,000), glyceraldehyde-3-phosphate dehydrogenase (Mr 36,000), carbonic anhydrase (Mr 29,000), bovine pancreatic trypsinogen (Mr 24,000), and soybean trypsin 25 inhibitor (Mr 20,100) (Sigma Chemical Co.). Isoelectric points were determined using internal control proteins with well-characterized isoelectric points. Following electrophoresis, gels were fixed in a solution containing 40% ethanol/10% acetic acid followed by treatment with a solution containing 0.5% glutaraidehyde. Gels were washed extensively and silver stained according to the method of Rabillioud et al. (Rabillioud et al. (1992) Electrophoresis 13:
429-439) and dried 3o between sheets of cellophane paper.

Silver-stained gels were imaged using a MasterScan Biological Imaging System (CSP, Inc., Billerica, MA) according to the manufacturer's instructions. Digital filtering algorithms were used to remove both uniform and non-uniform background without removing critical image data. Two-D scan (TM) two-dimensional gel analysis and database software (version 3 .1 ) using multiple Gaussian least-squares fitting algorithms were used to compute spot patterns into optimal-fit models of the data as reported by Olson et al. ( 1980) Anal.
Biochem. 169: 49-70.
Triangulation from the internal standards was used to precisely determine the molecular weight and isoelectric point of each target protein of interest. Interpretive densitometry was performed using specific software application modules to integrate the data into numeric and graphical 1o reports for each gel being analyzed.
Example 2 Identification of Cervical Cancer-associated Nuclear Matrix Proteins Having Differential Anyearance on 2-D Gels As described in the previous Example, 2-D gel electrophoresis patterns were obtained by 15 fractionating proteins isolated from either normal or cancerous cervical cells. Figure 1 a shows a typical cervical cancer-associated nuclear matrix protein pattern obtained from cervical cancer tissue. Figure 1 b shows a typical gel pattern produced by nuclear matrix proteins obtained from a normal cervical tissue sample. Approximately 600 proteins were resolved per gel. Most of the proteins observed were always present, irrespective of the type of cervical tissue under 2o investigation.
Comparison of Figures 1 and 2 reveals that, while most proteins in the cancer and non-cancer samples are identical, there are five proteins that are unique to the cervical cancer sample (labeled in Figure 1). The proteins, designated CvC-1 through CvC-5, were detected in 20 tissue samples obtained from patients diagnosed with cervical carcinoma but were not detected in 25 cervical tissue isolated from a group of 10 normal individuals. Table 2 identifies proteins, designated CvC-1 through CvC-5, by their approximate molecular weight and isoelectric point.
Both the molecular weight and isoelectric point values listed in Table I are approximate and accurate to within 2,000 Daltons for molecular weight and to within 0.2 pI
units for isoelectric point. A detailed analysis to identify proteins common to normal cervical tissue but absent from 3o cervical cancer tissue did not reveal any proteins that were specifically associated with normal cervical tissue.

Table 2: Cervical Cancer-associated Proteins PeptideMolecular WeightIsoelectric Cervical Cancer Normal Cervical Point CvC-1 69,408 5.78 + _ CvC-2 53,752 5.54 + _ CvC-3 47,887 5.60 + _ CvC-4 46,006 5.07 + _ CvC-5 44,864 6.6I + _ In addition, the expression of nuclear matrix proteins isolated from cervical cancer cell lines was investigated, the results of which are summarized in Table 3, below.
It is known that tumors of epithelial cell origin are characterized by the presence of stroma and other elements, such as those resulting from infiltrating inflammatory cells. Detection of nuclear matrix or matrix-associated proteins in tumor cell lines derived from cervical epithelial cell tumors reduces the possibility that the proteins are the result of stromal or other types of contamination of the nuclear matrix preparation.
2-D gel electrophoresis patterns were obtained from samples containing cervical cancer 1o cells derived from cervical cancer cell lines. Figure 2a shows a cervical cancer-associated nuclear matrix protein pattern obtained from the cervical cancer cell line C33A. In Figure 2a, tumor-associated proteins CvC-2 and CvC-5 are encircled and identified with numbers 2 and 5. Figure 2b shows a gel pattern produced by nuclear matrix proteins obtained from the cervical cancer cell line CaSki a normal cervical tissue sample. In Figure 2b, tumor associated proteins CvC-1 and CvC-3 are encircled and identified with numbers 1 and 3.
Four of the five tumor-associated proteins (CvCl to CvC-3 and CvC-5) were reproducibly detected in one or more cervical tumor cell lines (Figure 2, Table 3), confirming the epithelial origin of the proteins. Expression of the fifth protein, CvC-4, was variable but could be detected in the C33A tumor cell line (Table 3).
2o Table 3. Cervical Carcinoma-associated Protein Expression in Cervical Tumor Cell I fines Tumor cell line Histopathology of Nuclear matrix proteins expressed*
tumor or origin CvC-1 CvC-2 CvC-3 CvC-4 CvC-5 CaSKIt Epidermoid + try + - +

SiHa Squamous cell - tr +++ - +

HeLa Adenocarcinoma tr - +++ - +

ME-1801 Epidermoid - tr + - +

C33A Squamous cell + ++ - vary +

C4-I Squamous cell tr - +++ - +

C4-II Squamous cell - - - - tr HT-3t Epidermoid tr - + - tr * Nuclear matrix proteins were extracted from tumor cell lines obtained from the American Type Culture Collection using Fey and Penman extraction methodology.
Tumor cell lines arising from metastatic epidermoid carcinoma originating from cervix.
~ Indicates low level expression, detected by silver stain.
Indicates variable expression, detected by silver stain.
Two of the cervical cancer-associated proteins specific to cervical cancer cells were 1o isolated and processed for microsequence analysis.
Example 3 Characterization of Cervical Cancer-Associated Nuclear Matrix Protein Markers.
Two protein staining spots detectable on a 2-D gel corresponding to CvC-3 and CvC-5 were isolated, the protein harvested and subjected to microsequence analysis, as described hereinbelow.
For sequencing of the cervical cancer-associated polypeptides CvC-3 and CvC-5, the nuclear matrix fraction from HeLa cells were electrophoresed on two-dimensional gels as described above. Each gel was loaded with 300 micrograms of protein isolated by the nuclear 2o matrix protein isolation procedure, as described above. Following the second-dimension of electrophoresis, proteins were visualized by reverse staining. Briefly, gels were soaked in 200 mM imidazole for 10 minutes, rinsed for 1 minute in water, followed by 1-2 minutes in 300 mM
zinc chloride (Fernandez-Patron et al. (1992) BioTechniques 12: 564-573).
After the protein-containing spots began to appear, the gels were placed in water, and the relevant gels spots 2s excised. The isolated gel spots representing individual cervical cancer-associated polypeptides were pooled and destained by a 5 minute wash in 2% citric acid, followed by several washes in 100 mM Tris hydrochloride at pH 7.0 to raise the pH within the gel pieces.

Each set of pooled gel fragments was then diluted with an equal volume of 2X
sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (250mM Tris-Cl, 2% SDS, 20% glycerol, 0.01% bromophenol blue and 10% 13-mercaptoethanol, pH
6.8) and incubated at 75°C for 3 minutes. The gel fragment-containing samples were then cooled on ice and loaded onto a 4% polyacrylamide stacking/11% polyacrylamide separating SDS-PAGE gel, and electrophoresed in IX Tank Buffer (24mM Tris-HCI, 192 mM glycine, 1% SDS, pH 8.3) to focus the gel spots into bands. Molecular weight markers (BioRad #161-0304) were used on each gel to relate the observed molecular weights on the one-and two-dimensional gels.
Following electrophoresis, these gels were electroblotted onto Immobilon PVDF
membranes to (Oxford Glycosystems, Inc.) (Towbin et al. (1979) Proc. Nat'l. Acod. Sci.
USA 76: 4350-4354) as modified by Matsudaira (Matsudaira et al. (1987) .I. Biol. Chem. 262:
10035) for the mini-gel format. The membranes were then stained for 1 minute with Buffalo Black (0. I
% in 1 % acetic acid, 40% methanol) and rinsed with water. Regions of membrane containing polypeptide bands were excised with a clean scalpel.
15 The PVDF-bound polypeptides were then subjected to tryptic peptide mapping and microsequencing (Fernandez et al. {1994) Analytical Biochem. 218: 1 I2-I 17) at the Microchemistry Facility at the Worcester Foundation for Biomedical Research using a Hewlett Packard Model 1090M HPLC. Sequence determinations were made on an Applied Biosystems ProCise Sequenator, and most were confirmed by MALDI-TOF mass spectrometry of individual 2o peptides. Other peptides were identified by mass analysis alone, or mass analysis of carboxypeptidase-digested material.
Microseauence Analysis of CvC 3 Peptides.
Using the methodology described above, CvC-3 was isolated from approximately I20 two-dimensional gels of HeLa nuclear matrix and refocused on Immobilon-PVDF membrane 25 for microsequence analysis. Although only one protein was observed by silver staining the 2-D
gel location identified as CvC-3, refocusing of the protein on a one dimensional 1 I% minigel resulted in the resolution of two clearly separable protein bands. These proteins were labeled as CvC-3H and CvC-3L and submitted separately for microsequence analysis.
Analysis of the tryptic maps indicates that two different proteins were contained in the two bands seen on the 3o refocusing minigel, since little overlap was observed in the peak retention times of the two peptides.

Ten masses were detected by mass spectrometry from seven of the CvC-3H peaks.
Amino acid sequence was obtained for three peptides, two by Edman degradation and one by carboxypeptidase-MALDI-TOF analysis. The sequences obtained for these peptides, shown in Table 4 match a protein known as IEF SSP 9502 or "novel human nuclear phosphoprotein".
(Honore et. al. (1994) supra; GenBank Accession #L07758). The complete amino acid sequence for this protein, as derived from a gene sequence, is shown in SEQ. m No.: 10.
Seven other masses from peak fractions separated on the CvC-3H tryptic map also matched those of predicted tryptic fragments from this protein. Mass correlation data of tryptic peptides from CvC-3H are summarized in Table 4. The predicted molecular weight of the nuclear phosphoprotein, based l0 upon its nucleotide sequence is 55 kDa, whereas its observed molecular weight by 2-D gel analysis is 79 kDa (Honore et al. (1994) supra).
Table 4. Mass Correlation of CvC 3H derived Tryptic Peptides ObservedPredicted SEA.

PeakMass Mass DeltaSequence ID Protein Da Da No .
.

4 1 I 10,641109.25 0.13%PAASLAVHTDK 1 IEF SSP

5 834.62 835.92 0.16%FSGQIER 2 IEF SSP

7 1056.57 1057.26 0.07%RLIAEAKEK 3 IEF SSP

8 1187.45 1185.37 0.18%PSLVHSRDM 4 IEF SSP

1774.73 1766.93 0.44%VWDISTVSSVNEAFGR* 5 IEF SSP

10 1802.22 1805.02 0.16%LVLGSARNSSISGPFGSR 6 IEF SSP

11 2746.27 2743.02 0.12%SDKPff~'TLNAHNDEISGLDLSSOIK**7 IEF SSP

12 2412.23 2409.68 0.11%VOTLOFHPFEAQTLISGSYDK* 8 IEF SSP

12 2475.13 2483.98 0.36%MGVLFCSSCCPDLPFIYAFGGQK 9 IEF SSP

* Underlining reflects sequences confirmed by Edman degradation.
* * Bolded underlining reflects sequence confirmed by carboxypeptidase digestion.
In addition, seven masses were detected by mass spectrometry from four peaks derived from tryptic digestion of CvC-3L. One of these was directly sequenced and was found to be 2o identical to cytokeratin 17 (Troyanovsky et al. (1992), supra; GenBank Accession # Q04695).
Six other masses from fractions separated on the CvC-3L tryptic map also matched those of predicted tryptic fragments of human cytokeratin 17. The amino acid sequence for this protein, from Troyanovsky et al. (1992), supra, is shown in SEQ. m No.: 18. Mass correlation data of tryptic peptides from CvC-3L are summarized in Table 5. The apparent molecular weight of CvC-3L (47.9 kDa) is consistent with the detection of a full length molecule of cytokeratin 17 (predicted molecular weight, 48 kDa) in cervical tumors.
Table S. Mass Correlation of CvC.3L-derived Tryntic Peptides.

Observed Predicted SEO, Peak Mass Da Mass Da Delta Sequence ID No. Protein 4 995.46 994.03 0.14% DYSQYYR 11 Cytokeratin 4 1244.97 1242.34 0.21% NHEEEMNALR 12 Cytokeratin 9 15 I 8.031 S 16.670.09% LLEGEDAHLTQYK* 13 Cytokeratin 791.19 790.94 0.03 IL,NEMR 14 Cytokeratin % I 7 10 835.16 832.91 0.27% SEISELR 15 Cytokeratin I2 1144.21 1144.21 0.00% DAEDWFFSK 16 Cytokeratin 12 1187.57 1186.33 0.10% LSVEADINGLR 17 Cytokeratin * Underlining reflects sequences confirmed by Edman degradation.
MicroseAUence Analysis of CvGS Peptides.
The gel spot identified as CvC-5 was collected from HeLa nuclear matrix from the same to preparative two-dimensional gels that were used for the collection of CvC-3. Approximately 100 gel spots were collected as described and refocused on Immobilon-PVDF membrane for microsequence analysis. During the initial identification of tumor associated proteins it was noted that in some cervical tumors, two proteins appeared to migrate very closing together in the location identified as CvC-5. Only one protein was clearly apparent. However, when the expression of this protein was examined in cervical tumor cell lines, 3 of 8 cell lines showed the presence of at least two proteins in the area defined by CvC-5 (Table 3).
Without wishing to be bound by theory, one explanation for the apparent detection of only one protein in the CvC-5 gel spot in many tumors is that one of the proteins may be more abundant, thereby masking the presence of other closely migrating proteins. When CvC-5 gel spots were pooled and refocused onto a one dimensional minigel, only one diffusely stained protein band was detected.
The tryptic map of the diffuse band containing the polypeptide components of the CvC-5 gel spot contained approximately 30 resolved peaks. Mass analysis was performed on 12 of these peaks and 30 masses were obtained. Six amino acid sequences were obtained by automated Edman degradation, revealing the presence of three distinct polypeptides. The first of these is a protein known as TDP-43 or TAR DNA binding protein (Out ei. al. (1995) supra;
GenBank Accession # U23731). The complete amino acid sequence, as derived from the gene sequence for this protein, is shown in SEQ. ID. No. 26. The apparent molecular weight of 43 kDa suggests identification of the intact protein in cervical tumors. Six other masses from fractions separated on the CvC-5 tryptic map also matched those of predicted tryptic fragments from this protein.
Mass correlation data and peptide sequence data of tryptic peptides matching TDP-43 are shown in Table 6.
to Table 6. Mass Correlation of CvC S Derived Tryntic Peytides.
Observed Predicted SEO.

PeakMass Da Mass Da Delta Sequence ID Protein No .
.

12 1729.01 1726.79 0.13% FGGNPGGFGNQGGFGNSR 19 TDP43

13 655.72 653.78 0.30% WCDCK 20 TDP43 13 834.24 833.89 0.04% TTEQDLK 21 TDP43

14 682.63 681.79 0.12% GFGFVR 22 TDP43 16 1511.88 1511.66 0.01% LPNSKQSQDEPLR 23 TDP43 21 1280.01 1281.41 0.11% KMDETDASSAVK 24 TDP43 25 1342.84 1341.61 0.09% TSDLIVLGLPWK* 25 TDP43 * Underlining reflects sequences confirmed by Edman degradation.
Sequence information obtained for three peptides matched a nuclear pore protein known as nucleoporin or Nup358 (Wu et. al (1995) supra, Gen Bank Accession #
L41840). The complete amino acid sequence, as derived from the gene sequence, is shown in SEQ. ID. No.:34.
Mass correlation data for five additional masses identified from the CvC-5 tryptic map which matched predicted tryptic fragments of Nup358 are shown in Table 7. The location of the sequences matching Nup358 suggests our isolation of a C-terminal fragment of the intact protein (Mr 358 kDa) from cervical tumors.

Tavle 7. Mass Correlation of CvGS Derived Tryptic Peptides.
ObservedPredicted S

PeakMass Mass Delta Sequence ID Protein Da Da No .

9 613.14 614.66 0.25% NYYR* 27 nup358 613.20 614.66 0.24% NYYR* 28 nup358 11 702.22 701.78 0.06% VQEAQK 29 nup358 16 938.37 939.10 0.08% EVADCFK 30 nup358 17 2459.64 2458.54 0.04% HDGTGGOSIYGDKFEDENFDVK** 31 nup358 21 1419.00 1419.71 0.05% ITMELFXNIVPR** 32 nup358 21 2773.58 2771.11 0.09% HTGPGLLSMANOGQNTNNXXFVIXLK** 33 nup358 * Denotes a peptide that appeared in two adjacent HPLC fractions 5 ** Underlining reflects sequences confirmed by Edman degradation The third polypeptide identified in the CvC-5 gel spot is a fragment of lamin A (Fisher et.
al. (1986), supra; GenBank Accession #M13452). Two sequences matching lamin A
were obtained by Edman degradation (Table 8). Nine additional masses from fragments of the CvC-5 tryptic map match predicted masses of tryptic fragments from lamin A. Mass correlation data for to these additional masses were shown in Table 8. The amino acid sequence for this protein, (Fisher et. al. (1986) supra), is shown in SEQ. m No.: 46.
Table 8. Mass Correlation of CvC-5 derived Tryptic Peptides.
Observed Predicted S

PeakMass Da Mass Da DeltaSequence m No Protein .

7 667.10 666.69 0.06%EFESR 35 lamin A

8 569.50 568.63 0.15%TYSAK* 36 lamin A

8 585.78 587.63 0.31%LDNAR 37 lamin A

11 569.10 568.63 0.08%TYSAK* 38 lamin A

11 1025.18 1023.11 0.20%NIYSEELR 39 lamin A

12 805.83 803.91 0.24%TALSEKR 40 lamin A

17 1349.52 1347.56 0.15%LALDMEIHAYR** 41 lamin A

17 1009.78 1009.18 0.06%EMAEMRAR 42 lamin A

21 1912.74 1913.07 0.02% EELDFQKMYSEELR* 43 lamin A

22 1896.58 1894.13 0.13% MQQQLDEYQELLDIK** 44 lamin A

22 1913.03 1913.07 0.00% EELDFQKNIYSEELR* 45 lamin A

* Denotes a peptide that appeared in two adjacent HPLC fractions ** Underlining reflects sequences confirmed by Edman degradation Cervical cancer-associated proteins may be identified using well-known techniques based upon the partial amino acid sequences provided above. Thus, the cervical cancer-associated proteins detected according to methods of the invention may be referred to as comprising a continuous sequence shown in the above-noted sequence fragments. It is appreciated that the skilled artisan, in view of the foregoing disclosure, would be able to produce an antibody directed to against any cervical cancer-associated protein identified by the methods described herein.
Moreover, the skilled artisan, in view of the foregoing disclosure, would be able to produce nucleic acid sequences which encode the fragments described above, as well as nucleic acid sequences complementary thereto. In addition, the skilled artisan using conventional recombinant DNA methodologies, for example, by screening a cDNA library with such a nucleic acid sequence, would be able to isolate full length nucleic acid sequences encoding target cervical cancer-associated proteins. Such full length nucleic acid sequences, or fragments thereof, may be used to generate nucleic acid-based detection systems or therapeutics.
Example 4 Production ofAntibodies Which Bind Snecifical~
to Cervical Cancer-associated Proteins.
Once identified, a cervical cancer-associated protein, such as a CvC-1 through CvC-5, may be detected in a tissue or body fluid sample using numerous binding assays that are well known to those of ordinary skill in the art. For example, as discussed above, a cervical cancer-associated protein may be detected in either a tissue or body fluid sample using an antibody, for example, a monoclonal antibody, which bind specifically to an epitope disposed upon the cervical cancer-associated protein. In such detection systems, the antibody preferably is labeled with a detectable moiety.

Provided below is an exemplary protocol for the production of an anti-cervical cancer-associated monoclonal antibody. Other protocols also are envisioned.
Accordingly, the particular method of producing antibodies to target proteins is not envisioned to be an aspect of the invention.
Balb/c by J mice (Jackson Laboratory, Bar Harbor, ME) are injected inuaperitoneally with the target protein, e.g., CvC-3 protein isolated from HeLa cell nuclear matrix, every 2 weeks until the immunized mice obtain the appropriate serum titer. Thereafter, the mice are injected with 3 consecutive intravenous boosts. Freund's complete adjuvant (Gibco, Grand Island) is used in the first injection, incomplete Freund's in the second injection; and saline is used for subsequent intravenous injections. The animal is then sacrificed and its spleen removed.
Spleen cells (or lymph node cells) then are fused with a mouse myeloma line, e.g., using the method of Kohler et al. 1975) Nature Z56: 495.. Hybridomas producing antibodies that react with the target proteins then are cloned and grown as ascites. Hybridomas are screened by nuclear reactivity against the cell line that is the source of the immunogen, and by tissue immunohistochemistry using standard procedures known in the immunology art. Detailed descriptions of screening protocols, ascites production and immunoassays also are disclosed in published May 13, 1993.
Example S
Antibody-based Assay for Deteciin~ Cervical Cancer in an Individual.
The following assay has been developed for tissue samples, however, it is contemplated that similar assays for testing fluid samples may be developed without undue experimentation. A
typical assay may employ a commercial immunodetection kit, for example, the ABC Elite Kit from Vector Laboratories, Inc.
A biopsy sample, for example, a Pap smear is removed from the patient under investigation in accordance with the appropriate medical guidelines. The sample then is applied to a glass microscope slide and the sample fixed in cold acetone for 10 minutes.
Then, the slide is rinsed in distilled water and pretreated with a hydrogen peroxide containing solution (2 mL 30%
HZOZ and 30 mL cold methanol). The slide is then rinsed in a Buffer A
comprising Tris Buffered Saline (TBS) with 0.1°,% Tween and 0.1% Brij. A mouse anti-cervical cancer-associated protein monoclonal antibody in Buffer A is added to the slide and the slide then incubated for one hour at WO 98!09170 PCT/US97/14526 room temperature. The slide is then washed with Buffer A, and a secondary antibody (ABC Elite Kit, Vector Labs, Inc) in Buffer A is added to the slide. The slide is then incubated for 15 minutes at 37°C in a humidity chamber. The slides are washed again with Buffer A, and the ABC
reagent (ABC Elite Kit, Vector Labs, Inc.) is then added to the slide for amplification of the signal. The slide is then incubated for a further 15 minutes at 37°C in the humidity chamber.
The slide then is washed in distilled water, and a diamino benzenedine (DAB) substrate added to the slide for 4-5 minutes. The slide is then rinsed with distilled water, counterstained with hematoxylin, rinsed with 95% ethanol, rinsed with 100% ethanol, and then rinsed with xylene. A cover slip is then applied to the slide and the result observed by light microscopy.

WO 98/09170 PCT/US97l14526 EAUivalents.
The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: MATRITECH, INC.
(ii) TITLE OF INVENTION: METHODS AND COMPOSITIONS FOR THE
DETECTION OF CERVICAL CANCER
(iii) NUMBER OF SEQUENCES: 46 (iv) CORRESPONDENCE ADDRESS:
RICHES, MCKENZIE & HERBERT

TORONTO, CANADA

(v) COMPUTER READABLE FORM:
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(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 2,263,888 (B) FILING DATE: 1997-AUG-19 (C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
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(A) NAME: RICHES, MCKENZIE AND HERBERT
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(A) AUTHORS: HONORE, B
LEFFERS, H
MADSEN, P
CELIS, JE
(B) TITLE: CLONING OF A CDNA ENCODING A NOVEL HUMAN NUCLEAR

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Met Asn Arg Ser Arg Gln Val Thr Cys Val Ala Trp Val Arg Cys Gly Val Ala Lys Glu Thr Pro Asp Lys Val Glu Leu Ser Lys Glu Glu Val Lys Arg Leu Ile Ala Glu Ala Lys Glu Lys Leu Gln Glu Glu Gly Gly Gly Ser Asp Glu Glu Glu Thr Gly Ser Pro Ser Glu Asp Gly Met Gln Ser Ala Arg Thr Gln Ala Arg Pro Arg Glu Pro Leu Glu Asp Gly Asp Pro Glu Asp Asp Arg Thr Leu Asp Asp Asp Glu Leu Ala Glu Tyr Asp Leu Asp Lys Tyr Asp Glu Glu Gly Asp Pro Asp Ala Glu Thr Leu Gly Glu Ser Leu Leu Gly Leu Thr Val Tyr Gly Ser Asn Asp Gln Asp Pro $g Tyr Val Thr Leu Lys Asp Thr Glu Gln Tyr Glu Arg Glu Asp Phe Leu Ile Lys Pro Ser Asp Asn Leu Ile Val Cys Gly Arg Ala Glu Gln Asp Gln Cys Asn Leu Glu Val His Val Tyr Asn Gln Glu Glu Asp Ser Phe Tyr Val His His Asp Ile Leu Leu Ser Ala Tyr Pro Leu Ser Val Glu Trp Leu Asn Phe Asp Pro Ser Pro Asp Asp Ser Thr Gly Asn Tyr Ile Ala Val Gly Asn Met Thr Pro Val Ile Glu Val Trp Asp Leu Asp Ile Val Asp Ser Leu Glu Pro Val Phe Thr Leu Gly Ser Lys Leu Ser Lys Lys Lys Lys Lys Lys Gly Lys Lys Ser Ser Ser Ala Glu Gly His Thr Asp Ala Val Leu Asp Leu Ser Trp Asn Lys Leu Ile Arg Asn Val Leu Ala Ser Ala Ser Ala Asp Asn Thr Val Ile Leu Trp Asp Met Ser Leu Gly Lys Pro Ala Ala Ser Leu Ala Val His Thr Asp Lys Val Gln Thr Leu Gln Phe His Pro Phe Glu Ala Gln Thr Leu Ile Ser Gly Ser Tyr Asp Lys Ser Val Ala Leu Tyr Asp Cys Arg Ser Pro Asp Glu Ser His Arg Met Trp Arg Phe Ser Gly Gln Ile Glu Arg Val Thr Trp Asn His Phe Ser Pro Cys His Phe Leu Ala Ser Thr Asp Asp Gly Phe Val Tyr Asn Leu Asp Ala Arg Ser Asp Lys Pro Ile Phe Thr Leu Asn Ala His Asn Asp Glu Ile Ser Gly Leu Asp Leu Ser Ser Gln Ile Lys Gly Cys Leu Val Thr Ala Ser Ala Asp Lys Tyr Val Lys Ile Trp Asp Ile Leu Gly Asp Arg Pro Ser Leu Val His Ser Arg Asp Met Lys Met Gly Val Leu Phe Cys Ser Ser Cys Cys Pro Asp Leu Pro Phe Ile Tyr Ala Phe Gly Gly Gln Lys Glu Gly Leu Arg Val Trp Asp Ile Ser Thr Val Ser Ser Val Asn Glu Ala Phe Gly Arg Arg Glu Arg Leu Val Leu Gly Ser Ala Arg Asn Ser Ser Ile Ser Gly Pro Phe Gly Ser Arg Ser Ser Asp Thr Pro Met Glu Ser (2) INFORMATION FOR SEQ ID NO:11:
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Asp Ala Glu Asp Trp Phe Phe Ser Lys (2) INFORMATION FOR SEQ ID N0:17:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
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Leu Ser Val Glu Ala Asp Ile Asn Gly Leu Arg (2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 432 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL:
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OTHER INFORMATION: CYTOKERATIN 17 (x) PUBLICATION INFORMATION
(A) AUTHORS: TROYANOVSKY, SM
LEUBE, RE
FRANKE, WW
(B) TITLE: CHARACTERIZATION OF THE HUMAN GENE ENCODING CYTOKERATIN

(C) JOURNAL: EUR J CELL BIOL
(D) VOLUME: 59 (E) ISSUE: 1 (F) PAGES: 127-37 (G) DATE: 1992-OCT
(H) DOCUMENT NUMBER:
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(K) RELEVANT RESIDUES IN SEQ ID NO.: 1-432 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:
Met Thr Thr Ser Ile Arg Gln Phe Thr Ser Ser Ser Ser Ile Lys Gly Ser Ser Gly Leu Gly Gly Gly Ser Ser Arg Thr Ser Cys Arg Leu Ser Gly Gly Leu Gly Ala Gly Ser Cys Arg Leu Gly Ser Ala Gly Gly Leu Gly Ser Thr Leu Gly Gly Ser Ser Tyr Ser Ser Cys Tyr Ser Phe Gly Ser Gly Gly Gly Tyr Gly Ser Ser Phe Gly Gly Val Asp Gly Leu Leu Ala Gly Gly Glu Lys Ala Thr Met Gln Asn Leu Asn Asp Arg Leu Ala Ser Tyr Leu Asp Lys Val Arg Ala Leu Glu Glu Ala Asn Thr Glu Leu Glu Val Lys Ile Arg Asp Trp Tyr Gln Arg Gln Ala Pro Gly Pro Ala Arg Asp Tyr Ser Gln Tyr Tyr Arg Thr Ile Glu Glu Leu Gln Asn Lys Ile Leu Thr Ala Thr Val Asp Asn Ala Asn Ile Leu Leu Gln Ile Asp Asn Ala Arg Leu Ala Ala Asp Asp Phe Arg Thr Lys Phe Glu Thr Glu Gln Ala Leu Arg Leu Ser Val Glu Ala Asp Ile Asn Gly Leu Arg Arg Val Leu Asp Glu Leu Thr Leu Ala Arg Ala Asp Leu Glu Met Gln Ile Glu Asn Leu Lys Glu Glu Leu Ala Tyr Leu Lys Lys Asn His Glu Glu Glu Met Asn Ala Leu Arg Gly Gln Val Gly Gly Glu Ile Asn Val Glu Met Asp Ala Ala Pro Gly Val Asp Leu Ser Arg Ile Leu Asn Glu Met Arg Asp Gln Tyr Glu Lys Met Ala Glu Lys Asn Arg Lys Asp Ala Glu Asp Trp Phe Phe Ser Lys Thr Glu Glu Leu Asn Arg Glu Val Ala Thr Asn Ser Glu Leu Val Gln Ser Gly Lys Ser Glu Ile Ser Glu Leu Arg Arg Thr Met Gln Ala Leu Glu Ile Glu Leu Gln Ser Gln Leu Ser Met Lys Ala Ser Leu Glu Gly Asn Leu Ala Glu Thr Glu Asn Arg Tyr Cys Val Gln Leu Ser Gln Ile Gln Gly Leu Ile Gly Ser Val Glu Glu Gln Leu Ala Gln Leu Arg Cys Glu Met Glu Gln Gln Asn Gln Glu Tyr Lys Ile Leu Leu Asp Val Lys Thr Arg Leu Glu Gln Glu Ile Ala Thr Tyr Arg Arg Leu Leu Glu Gly Glu Asp Ala His Leu Thr Gln Tyr Lys Lys Glu Pro Val Thr Thr Arg Gln Val Arg Thr Ile Val Glu Glu Val Gln Asp Gly Lys Val Ile Ser Ser Arg Glu Gln Val His Gln Thr Thr Arg (2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
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Phe Gly Gly Asn Pro Gly Gly Phe Gly Asn Gln Gly Gly Phe Gly Asn Ser Arg (2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL:
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:
Trp Cys Asp Cys Lys (2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL:
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(1x) FEATURE
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LOCATION:
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OTHER INFORMATION: SIMILAR TO RESIDUES 115-121 OF SEQ ID N0:26 (x) PUBLICATION INFORMATION
(A) AUTHORS:
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~1 (H) DOCUMENT NUMBER:
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:
Thr Thr Glu Gln Asp Leu Lys (2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL:
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
Gly Phe Gly Phe Val Arg (2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
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OTHER INFORMATION: SIMILAR TO RESIDUES 177-189 OF SEQ ID N0:26 (x) PUBLICATION INFORMATION
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
Leu Pro Asn Ser Lys Gln Ser Gln Asp Glu Pro Leu Arg (2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
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(vi) ORIGINAL SOURCE:
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NAME/KEY:
LOCATION:
IDENTIFICATION METHOD:
OTHER INFORMATION: SIMILAR TO RESIDUES 84-95 OF SEQ ID N0:26 (x) PUBLICATION INFORMATION
(A) AUTHORS:
(B) TITLE:
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:

Lys Met Asp Glu Thr Asp Ala Ser Ser Ala Val Lys (2) INFORMATION FOR SEQ ID N0:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
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NAME/KEY:
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IDENTIFICATION METHOD:
OTHER INFORMATION: SIMILAR TO RESIDUES 103-114 OF SEQ ID N0:26 (x) PUBLICATION INFORMATION
(A) AUTHORS:
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:
Thr Ser Asp Leu Ile Val Leu Gly Leu Pro Trp Lys ~$
(2) INFORMATION FOR SEQ ID N0:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 414 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
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(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE
NAME/KEY:
LOCATION:
IDENTIFICATION METHOD:
OTHER INFORMATION: TDP-43 (x) PUBLICATION INFORMATION
(A) AUTHORS: OU, SH
WL1, F
HARRICH, D
GARCIA-MARTINEZ, LF
GAYNOR, RB
(B) TITLE: CLONING AND CHARACTERIZATION OF A NOVEL CELLULAR PROTEIN, TDP-43, THAT BINDS TO HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 TAR DNA SEQUENCE MOTIFS
(C) JOURNAL: J VIROL
(D) VOLUME: 69 (E) ISSUE: 6 (F) PAGES: 3584-96 (G) DATE: 1995-JUN
(H) DOCUMENT NUMBER:
(I) FILING DATE:
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(K) RELEVANT RESIDUES IN SEQ ID NO.: 1-414 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:26:

Met Ser Glu Tyr Ile Arg Val Thr Glu Asp Glu Asn Asp Glu Pro Ile Glu Ile Pro Ser Glu Asp Asp Gly Thr Val Leu Leu Ser Thr Val Thr Ala Gln Phe Pro Gly Ala Cys Gly Leu Arg Tyr Arg Asn Pro Val Ser Gln Cys Met Arg Gly Val Arg Leu Val Glu Gly Ile Leu His Ala Pro Asp Ala Gly Trp Gly Asn Leu Val Tyr Val Val Asn Tyr Pro Lys Asp Asn Lys Arg Lys Met Asp Glu Thr Asp Ala Ser Ser Ala Val Lys Val Lys Arg Ala Val Gln Lys Thr Ser Asp Leu Ile Val Leu Gly Leu Pro Trp Lys Thr Thr Glu Gln Asp Leu Lys Glu Tyr Phe Ser Thr Phe Gly Glu Val Leu Met Val Gln Val Lys Lys Asp Leu Lys Thr Gly His Ser Lys Gly Phe Gly Phe Val Arg Phe Thr Glu Tyr Glu Thr Gln Val Lys Val Met Ser Gln Arg His Met Ile Asp Gly Arg Trp Cys Asp Cys Lys Leu Pro Asn Ser Lys Gln Ser Gln Asp Glu Pro Leu Arg Ser Arg Lys Val Phe Val Gly Arg Cys Thr Glu Asp Met Thr Glu Asp Glu Leu Arg Glu Phe Phe Ser Gln Tyr Gly Asp Val Met Asp Val Phe Ile Pro Lys Pro Phe Arg Ala Phe Ala Phe Val Thr Phe Ala Asp Asp Gln Ile Ala Gln Ser Leu Cys Gly Glu Asp Leu Ile Ile Lys Gly Ile Ser Val His Ile Ser Asn Ala Glu Pro Lys His Asn Ser Asn Arg Gln Leu Glu Arg Ser Gly Arg Phe Gly Gly Asn Pro Gly Gly Phe Gly Asn Gln Gly Gly Phe Gly Asn Ser Arg Gly Gly Gly Ala Gly Leu Gly Asn Asn Gln Gly Ser Asn Met Gly Gly Gly Met Asn Phe Gly Ala Phe Ser Ile Asn Pro Ala Met Met Ala Ala Ala Gln Ala Ala Leu Gln Ser Ser Trp Gly Met Met Gly Met Leu Ala Ser Gln Gln Asn Gln Ser Gly Pro Ser Gly Asn Asn Gln Asn Gln Gly Asn Met Gln Arg Glu Pro Asn Gln Ala Phe Gly Ser Gly Asn Asn Ser Tyr Ser Gly Ser Asn Ser Gly Ala Ala Ile Gly Trp Gly Ser Ala Ser Asn Ala Gly Ser Gly Ser Gly Phe Asn Gly Gly Phe Gly Ser Ser Met Asp Ser Lys Ser Ser Gly Trp Gly Met (2) INFORMATION FOR SEQ ID N0:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
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(viii) POSITION IN GENOME:
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NAME/KEY:
LOCATION:
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OTHER INFORMATION: SIMILAR TO RESIDUES 2967-2970 OF SEQ ID N0:34 (x) PUBLICATION INFORMATION
(A) AUTHORS:

.
(B) TITLE:
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:
Asn Tyr Tyr Arg (2) INFORMATION FOR SEQ ID N0:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
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NAME/KEY:
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:
Asn Tyr Tyr Arg (2) INFORMATION FOR SEQ ID N0:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
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NAME/KEY:
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IDENTIFICATION METHOD:
OTHER INFORMATION: SIMILAR TO RESIDUES 2760-2765 OF SEQ ID N0:34 (x) PUBLICATION INFORMATION
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:
Val Gln Glu Ala Gln Lys (2) INFORMATION FOR SEQ ID N0:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
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i gl (K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:
Glu Val Ala Asp Cys Phe Lys (2) INFORMATION FOR SEQ ID N0:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (1ii) HYPOTHETICAL:
(iv) ANTI-SENSE:
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:31:
His Asp Gly Thr Gly Gly Gln Ser Ile Tyr Gly Asp Lys Phe Glu Asp Glu Asn Phe Asp Val Lys (2) INFORMATION FOR SEQ ID N0:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
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(viii) POSITION IN GENOME:
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NAME/KEY:
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OTHER INFORMATION: SIMILAR TO RESIDUES 3080-3091 OF SEQ ID N0:34 (x) PUBLICATION INFORMATION
(A) AUTHORS:
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i (xi) SEQUENCE DESCRIPTION: SEQ ID N0:32:
Ile Thr Met Glu Leu Phe Xaa Asn Ile Val Pro Arg (2) INFORMATION FOR SEQ ID N0:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
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OTHER INFORMATION: SIMILAR TO RESIDUES 3152-3177 OF SEQ ID N0:34 (x) PUBLICATION INFORMATION
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:33:

i His Thr Gly Pro Gly Leu Leu Ser Met Ala Asn Gln Gly Gln Asn Thr Asn Asn Xaa Xaa Phe Val Ile Xaa Leu Lys (2) INFORMATION FOR SEQ ID N0:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3224 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
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(A) CHROMOSOME/SEGMENT:
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(C) UNITS:
(ix) FEATURE
NAME/KEY:
LOCATION:
IDENTIFICATION METHOD:
OTHER INFORMATION: NUP358 (x) PUBLICATION INFORMATION
(A) AUTHORS: WU, J
MATUNIS, MJ
KRAEMER, D
BLOBEL G
COUTAVAS, E
(B) TITLE: NUP358, A CYTOPLASMICALLY EXPOSED NUCLEOPORIN WITH
PEPTIDE REPEATS, RAN-GTP BINDING SITES, ZINC FINGERS, A
CYCLOPHILIN A HOMOLOGOUS DOMAIN, AND A LEUCINE-RICH
REGION
(C) JOURNAL: J BIOL CHEM
(D) VOLUME: 270 (E) ISSUE: 23 (F) PAGES: 14209-13 (G) DATE: 1995-JUN-9 (H) DOCUMENT NUMBER:
(I) FILING DATE:

g$
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(K) RELEVANT RESIDUES IN SEQ ID NO.: 1-3224 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:34:
Met Arg Arg Ser Lys Ala Asp Val Glu Arg Tyr Ile Ala Ser Val Gln Gly Ser Thr Pro Ser Pro Arg Gln Lys Ser Met Lys Gly Phe Tyr Phe Ala Lys Leu Tyr Tyr Glu Ala Lys Glu Tyr Asp Leu Ala Lys Lys Tyr Ile Cys Thr Tyr Ile Asn Val Gln Glu Arg Asp Pro Lys Ala His Arg Phe Leu Gly Leu Leu Tyr Glu Leu Glu Glu Asn Thr Asp Lys Ala Val Glu Cys Tyr Arg Arg Ser Val Glu Leu Asn Pro Thr Gln Lys Asp Leu Val Leu Lys Ile Ala Glu Leu Leu Cys Lys Asn Asp Val Thr Asp Gly Arg Ala Lys Tyr Trp Leu Glu Arg Ala Ala Lys Leu Phe Pro Gly Ser Pro Ala Ile Tyr Lys Leu Lys Glu Gln Leu Leu Asp Cys Glu Gly Glu Asp Gly Trp Asn Lys Leu Phe Asp Leu Ile Gln Ser Glu Leu Tyr Val Arg Pro Asp Asp Val His Val Asn Ile Arg Leu Val Glu Val Tyr Arg Ser Thr Lys Arg Leu Lys Asp Ala Val Ala His Cys His Glu Ala Glu Arg Asn Ile Ala Leu Arg Ser Ser Leu Glu Trp Asn Ser Cys Val Val Gln Thr Leu Lys Glu Tyr Leu Glu Ser Leu Gln Cys Leu Glu Ser Asp Lys Ser Asp Trp Arg Ala Thr Asn Thr Asp Leu Leu Leu Ala Tyr Ala Asn Leu Met Leu Leu Thr Leu Ser Thr Arg Asp Val Gln Glu Ser Arg Glu Leu Leu Gln Ser Phe Asp Ser Ala Leu Gln Ser Val Lys Ser Leu Gly Gly Asn Asp Glu Leu Ser Ala Thr Phe Leu Glu Met Lys Gly His Phe Tyr Met His Ala Gly Ser Leu Leu Leu Lys Met Gly Gln His Ser Ser Asn Val Gln Trp Arg Ala Leu Ser Glu Leu Ala Ala Leu Cys Tyr Leu Ile Ala Phe Gln Val Pro Arg Pro Lys Ile Lys Leu Ile Lys Gly Glu Ala Gly Gln Asn Leu Leu Glu Met Met Ala Cys Asp Arg Leu Ser Gln Ser Gly His Met Leu Leu Asn Leu Ser Arg Gly Lys Gln Asp Phe Leu Lys Glu Ile Val Glu Thr Phe Ala Asn Lys Ser Gly Gln Ser Ala Leu Tyr Asp Ala Leu Phe Ser Ser Gln Ser Pro Lys Asp Thr Ser Phe Leu Gly Ser Asp Asp Ile Gly Asn Ile Asp Val Arg Glu Pro Glu Leu Glu Asp Leu Thr Arg Tyr Asp Val Gly Ala Ile Arg Ala His Asn Gly Ser Leu Gln His Leu Thr Trp Leu Gly Leu Gln Trp Asn Ser Leu Pro Ala Leu Pro Gly Ile Arg Lys Trp Leu Lys Gln Leu Phe His His Leu Pro His Glu Thr Ser Arg Leu Glu Thr Asn Ala Pro Glu Ser Ile Cys Ile Leu Asp Leu Glu Val Phe Leu Leu Gly Val Val Tyr Thr Ser His Leu Gln Leu Lys Glu Lys Cys Asn Ser His His Ser Ser Tyr Gln Pro Leu Cys Leu Pro Leu Pro Val Cys Lys Gln Leu Cys Thr Glu Arg Gln Lys Ser Trp Trp Asp Ala Val Cys Thr Leu Ile His Arg Lys Ala Val Pro Gly Asn Val Ala Lys Leu Arg Leu Leu Val Gln His Glu Ile Asn Thr Leu Arg Ala Gln Glu Lys His Gly Leu Gln Pro Ala Leu Leu Val g7 His Trp Ala Glu Cys Leu Gln Lys Thr Gly Ser Gly Leu Asn Ser Phe Tyr Asp Gln Arg Glu Tyr Ile Gly Arg Ser Val His Tyr Trp Lys Lys Val Leu Pro Leu Leu Lys Ile Ile Lys Lys Lys Asn Ser Ile Pro Glu Pro Ile Asp Pro Leu Phe Lys His Phe His Ser Val Asp Ile Gln Ala Ser Glu Ile Val Glu Tyr Glu Glu Asp Ala His Ile Thr Phe Ala Ile Leu Asp Ala Val Asn Gly Asn Ile Glu Asp Ala Val Thr Ala Phe Glu Ser Ile Lys Ser Val Val Ser Tyr Trp Asn Leu Ala Leu Ile Phe His Arg Lys Ala Glu Asp Ile Glu Asn Asp Ala Leu Ser Pro Glu Glu Gln Glu Glu Cys Lys Asn Tyr Leu Arg Lys Thr Arg Asp Tyr Leu Ile Lys Ile Ile Asp Asp Ser Asp Ser Asn Leu Ser Val Val Lys Lys Leu Pro Val Pro Leu Glu Ser Val Lys Glu Met Leu Asn Ser Val Met Gln Glu Leu Glu Asp Tyr Ser Glu Gly Gly Pro Leu Tyr Lys Asn Gly Ser Leu Arg Asn Ala Asp Ser Glu Ile Lys Arg Ser Thr Pro Ser Pro Thr Arg Tyr Ser Leu Ser Pro Ser Lys Ser Tyr Lys Tyr Ser Pro Lys Thr Pro Pro Arg Trp Ala Glu Asp Gln Asn Ser Leu Leu Lys Met Ile Cys Gln Gln Val Glu Ala Ile Lys Lys Glu Met Gln Glu Leu Lys Leu Asn Ser Ser Asn Ser Ala Ser Pro His Arg Trp Pro Thr Glu Asn Tyr Gly Pro Asp Ser Val Pro Asp Gly Tyr Gln Gly Ser Gln Thr Phe His Gly Ala Pro Leu Thr Val Ala Thr Thr Gly Pro Ser Val Tyr Tyr Ser Gln Ser 8g Pro Ala Tyr Asn Ser Gln Tyr Leu Leu Arg Pro Ala Ala Asn Val Thr Pro Thr Lys Gly Pro Val Tyr Gly Met Asn Arg Leu Pro Pro Gln Gln His Ile Tyr Ala Tyr Pro Gln Gln Met His Thr Pro Pro Val Gln Ser Ser Ser Ala Cys Met Phe Ser Gln Glu Met Tyr Gly Pro Pro Ala Leu Arg Phe Glu Ser Pro Ala Thr Gly Ile Leu Ser Pro Arg Gly Asp Asp Tyr Phe Asn Tyr Asn Val Gln Gln Thr Ser Thr Asn Pro Pro Leu Pro Glu Pro Gly Tyr Phe Thr Lys Pro Pro Ile Ala Ala His Ala Ser Arg Ser Ala Glu Ser Lys Thr Ile Glu Phe Gly Lys Thr Asn Phe Val Gln Pro Met Pro Gly Glu Gly Leu Arg Pro Ser Leu Pro Thr Gln Ala His Thr Thr Gln Pro Thr Pro Phe Lys Phe Asn Ser Asn Phe Lys Ser Asn Asp Gly Asp Phe Thr Phe Ser Ser Pro Gln Val Val Thr Gln Pro Pro Pro Ala Ala Tyr Ser Asn Ser Glu Ser Leu Leu Gly Leu Leu Thr Ser Asp Lys Pro Leu Gln Gly Asp Gly Tyr Ser Gly Ala Lys Pro Ile Pro Gly Gly Gln Thr Ile Gly Pro Arg Asn Thr Phe Asn Phe Gly Ser Lys Asn Val Ser Gly Ile Ser Phe Thr Glu Asn Met Gly Ser Ser Gln Gln Lys Asn Ser Gly Phe Arg Arg Ser Asp Asp Met Phe Thr Phe His Gly Pro Gly Lys Ser Val Phe Gly Thr Pro Thr Leu Glu Thr Ala Asn Lys Asn His Glu Thr Asp Gly Gly Ser Ala His Gly Asp Asp Asp Asp Asp Gly Pro His Phe Glu Pro Val Val Pro Leu Pro Asp Lys Ile Glu Val Lys Thr Gly Glu Glu Asp Glu Glu Glu Phe Phe Cys Asn Arg Ala Lys Leu Phe Arg Phe Asp Val Glu Ser Lys Glu Trp Lys Glu Arg Gly Ile Gly Asn Val Lys Ile Leu Arg His Lys Thr Ser Gly Lys Ile Arg Leu Leu Met Arg Arg Glu Gln Val Leu Lys Ile Cys Ala Asn His Tyr Ile Ser Pro Asp Met Lys Leu Thr Pro Asn Ala Gly Ser Asp Arg Ser Phe Val Trp His Ala Leu Asp Tyr Ala Asp Glu Leu Pro Lys Pro Glu Gln Leu Ala Ile Arg Phe Lys Thr Pro Glu Glu Ala Ala Leu Phe Lys Cys Lys Phe Glu Glu Ala Gln Ser Ile Leu Lys Ala Pro Gly Thr Asn Val Ala Met Ala Ser Asn Gln Ala Val Arg Ile Val Lys Glu Pro Thr Ser His Asp Asn Lys Asp Ile Cys Lys Ser Asp Ala Gly Asn Leu Asn Phe Glu Phe Gln Val Ala Lys Lys Glu Gly Ser Trp Trp His Cys Asn Ser Cys Ser Leu Lys Asn Ala Ser Thr Ala Lys Lys Cys Val Ser Cys Gln Asn Leu Asn Pro Ser Asn Lys Glu Leu Val Gly Pro Pro Leu Ala Glu Thr Val Phe Thr Pro Lys Thr Ser Pro Glu Asn Val Gln Asp Arg Phe Ala Leu Val Thr Pro Lys Lys Glu Gly His Trp Asp Cys Ser Ile Cys Leu Val Arg Asn Glu Pro Thr Val Ser Arg Cys Ile Ala Cys Gln Asn Thr Lys Ser Ala Asn Lys Ser Gly Ser Ser Phe Val His Gln Ala Ser Phe Lys Phe Gly Gln Gly Asp Leu Pro Lys Pro Ile Asn Ser Asp Phe Arg Ser Val Phe Ser Thr Lys Glu Gly Gln Trp Asp Cys Ser Ala Cys Leu Val Gln Asn Glu Gly Ser Ser Thr Lys Cys Ala Ala Cys Gln Asn Pro Arg Lys Gln Ser Leu Pro Ala Thr Ser Ile Pro Thr Pro Ala Ser Phe Lys Phe Gly Thr Ser Glu Thr Ser Lys Thr Leu Lys Ser Gly Phe Glu Asp Met Phe Ala Lys Lys Glu Gly Gln Trp Asp Cys Ser Ser Cys Leu Val Arg Asn Glu Ala Asn Ala Thr Arg Cys Val Ala Cys Gln Asn Pro Asp Lys Pro Ser Pro Ser Thr Ser Val Pro Ala Pro Ala Ser Phe Lys Phe Gly Thr Ser Glu Thr Ser Lys Ala Pro Lys Ser Gly Phe Glu Gly Met Phe Thr Lys Lys Glu Gly Gln Trp Asp Cys Ser Val Cys Leu Val Arg Asn Glu Ala Ser Ala Thr Lys Cys Ile Ala Cys Gln Asn Pro Gly Lys Gln Asn Gln Thr Thr Ser Ala Val Ser Thr Pro Ala Ser Ser Glu Thr Ser Lys Ala Pro Lys Ser Gly Phe Glu Gly Met Phe Thr Lys Lys Glu Gly Gln Trp Asp Cys Ser Val Cys Leu Val Arg Asn Glu Ala Ser Ala Thr Lys Cys Ile Ala Cys Gln Asn Pro Gly Lys Gln Asn Gln Thr Thr Ser Ala Val Ser Thr Pro Ala Ser Ser Glu Thr Ser Lys Ala Pro Lys Ser Gly Phe Glu Gly Met Phe Thr Lys Lys Glu Gly Gln Trp Asp Cys Ser Val Cys Leu Val Arg Asn Glu Ala Ser Ala Thr Lys Cys Ile Ala Cys Gln Cys Pro Ser Lys Gln Asn Gln Thr Thr Ala Ile Ser Thr Pro Ala Ser Ser Glu Ile Ser Lys Ala Pro Lys Ser Gly Phe Glu Gly Met Phe Ile Arg Lys Gly Gln Trp Asp Cys Ser Val Cys Cys Val Gln Asn Glu Ser Ser Ser Leu Lys Cys Val Ala Cys Asp Ala Ser Lys Pro Thr His Lys Pro Ile Ala Glu Ala Pro Ser Ala Phe Thr Leu Gly Ser Glu Met Lys Leu His Asp Ser Ser Gly Ser Gln Val Gly Thr Gly Phe Lys Ser Asn Phe Ser Glu Lys Ala Ser Lys Phe Gly Asn Thr Glu Gln Gly Phe Lys Phe Gly His Val Asp Gln Glu Asn Ser Pro Ser Phe Met Phe Gln Gly Ser Ser Asn Thr Glu Phe Lys Ser Thr Lys Glu Gly Phe Ser Ile Pro Val Ser Ala Asp Gly Phe Lys Phe Gly Ile Ser Glu Pro Gly Asn Gln Glu Lys Lys Ser Glu Lys Pro Leu Glu Asn Gly Thr Gly Phe Gln Ala Gln Asp Ile Ser Gly Gln Lys Asn Gly Arg Gly Val Ile Phe Gly Gln Thr Ser Ser Thr Phe Thr Phe Ala Asp Leu Ala Lys Ser Thr Ser Gly Glu Gly Phe Gln Phe Gly Lys Lys Asp Pro Asn Phe Lys Gly Phe Ser Gly Ala Gly Glu Lys Leu Phe Ser Ser Gln Tyr Gly Lys Met Ala Asn Lys Ala Asn Thr Ser Gly Asp Phe Glu Lys Asp Asp Asp Ala Tyr Lys Thr Glu Asp Ser Asp Asp Ile His Phe Glu Pro Val Val Gln Met Pro Glu Lys Val Glu Leu Val Thr Gly Glu Glu Asp Glu Lys Val Leu Tyr Ser Gln Arg Val Lys Leu Phe Arg Phe Asp Ala Glu Val Ser Gln Trp Lys Glu Arg Gly Leu Gly Asn Leu Lys Ile Leu Lys Asn Glu Val Asn Gly Lys Leu Arg Met Leu Met Arg Arg Glu Gln Val Leu Lys Val Cys Ala Asn His Trp Ile Thr Thr Thr Met Asn Leu Lys Pro Leu Ser Gly Ser Asp Arg Ala Trp Met Trp Leu Ala Ser Asp Phe Ser Asp Gly Asp Ala Lys Leu Glu Gln Leu Ala Ala Lys Phe Lys Thr Pro Glu Leu Ala Glu Glu Phe Lys Gln Lys Phe Glu Glu Cys Gln Arg Leu Leu Leu Asp Ile Pro Leu Gln Thr Pro His Lys Leu Val Asp Thr Gly Arg Ala Ala Lys Leu Ile Gln Arg Ala Glu Glu Met Lys Ser Gly Leu Lys Asp Phe Lys Thr Phe Leu Thr Asn Asp Gln Thr Lys Val Thr Glu Glu Glu Asn Lys Gly Ser Gly Thr Gly Ala Ala Gly Ala Ser Asp Thr Thr Ile Lys Pro Asn Pro Glu Asn Thr Gly Pro Thr Leu Glu Trp Asp Asn Tyr Asp Leu Arg Glu Asp Ala Leu Asp Asp Ser Val Ser Ser Ser Ser Val His Ala Ser Pro Leu Ala Ser Ser Pro Val Arg Lys Asn Leu Phe Arg Phe Gly Glu Ser Thr Thr Gly Phe Asn Phe Ser Phe Lys Ser Ala Leu Ser Pro Ser Lys Ser Pro Ala Lys Leu Asn Gln Ser Gly Thr Ser Val Gly Thr Asp Glu Glu Ser Asp Val Thr Gln Glu Glu Glu Arg Asp Gly Gln Tyr Phe Glu Pro Val Val Pro Leu Pro Asp Leu Val Glu Val Ser Ser Gly Glu Glu Asn Glu Gln Val Val Phe Ser His Arg Ala Lys Leu Tyr Arg Tyr Asp Lys Asp Val Gly Gln Trp Lys Glu Arg Gly Ile Gly Asp Ile Lys Ile Leu Gln Asn Tyr Asp Asn Lys Gln Val Arg Ile Val Met Arg Arg Asp Gln Val Leu Lys Leu Cys Ala Asn His Arg Ile Thr Pro Asp Met Thr Leu Gln Asn Met Lys Gly Thr Glu Arg Val Trp Leu Trp Thr Ala Cys Asp Phe Ala Asp Gly Glu Arg Lys Val Glu His Leu Ala Val Arg Phe Lys Leu Gln Asp Val Ala Asp Ser Phe Lys Lys Ile Phe Asp Glu Ala Lys Thr Ala Gln Glu Lys Asp Ser Leu Ile Thr Pro His Val Ser Arg Ser Ser Thr Pro Arg Glu Ser Pro Cys Gly Lys Ile Ala Val Ala Val Leu Glu Glu Thr Thr Arg Glu Arg Thr Asp Val Ile Gln Gly Asp Asp Val Ala Asp Ala Thr Ser Glu Val Glu Val Ser Ser Thr Ser Glu Thr Thr Pro Lys Ala Val Val Ser Pro Pro Lys Phe Val Phe Gly Ser Glu Ser Val Lys Ser Ile Phe Ser Ser Glu Lys Ser Lys Pro Phe Ala Phe Gly Asn Ser Ser Ala Thr Gly Ser Leu Phe Gly Phe Ser Phe Asn Ala Pro Leu Lys Ser Asn Asn Ser Glu Thr Ser Ser Val Ala Gln Ser Gly Ser Glu Ser Lys Val Glu Pro Lys Lys Cys Glu Leu Ser Lys Asn Ser Asp Ile Glu Gln Ser Ser Asp Ser Lys Val Lys Asn Leu Phe Ala Ser Phe Pro Thr Glu Glu Ser Ser Ile Asn Tyr Thr Phe Lys Thr Pro Glu Lys Ala Lys Glu Lys Lys Lys Pro Glu Asp Ser Pro Ser Asp Asp Asp Val Leu Ile Val Tyr Glu Leu Thr Pro Thr Ala Glu Gln Lys Ala Leu Ala Thr Lys Leu Lys Leu Pro Pro Thr Phe Phe Cys Tyr Lys Asn Arg Pro Asp Tyr Val Ser Glu Glu Glu Glu Asp Asp Glu Asp Phe Glu Thr Ala Val Lys Lys Leu Asn Gly Lys Leu Tyr Leu Asp Gly Ser Glu Lys Cys Arg Pro Leu Glu Glu Asn Thr Ala Asp Asn Glu Lys Glu Cys Ile Ile Val Trp Glu Lys Lys Pro Thr Val Glu Glu Lys Ala Lys Ala Asp Thr Leu Lys Leu Pro Pro Thr Phe Phe Cys Gly Val Cys Ser Asp Thr Asp Glu Asp Asn Gly Asn Gly Glu Asp Phe Gln Ser Glu Leu Gln Lys Val Gln Glu Ala Gln Lys Ser Gln Thr Glu Glu Ile Thr Ser Thr Thr Asp Ser Val Tyr Thr Gly Gly Thr Glu Val Met Val Pro Ser Phe Cys Lys Ser Glu Glu Pro Asp Ser Ile Thr Lys Ser Ile Ser Ser Pro Ser Val Ser Ser Glu Thr Met Asp Lys Pro Val Asp Leu Ser Thr Arg Lys Glu Ile Asp Thr Asp Ser Thr Ser Gln Gly Glu Ser Lys Ile Val Ser Phe Gly Phe Gly Ser Ser Thr Gly Leu Ser Phe Ala Asp Leu Ala Ser Ser Asn Ser Gly Asp Phe Ala Phe Gly Ser Lys Asp Lys Asn Phe Gln Trp Ala Asn Thr Gly Ala Ala Val Phe Gly Thr Gln Ser Val Gly Thr Gln Ser Ala Gly Lys Val Gly Glu Asp Glu Asp Gly Ser Asp Glu Glu Val Val His Asn Glu Asp Ile His Phe Glu Pro Ile Val Ser Leu Pro Glu Val Glu Val Lys Ser Gly Glu Glu Asp Glu Glu Ile Leu Phe Lys Glu Arg Ala Lys Leu Tyr Arg Trp Asp Arg Asp Val Ser Gln Trp Lys Glu Arg Gly Val Gly Asp Ile Lys Ile Leu Trp His Thr Met Lys Asn Tyr Tyr Arg Ile Leu Met Arg Arg Asp Gln Val Phe Lys Val Cys Ala Asn His Val Ile Thr Lys Thr Met Glu Leu Lys Pro Leu Asn Val Ser Asn Asn Ala Leu Val Trp Thr Ala Ser Asp Tyr Ala Asp Gly Glu Ala Lys Val Glu Gln Leu Ala Val Arg Phe Lys Thr Lys Glu Val Ala Asp Cys Phe Lys Lys Thr Phe Glu Glu Cys Gln Gln Asn Leu Met Lys Leu Gln Lys Gly His Val Ser Leu Ala Ala Glu Leu Ser Lys Glu Thr Asn Pro Val Val Phe Phe Asp Val Cys Ala Asp Gly Glu Pro Leu Gly Arg Ile Thr Met Glu Leu Phe Ser Asn Ile Val Pro Arg Thr Ala Glu Asn Phe Arg Ala Leu Cys Thr Gly Glu Lys Gly Phe Gly Phe Lys Asn Ser Ile Phe His Arg Val Ile Pro Asp Phe Val Cys Gln Gly Gly Asp Ile Thr Lys His Asp Gly Thr Gly Gly Gln Ser Ile Tyr Gly Asp Lys Phe Glu Asp Glu Asn Phe Asp Val Lys His Thr Gly Pro Gly Leu Leu Ser Met Ala Asn Gln Gly Gln Asn Thr Asn Asn Ser Gln Phe Val Ile Thr Leu Lys Lys Ala Glu His Leu Asp Phe Lys His Val Val Phe Gly Phe Val Lys Asp Gly Met Asp Thr Val Lys Lys Ile Glu Ser Phe Gly Ser Pro Lys Gly Ser Val Cys Arg Arg Ile Thr Ile Thr Glu Cys Gly Gln Ile (2) INFORMATION FOR SEQ ID N0:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL:
(iv) ANTI-SENSE:

(v) FRAGMENT TYPE:
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(viii) POSITION IN GENOME:
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(ix) FEATURE
NAME/KEY:
LOCATION:
IDENTIFICATION METHOD:
OTHER INFORMATION: SIMILAR TO RESIDUES 87-91 OF SEQ ID N0:46 (x) PUBLICATION INFORMATION
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:35:
Glu Phe Glu Ser Arg (2) INFORMATION FOR SEQ ID N0:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:

(vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE
NAME/KEY:
LOCATION:
IDENTIFICATION METHOD:
OTHER INFORMATION: SIMILAR TO RESIDUES 117-121 OF SEQ ID N0:46 (x) PUBLICATION INFORMATION
(A) AUTHORS:
(B) TITLE:
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(D) VOLUME:
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:36:
Thr Tyr Ser Ala Lys (2) INFORMATION FOR SEQ ID N0:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(vii) IMMEDIATE SOURCE:

(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE
NAME/KEY:
LOCATION:
IDENTIFICATION METHOD:
OTHER INFORMATION: SIMILAR TO RESIDUES 122-126 OF SEQ ID NO: 46 (x) PUBLICATION INFORMATION
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(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:37:
Leu Asp Asn Ala Arg (2) INFORMATION FOR SEQ ID N0:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:

(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE
NAME/KEY:
LOCATION:
IDENTIFICATION METHOD:
OTHER INFORMATION: SIMILAR TO RESIDUES 117-121 OF SEQ ID N0:46 (x) PUBLICATION INFORMATION
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
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(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:38:
Thr Tyr Ser Ala Lys (2) INFORMATION FOR SEQ ID N0:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:

1~~
(ix) FEATURE
NAME/KEY:
LOCATION:
IDENTIFICATION METHOD:
OTHER INFORMATION: SIMILAR TO RESIDUES 60-67 OF SEQ ID N0:46 (x) PUBLICATION INFORMATION
(A) AUTHORS:
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(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:39:
Asn Ile Tyr Ser Glu Glu Leu Arg (2) INFORMATION FOR SEQ ID N0:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE
NAME/KEY:

LOCATION:
IDENTIFICATION METHOD:
OTHER INFORMATION: SIMILAR TO RESIDUES 1-7 OF SEQ ID N0:46 (x) PUBLICATION INFORMATION
(A) AUTHORS:
(B) TITLE:
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(D) VOLUME:
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(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:40:
Thr Ala Leu Ser Glu Lys Arg (2) INFORMATION FOR SEQ ID N0:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE
NAME/KEY:
LOCATION:
IDENTIFICATION METHOD:
OTHER INFORMATION: SIMILAR TO RESIDUES 218-228 OF SEQ ID NO: 46 (x) PUBLICATION INFORMATION
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
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(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:41:
Leu Ala Leu Asp Met Glu Ile His Ala Tyr Arg (2) INFORMATION FOR SEQ ID N0:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE
NAME/KEY:
LOCATION:
IDENTIFICATION METHOD:
OTHER INFORMATION: SIMILAR TO RESIDUES 195-202 OF SEQ ID N0:46 (x) PUBLICATION INFORMATION

(A) AUTHORS:
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(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:42:
Glu Met Ala Glu Met Arg Ala Arg (2) INFORMATION FOR SEQ ID N0:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE
NAME/KEY:
LOCATION:
IDENTIFICATION METHOD:
OTHER INFORMATION: SIMILAR TO RESIDUES 53-67 OF SEQ ID N0:46 (x) PUBLICATION INFORMATION
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:

(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:43:
Glu Glu Leu Asp Phe Gln Lys Asn Ile Tyr Ser Glu Glu Leu Arg (2) INFORMATION FOR SEQ ID N0:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE
NAME/KEY:
LOCATION:
IDENTIFICATION METHOD:
OTHER INFORMATION: SIMILAR TO RESIDUES 203-217 OF SEQ ID N0:46 (x) PUBLICATION INFORMATION
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
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(F) PAGES:

( G ) DATE
(H) DOCUMENT NUMBER:
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(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:44:
Met Gln Gln Gln Leu Asp Glu Tyr Gln Glu Leu Leu Asp Ile Lys (2) INFORMATION FOR SEQ ID N0:45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE
NAME/KEY:
LOCATION:
IDENTIFICATION METHOD:
OTHER INFORMATION: SIMILAR TO RESIDUES 53-67 OF SEQ ID N0:46 (x) PUBLICATION INFORMATION
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:

(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:45:
Glu Glu Leu Asp Phe Gln Lys Asn Ile Tyr Ser Glu Glu Leu Arg (2) INFORMATION FOR SEQ ID N0:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 515 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE
NAME/KEY:
LOCATION:
IDENTIFICATION METHOD:
OTHER INFORMATION: LAMIN A
(x) PUBLICATION INFORMATION
(A) AUTHORS: FISHER, DZ
CHAUDHARY, N
BLOBEL, G
(B) TITLE: CDNA SEQUENCING OF NUCLEAR LAMINS A AND C REVEALS
PRIMARY AND SECONDARY STRUCTURAL HOMOLOGY TO
INTERMEDIATE FILAMENT PROTEINS
(C) JOURNAL: PROC NATL ACRD SCI U S A
(D) VOLUME: 83 (E) ISSUE: 17 (F) PAGES: 6450-4 (G) DATE: 1986-SEP
(H) DOCUMENT NUMBER:

1~~
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.: 1-515 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:46:
Thr Ala Leu Ser Glu Lys Arg Thr Leu Glu Gly Glu Leu His Asp Leu Arg Gly Gln Val Ala Lys Leu Glu Ala Ala Leu Gly Glu Ala Lys Lys Gln Leu Gln Asp Glu Met Leu Arg Arg Val Asp Ala Glu Asn Arg Leu Gln Thr Met Lys Glu Glu Leu Asp Phe Gln Lys Asn Ile Tyr Ser Glu Glu Leu Arg Glu Thr Lys Arg Arg His Glu Thr Arg Leu Val Glu Ile Asp Asn Gly Lys Gln Arg Glu Phe Glu Ser Arg Leu Ala Asp Ala Leu Gln Glu Leu Arg Ala Gln His Glu Asp Gln Val Glu Gln Tyr Lys Lys Glu Leu Glu Lys Thr Tyr Ser Ala Lys Leu Asp Asn Ala Arg Gln Ser Ala Glu Arg Asn Ser Asn Leu Val Gly Ala Ala His Glu Glu Leu Gln Gln Ser Arg Ile Arg Ile Asp Ser Leu Ser Ala Gln Leu Ser Gln Leu Gln Lys Gln Leu Ala Ala Lys Glu Ala Lys Leu Arg Asp Leu Glu Asp Ser Leu Ala Arg Glu Arg Asp Thr Ser Arg Arg Leu Leu Ala Glu Lys Glu Arg Glu Met Ala Glu Met Arg Ala Arg Met Gln Gln Gln Leu Asp Glu Tyr Gln Glu Leu Leu Asp Ile Lys Leu Ala Leu Asp Met Glu Ile His Ala Tyr Arg Lys Leu Leu Glu Gly Glu Glu Glu Arg Leu Arg Leu Ser Pro Ser Pro Thr Ser Gln Arg Ser Arg Gly Arg Ala Ser Ser His Ser Ser Gln Thr Gln Gly Gly Gly Ser Val Thr Lys Lys Arg Lys Leu Glu Ser Thr Glu Ser Arg Ser Ser Phe Ser Gln His Ala Arg Thr Ser Gly Arg Val Ala Val Glu Glu Val Asp Glu Glu Gly Lys Phe Val Arg Leu Arg Asn Lys Ser Asn Glu Asp Gln Ser Met Gly Asn Trp Gln Ile Lys Arg Gln Asn Gly Asp Asp Pro Leu Leu Thr Tyr Arg Phe Pro Pro Lys Phe Thr Leu Lys Ala Gly Gln Val Val Thr Ile Trp Ala Ala Gly Ala Gly Ala Thr His Ser Pro Pro Thr Asp Leu Val Trp Lys Ala Gln Asn Thr Trp Gly Cys Gly Asn Ser Leu Arg Thr Ala Leu Ile Asn Ser Thr Gly Glu Glu Val Ala Met Arg Lys Leu Val Arg Ser Val Thr Val Val Glu Asp Asp Glu Asp Glu Asp Gly Asp Asp Leu Leu His His His His Gly Ser His Cys Ser Ser Ser Gly Asp Pro Ala Glu Tyr Asn Leu Arg Ser Arg Thr Val Leu Cys Gly Thr Cys Gly Gln Pro Ala Asp Lys Ala Ser Ala Ser Gly Ser Gly Ala Gln Val Gly Gly Pro Ile Ser Ser Gly Ser Ser Ala Ser Ser Val Thr Val Thr Arg Ser Tyr Arg Ser Val Gly Gly Ser Gly Gly Gly Ser Phe Gly Asp Asn Leu Val Thr Arg Ser Tyr Leu Leu Gly Asn Ser Ser Pro Arg Thr Gln Ser Pro Gln Asn Cys Ser Ile Met 584BAF8395/27.744645-2

Claims (42)

1. A method of detecting cervical cancer in a human, the method comprising:
detecting in a sample isolated from said human the presence of a protein comprising an amino acid 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:9; and SEQ ID NO:10, the presence of said protein being indicative of cervical cancer in said human.

2. The method of claim 1, wherein said protein comprises the amino acid sequence set forth in SEQ ID NO:1.

3. The method of claim 1, wherein said protein comprises the amino acid sequence set forth in SEQ ID NO:2.

4. The method of claim 1, wherein said protein comprises the amino acid sequence set forth in SEQ ID NO:3.

5. The method of claim 1, wherein said protein comprises the amino acid sequence set forth in SEQ ID NO:4.

6. The method of claim 1, wherein said protein comprises the amino acid sequence set forth in SEQ ID NO:5.

7. The method of claim 1, wherein said protein comprises the amino acid sequence set forth in SEQ ID NO:6.

8. The method of claim 1, wherein said protein comprises the amino acid sequence sec forth in SEQ ID NO:7.

9. The method of claim 1, wherein said protein comprises the amino acid sequence set forth in SEQ ID NO:8.

10. The method of claim 1, wherein said protein comprises the amino acid sequence set forth in SEQ ID NO:9.

11. The method of claim 1, wherein said protein comprises the amino acid sequence set forth in SEQ ID NO:10.

110 13. The method of claim 1, wherein said sample is a tissue or body fluid sample.

13. The method of claim 1, wherein said sample is a biopsy sample.

14. The method of claim 1, wherein said sample is a cervical cell sample.

15. The method of claim 1, wherein said sample is a Papanicolaou smear.

16. A method of detecting cervical cancer in a human. the method comprising the steps of:
(a) contacting a sample derived from said human with a binding moiety that binds specifically to a cervical cancer-associated protein to produce a binding moiety-cervical cancer-associated protein complex, wherein said binding moiety is characterized as being capable of binding specifically to a protein comprising the amino acid sequence set forth in SEQ ID NO:10; and (b) detecting the presence of said complex, which if present is indicative of the presence of cervical cancer in said human.

17. The method of claim 16, wherein said cervical cancer-associated protein is further characterized as being detectable at a higher level in a human cervical cancer cell than in a normal human cervical cell. as determined by two dimensional gel electrophoresis.

18. The method of claim 16, wherein said sample is a tissue or body fluid sample.

19. The method of claim 16, wherein said sample is a biopsy sample.

20. The method of claim 16, wherein said sample is a Papanicolaou smear.

21. The method of claim 16, wherein said sample is a cervical cell sample.

22. The method of claim 16, wherein said binding moiety is selected from the group consisting of an antibody, an antibody fragment, and a biosynthetic antibody binding site.

23. The method of claim 16, wherein said binding moiety is an antibody.

24. The method of claim 23, wherein said antibody is a monoclonal antibody.

25. The method of claim 23, wherein said antibody is labelled with a detectable moiety.

26. The method of claim 24, wherein said monoclonal antibody is labelled with a detectable moiety.

27. A method of detecting cervical cancer in a human, comprising:
detecting the presence of a nucleic acid molecule in a tissue or body fluid sample of the human thereby to indicate the presence of a cervical cancer in a human, the nucleic acid molecule comprising a nucleotide sequence encoding a cervical-cancer associated protein comprising an amino acid 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:9, and SEQ ID
NO:10.

28. The method of claim 27, comprising the additional step of reacting the sample with a labelled hybridization probe capable of hybridizing specifically to at least a portion of the nucleic acid molecule.

29. The method of claim 27, wherein the nucleic acid molecule is capable of binding specifically to a protein associated with metastasized cervical cancer, and wherein the presence of the nucleic acid molecule is indicative of the presence of metastasized cervical cancer.

30. A kit for detecting the presence of cervical cancer or for evaluating the efficacy of a therapeutic treatment of a cervical cancer, the kit comprising in combination:
(a) a receptacle for receiving a human tissue or body fluid sample; and (b) a binding moiety capable of binding specifically to a cervical cancer-associated protein comprising an amino acid 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:9, and SEQ ID NO:10.

31. The kit of claim 30, further comprising a reference sample indicative of a normal cervical cell.

32. The kit of claim 30, wherein said binding moiety is selected from the group consisting of an antibody, an antibody fragment and a biosynthetic antibody binding site.

33. The method of claim 27, wherein the nucleic acid molecule is a ribonucleic acid.

34. A method for detecting cervical cancer in a human, the method comprising:
detecting, in a tissue or body fluid sample from the human, a ribonucleic acid molecule that encodes a protein comprising the amino acid sequence set forth in SEQ ID NO:10, wherein the ribonucleic acid molecule, if present in the sample, is indicative of the presence of cervical cancer in the human.

35. A method for detecting cervical cancer in a human, the method comprising:
(a) contacting a tissue or body fluid sample from the human with a binding moiety selected from the group consisting of a nucleic acid and a peptide nucleic acid that binds to a target nucleic acid indicative of cervical cancer, if present in the tissue or body fluid sample, to produce a complex comprising the binding moiety and the target nucleic acid, wherein the binding moiety binds specifically to a nucleic acid encoding a protein having an amino acid sequence set forth in SEQ ID NO:10; and (b) detecting the complex, which if present in the sample is indicative of cervical cancer in the human.

36. The method of claim 35, wherein the binding moiety comprises a detectable label.

37. The method of claim 36, wherein the detectable label comprises a radioisotope.

38. The method of claim 36, wherein the detectable label comprises a fluorescent compound.

39. The method of claim 36, wherein the detectable label comprises an enzyme.

40. The method of claim 35, wherein the binding moiety is a nucleic acid from 8 to 100 nucleotides in length.

41. The method of claim 35, wherein the binding moiety is a nucleic acid from 15 to 50 nucleotides in length.

42. The method of claim 35, wherein the method comprises the additional step of performing a polymerase chain reaction to amplify the target nucleic acid.