WO2001051660A2

WO2001051660A2 - Method and kit for diagnosis of neoplastic transformation

Info

Publication number: WO2001051660A2
Application number: PCT/IB2001/000088
Authority: WO
Inventors: Linda A. Kowalski
Original assignee: Vera Genics Ltd.
Priority date: 2000-01-14
Filing date: 2001-01-12
Publication date: 2001-07-19
Also published as: WO2001051660A3; AU2543301A

Abstract

This invention discloses a method and assay kit for the diagnosis of neoplastic transformation in a cell sample, tissue sample, or bodily fluid sample of a patient. Specifically, the detection of cyclooxygenase-1 (COX-1) and/or cyclooxygenase-2 (COX-2) expression and/or biological activity, particularly in conjunction with the detection of the presence of an oncogenic virus, is used as an indicator of neoplastic transformation or a potential for neoplastic transformation in a sample. The invention also includes a method for evaluating the carcinogenicity of a compound by evaluating COX-1 and/or COX-2 expression and/or biological activity in a test cell before and after contact with a putative carcinogen. Diagnostic assay kits suitable for use with the present methods are also disclosed.

Description

METHOD AND KIT FOR DIAGNOSIS OF NEOPLASTIC TRANSFORMATION

Field of the Invention The present invention relates to a method and assay kit for the diagnosis of neoplastic transformation in a cell sample, tissue sample, or bodily fluid sample of a patient. Specifically, the present invention relates to the detection of cyclooxygenase-1 (COX-1) and/or cyclooxygenase-2 (COX-2), particularly in conjunction with the detection of the presence of an oncogenic virus, as an indicator of neoplastic transformation or a potential for neoplastic transformation.

Background of the Invention Neoplasia, or a process of rapid cellular proliferation resulting in new, abnormal growth, is a characteristic of many diseases which can be serious, and sometimes, life- threatening. Typically, neoplastic growth of cells and tissues is characterized by greater than normal proliferation of cells, wherein the cells continue to grow even after the instigating factor (e.g., tumor promoter, carcinogen, virus) is no longer present. The cellular growth tends to show a lack of structural organization and/or coordination with the normal tissue and usually creates a mass of tissue (e.g. , a tumor) which may be benign or malignant. Malignant cellular growth, or malignant tumors, are a leading cause of death worldwide, and the development of effective therapy for neoplastic disease is the subject of a large body of research. The ability for early diagnosis of neoplastic growth, and more preferably, for the diagnosis of a potential for development of neoplastic growth, would allow for early treatment of the disease, resulting in higher or prolonged survival rates for neoplastic disease. Therefore, there is a need in the art for the development of diagnostic assays which can identify patients at risk of developing, or in the early stages of developing, neoplastic disease.

Although neoplastic disease can be the result of a variety of environmental, hereditary and apparently spontaneously arising factors, several malignant neoplasias are associated with viral infection. The first tumor-producing virus was discovered in 1908 by Ellerman and Bang, who demonstrated that seemingly spontaneous leukemias of chickens could be transmitted to other chickens by cell-free filtrates. In 1911, Rous found that a chicken sarcoma, a solid tumor, can be similarly transmitted. The viruses responsible for these malignancies were identified as retroviruses. In 1932 and 1934, DNA-containing viruses were shown to produce a cutaneous fibroma and a papilloma of wild rabbits (Shope, 1932) and the renal adenocarcinoma of the frog (Lucke, 1934), respectively. Since these early studies, it has been demonstrated that several DNA and RNA viruses can have oncogenic activity in various mammalian species, including humans. Such viruses include polyoma virus, simian virus 40 (SN40), adenoviruses, papilloma viruses and herpesviruses. Many of these viruses directly or indirectly induce neoplastic transformation via viral oncogenes, some of which are highly homologous to cellular protooncogenes. Unfortunately, the viral etiology of a cancer can easily go unrecognized for several reasons, including: (1) the cancer can be caused as a rare effect by ubiquitous viruses, which can often be considered to be innocuous bystanders; (2) some oncogenic viruses have heterogeneous viral particles and infect cells without inducing cancer; (3) the disease may not overtly develop until long after viral infection; and, (4) the cancers may not seem related to a contagious factor because the method of transmission of the virus is not apparent (e.g., through an embryo or milk). Since diagnosis of infection by an oncogenic virus may be an insufficient predictor of potential neoplastic transformation, and since diagnosis of overt malignant neoplasia may be too late for many patients, there remains a need in the art for a method for diagnosing virus- associated neoplastic transformation, or a potential therefor.

The cyclooxygenase 1 and 2 genes (COX- 1 and -2, respectively) code for membrane- bound proteins which convert arachidonic acid (or other lipids) to prostaglandins (e.g., PGF₂, PGE₂, PGD₂), prostacyclin and thromboxane A**., which are compounds involved in inflammation, pain, fever and blood clotting. COX-1 is present in most cell types at housekeeping levels and is not further induced by xenobiotics. COX-2 is virtually undetectable in normal tissues except for brain, testes, tracheal epithelia, kidney macula densa and the pregnant uterus, where it is expressed constitutively (Dubois, RΝ, Abramson, SB, Crofford, L, Gupta, RA, Simon, LS, Nan de Putte, LBA, Lipsky, PF (1998) Cyclooxygenase in biology and disease. FASEBJ12: 1063-1073). hi tissue in which it is not normally expressed, COX-2 is induced by tumor promoters, growth factors, cytokines, hormones, bacterial endotoxins and carcinogens (Smith, WL, Garavito, MR, DeWitt, DL (1996) ProstaglandinEndoperoxideH Synthases (Cyclooxygenases)-l and-2.J. Biol. Chem. 271: 33157-33160). Up-regulation of COX-2 has been reported for many spontaneous and hereditary neoplastic conditions. For example, human gastric (Ristimaki, A, Honkanen, Ν, Jankala, H, Sipponen, P, Harkonen, M (1997) Expression of cyclooxygenase-2 in human gastric carcinoma. Cancer Res., 57: 1296-80), breast (Parrett, ML, Harris, RE, Joarder, FS, Ross, MS, Clausen, KP, Robertson, FM (1997) Cyclooxygenase-2 gene expression in human breast cancer. Int. J. Oncol. 10: 503-7), colorectal (Eberhart, CF, Coffey, RJ, Radhika, A, Giaediello, FM, Ferrenbach, S. et al (1994) Upregulation of cyclooxygenase-2 gene expression in human colorectal adenomas and adenocarcinomas. Gastroenterology 107: 1183-88), and esophageal tumors (Zimmerman, KC, Sarbia, M, Weber, AA, Borchard, F, Gabbert, HE, Schror, K. (1999) Cyclooxygenase-2 expression in human esophageal carcinoma. Cancer Res. 59: 198-204) express higher levels of COX-2. Increases in COX-2 have also been observed in chemical carcinogenesis caused by benzo[a]pyrene (Kelley, D J, Mestre, JR, Subbaramaiah, K, Sacks, PG, Schantz, SP, Tanabe, T, ioue, H, Ramonetti, JT, Dannenberg, AJ (1997) Benz[a]pyrene up-regulates cyclooxygenase-2 gene expression in oral epithelial cells. Carcinog. 18: 795-799); 2,3,7,8 tetrachlorodibenzo-jo-dioxin (Wolfle, D, Marotzki, S., Dartsch, D., Schafer, W, Marquardt, H. (2000) Induction of cyclooxygenase expression and enhancement of malignant cell transformation by 2,3,7,8-tetrachlorodibenzo p-dioxin. Carcinog. 21: 15-21), and 4-(methylnitrosoamine)-4-(3- pyridyl)-l-butanone (El-Bayoumy, K, Iatropoulos, M, Amin, S, Hoffman, D, Wynder, EL, (1999) Increased expression of cyclooxygenase-2 in rat lung tumors induced by the tobacco-specific nitrosamine 4-(methylnitrosamine)-4-(3-pyridyl)-l-butanone: The impact of a high fat diet. Cancer Res. 59: 1400-1403). However, prior to the present invention, however, it is believed that COX-2 has not been associated with viral carcinogenesis.

Most studies of neoplastic conditions have not indicated any upregulation of COX-1 at all, although Bauer et al. reported upregulation of both COX-1 and COX-2 in mouse lung tumors (Bauer, A.K., et al (2000) Carcinogenesis 21:543-550). As noted earlier, COX-1 is normally maintained at a constant "housekeeping" level and is not further induced by xenobiotics, hormones or molecules which can induce COX-2 expression. Indeed, most authors report upregulation of COX-2 but not of COX-1. Upregulation of COX-2, but a control level or undetectable level of COX-1, was reported for the following neoplastic conditions, as examples: transformed mouse mammary epithelial cells (Subbaramaiah, et al (1996) Cancer Research 56:4424-4429), human colorectal tumor cells (Kawahito, S.H., et al (1995) Cancer Research 55:3785-3789 and Eberhart, C.E. et al (1994) Gastroenterology 107: 1183-1188), human hepatocellular carcinoma (Sakisaka,K.H., etal (1999), Hepatology 29: 688-696), laryngeal papilloma (Robinson, A.B. et al (1999) Laryngoscope 109: 1137- 1141). Since the filing of the priority applications for this invention (U.S. Provisional Application Serial No. 60/176,147, filed January 14, 2000, and U.S. Application Serial No. 09/502,355, filed Feb. 11, 2000, both of which are incorporated herein by reference in their entireties) upregulation of COX-2, but not COX-1, has been reported in metastatic cervical tumors (Ryu, H. et al (Published electronically Feb 22, 2000, published in journal March, 2000), Gynecologic Oncology 76: 320-325).

Therefore, it is not believed that COX-1 has been proposed as a potential target for therapeutic intervention or diagnostic assays in neoplastic conditions. The identification of COX-2 upregulation has led many researchers to focus on COX-2 as a target for therapeutic intervention in neoplastic conditions (see for review: Levy, GN (1997) Prostaglandin H synthases, nonsteroidal anti-inflammatory drugs and colon cancer. FASEB J. 11: 235-247). For example, U.S. Patent No. 5,972,986 discloses the use of COX-2 inhibitors in the treatment and prevention of neoplasia. U.S. PatentNo. 5,543,297 discloses the human COX- 2 cDNA and its use in assays which measure COX-2 activity for the purpose of identifying COX-2 inhibitors, where such inhibitors could be used as an antiinflammatory, antipyretic, analgesic, and/or anti-cancer agents, as well as agents to inhibit hormone-induced uterine contractions. The use of Cox-2 as a prognostic marker (i.e., as a predictor of the probable course or outcome of neoplastic disease) has also been investigated (Achiwa et al., 1999, Clin Cancer Res 5(5):1001-1005). Such use of Cox-2 was made after the diagnosis of neoplastic disease had been established, and the study generally found no relationship between Cox-2 expression and clinical outcomes overall, although a correlation between Cox-2 expression and shortened survival in patients having early stage disease was observed. Kondo et al. also investigated whether Cox-2 expression in patients with hepatocellular carcinomas (HCC) could be prognostic. However, Kondo et al. found no correlation between Cox-2 in HCC and prognosis. It was determined that Cox-2 expression in non-tumor tissue in patients with hepatocellular carcinomas HCC could be correlated with the presence of active inflammation and a shorter disease-free survival rate in the patients, suggesting that Cox-2 in non-tumor tissue could be playing a role in relapse of HCC after surgery (Kondo et al., 1999, Clin Cancer Res 5(12):4005-4012). Despite the extensive investigation of COX-2 as a target for therapeutic intervention in neoplastic and inflammatory disease, and the few investigations into the use of Cox-2 expression as a prognostic marker for neoplastic disease, to the present inventor ' s knowledge, neither Cox-1 nor Cox-2 have, prior to the present invention, been used as a diagnostic marker for neoplastic disease. Moreover, prior to the present invention, it was not known that an increase in Cox-1 and/or Cox-2 expression could be correlated with viral carcinogenesis (i.e., neoplastic transformation as a result of viral infection).

Summary of the Invention The present invention generally relates to a method for diagnosing neoplastic transformation or a potential for neoplastic transformation in a patient. The method includes the steps of: (a) obtaining from a patient a sample selected from the group consisting of a cell sample, a tissue sample, and a bodily fluid sample; (b) establishing a baseline level of expression or biological activity of at least one cyclooxygenase for the sample, wherein the cyclooxygenase is cyclooxygenase-1 (COX-1) and or cyclooxygenase-2 (COX-2); (c) detecting expression or biological activity of the cyclooxygenase in the sample; (d) comparing the expression or biological activity of the cyclooxygenase as determined in step (c) to the baseline level of expression or biological activity of the cyclooxygenase established in step (b); and, (e) making a diagnosis of the patient. Detection of increased cyclooxygenase expression or biological activity as compared to the baseline level of cyclooxygenase expression or biological activity, indicates a positive diagnosis of neoplastic transformation or a potential therefor in the patient. A bodily fluid can include, but is not limited to, mucous, seminal fluid, saliva, breast milk, bile and urine. In one embodiment, the sample is from a tissue selected from the group consisting of cervix, uterus and prostate, and a positive diagnosis is indicative of cervical cancer, uterine cancer or prostate cancer, respectively.

Detection of cyclooxygenase expression can include, but are not limited to, detecting Cox-1 and/or Cox-2 mRNA transcription, detecting COX-1 and/or COX-2 translation, detecting COX-1 and/or COX-2 biological activity, and/or detecting production of a COX-1 and/or COX-2 biochemical endproduct. Methods of detecting Cox-1 and/or Cox-2 transcription include, but are not limited to, reverse transcriptase-PCR (RT-PCR), in situ hybridization, Northern blot, sequence analysis and/or detection of a reporter gene. Methods of detecting COX-1 and/or COX-2 translation include, but are not limited to, immunoblot, enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA), and/or immunoprecipitation. Methods of detecting COX-1 and/or COX-2 biological activity include, but are not limited to, an assay for detection of prostaglandin E2 (PGE2) activity and/or an assay for detection of 15-R-hydroxyeicosatetraenoic acid (15-R-HETE) activity. COX- 1 and/or COX-2 biological endproducts that can be detected include, but are not limited to, prostaglandin, prostacyclin and/or thromboxane A₂.

In one embodiment, detection of at least about a 1.5-fold increase in cyclooxygenase expression or biological activity in the sample as compared to the baseline level, indicates a positive diagnosis for the sample. In another embodiment, detection of at least about a 3- fold increase in cyclooxygenase expression or biological activity in the sample as compared to the baseline level, indicates a positive diagnosis for the sample.

The baseline level of cyclooxygenase expression or biological activity can be established by a method which includes, but is not limited to: (1) establishing a baseline level of cyclooxygenase expression or biological activity in an autologous control sample from the patient, wherein the autologous sample is of a same cell type, tissue type or bodily fluid type as the sample of step (a); (2) establishing a baseline level of cyclooxygenase expression or biological activity that is an average from at least two previous detections of cyclooxygenase expression or biological activity in a previous sample from the patient, wherein each of the previous samples were of a same cell type, tissue type or bodily fluid type as the sample of step (a), and wherein the previous evaluations resulted in a negative diagnosis; and, (3) establishing a baseline level of cyclooxygenase expression or biological activity from control samples of a same cell type, tissue type or bodily fluid type as the sample of step (a), the control samples having been obtained from a population of matched individuals.

In one embodiment of the present invention, the method can include, after the step (a) of obtaining and prior to step (e) of making a diagnosis, a step of detecting whether the sample carries an oncogenic RNA or DNA virus. In this embodiment, detection of the oncogenic RNA or DNA virus, in combination with detection of increased cyclooxygenase (i.e., COX-1 and/or COX-2) expression or biological activity as compared to the baseline level of cyclooxygenase expression or biological activity, indicates a positive diagnosis of virus-associated neoplastic transformation or a potential therefor. Oncogenic DNA viruses that can be detected by the present method include, but are not limited to, Papovaviruses, Herpesviruses, Hepadnaviruses andPoxviruses; and more preferably, human papilloma virus, polyoma virus and Simian virus 40 (SN40); and most preferably, human papilloma virus. Oncogenic RΝA viruses that can be detected by the method of the present invention include, but are not limited to, human lymphotropic virus and human immunodeficiency virus. The step of detecting whether the sample carries an oncogenic RΝA or DΝA virus can be performed by a method which includes, but is not limited to, PCR, RT-PCR, Northern blot, Southern blot, sequence analysis, and/or in situ hybridization. Other methods for detecting whether the sample carries an oncogenic RNA or DNA virus include, but are not limited to immunoblot, enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA), and/or immunoprecipitation.

Another embodiment of the present invention relates to a method for diagnosing neoplastic transformation or a potential for neoplastic transformation associated with an oncogenic virus. The method includes the steps of: (a) obtaining from a patient, who has been identified as having been infected with an oncogenic virus, a sample to be evaluated for neoplastic transformation, the sample being selected from the group of a cell sample, a tissue sample, and a bodily fluid sample; and, (b) detecting the expression or biological activity of at least one cyclooxygenase in the sample. The cyclooxygenase is COX-1 and or COX-2. Detection of increased cyclooxygenase expression or biological activity over a baseline cyclooxygenase expression or biological activity indicates a positive diagnosis of virus- associated neoplastic transformation. Specific aspects of this embodiment of the invention are as set forth above.

Yet another embodiment of the present invention relates to a method for diagnosing neoplastic transformation or a potential for neoplastic transformation associated with an oncogenic virus. The method includes the steps of: (a) obtaining from a patient a sample selected from the group of a cell sample, a tissue sample, and a bodily fluid sample; (b) detecting whether the sample carries an oncogenic RNA or DNA virus; and, (c) detecting the expression or biological activity of at least one cyclooxygenase in the sample. The cyclooxygenase is COX-1 and/or COX-2. Detection of: (1) the oncogenic virus in the sample, and (2) increased cyclooxygenase expression or biological activity, over a baseline cyclooxygenase expression or biological activity, indicates a positive diagnosis of virus- associated neoplastic transformation or a potential therefor. Specific aspects of this embodiment of the invention are as set forth above.

Another embodiment of the present invention relates to a method for diagnosing cervical, uterine or prostate cancer or a potential for development of cervical, uterine or prostate cancer. This method includes the steps of: (a) obtaining from a patient a sample from a tissue selected from the group consisting of cervix, uterus and prostate tissue to be evaluated for neoplastic transformation, the sample selected from the group consisting of a cell sample, a tissue sample, and a bodily fluid sample; and, (b) detecting the expression or biological activity of at least one cyclooxygenase in the sample. The cyclooxygenase is COX-1 and/or COX-2. Detection of increased cyclooxygenase expression or biological activity over a baseline level of cyclooxygenase expression or biological activity the cervix, uterine or prostate sample indicates a positive diagnosis of cervical, uterine or prostate cancer, respectively, or a potential for development of cervical, uterine or prostate cancer, respectively, h a preferred embodiment, the method further comprises a step of detecting whether the sample carries papilloma virus. Specific aspects of this embodiment of the invention are as set forth above.

Yet another embodiment of the present invention relates to an assay kit for diagnosing neoplastic transformation or a potential for neoplastic transformation in a patient. The assay kit includes: (a) a means for detecting the presence of an oncogenic virus in a sample obtained from a patient, wherein the sample is selected from the group of a cell sample, a tissue sample and a bodily fluid sample; and, (b) a means for detecting the expression or biological activity of at least one cyclooxygenase in the sample. The cyclooxygenase is COX-1 and/or COX-2. In one aspect of this embodiment, the assay kit can be configured to test a bodily fluid selected from the group of mucous, seminal fluid, saliva, breast milk, bile and urine. In one aspect of this embodiment of the present invention, the means for detecting of part (a) and/or part (b) can be conjugated to a detectable marker. In another aspect of this embodiment of the present invention, the means for detecting of part (a) and/or part (b) can be immobilized on a substrate. Specific aspects of this embodiment of the invention are as set forth above. this embodiment of the present invention, the means for detecting the presence of an oncogenic virus can include, but is not limited to: a probe that hybridizes under stringent hybridization conditions to a nucleic acid molecule from the virus; PCR primers which amplify a nucleic acid molecule from the virus; an antibody that selectively binds to an oncogenic viral protein in the sample and/or a viral antigen that can be bound by anti-virus antibodies in the sample. The means for detecting cyclooxygenase expression or biological activity can include, but is not limited to: a probe that hybridizes under stringent hybridization conditions to a nucleic acid molecule encoding the cyclooxygenase or a fragment thereof; RT-PCR primers for amplification of mRNA encoding the cyclooxygenase or a fragment thereof; an antibody that selectively binds to the cyclooxygenase; a probe that hybridizes under stringent hybridization conditions to a nucleic acid molecule encoding a cyclooxygenase biochemical endproduct or a fragment thereof; RT-PCR primers for amplification of mRNA encoding a cyclooxygenase biochemical endproduct or a fragment thereof; and/or an antibody that selectively binds to a cyclooxygenase biochemical endproduct. The cyclooxygenase biological endproduct can include, but is not limited to a prostaglandin, prostacyclin and thromboxane A₂. Specific aspects of this embodiment of the invention are as set forth above.

Another embodiment of the present invention relates to a method for evaluating the carcinogenicity of a compound. The method includes the steps of: (a) contacting with the compound being tested for carcinogenicity a test cell; and, (b) detecting the expression or biological activity of at least one cyclooxygenase by the test cell, wherein an increase in cyclooxygenase expression in the test cell after contact with the compound as compared to before contact with the compound indicates that the compound is carcinogenic. The cyclooxygenase is COX-1 and/or COX-2. Preferably, the test cell is mammalian, including, but not limited to, mouse C127 cells, C3Wl0TYz cells, human newborn foreskin keratinocytes and primary fibroblast cultures of C57BL/6J mice. Methods for detection of cyclooxygenase expression or biological activity can include, but are not limited to those described above.

In one aspect of this method, the test cell is transfected with a recombinant nucleic acid molecule from a DNA or RNA virus encoding a transforming protein, wherein the test cell maintains a copy number of the recombinant nucleic acid molecule of within at least about 50% over 5 passages of the test cell. In a preferred embodiment, the recombinant nucleic acid molecule is from a papilloma virus, and more preferably, from a bovine papilloma virus (BPN). In even more preferred embodiments, the recombinant nucleic acid molecule is from the group consisting of the BPN E5, E6 and E7 open reading frames; is selected from the group of the BPN E6 open reading frame and the BPN E7 open reading frame; or is a BPN genome in which a viral gene selected from the El and E2 open reading frames has been removed or made non-functional.

Detailed Description of the Invention The present invention generally relates to a method and kit for diagnosing neoplastic transformation, or a potential for neoplastic transformation, in a patient. In one embodiment, the present invention relates to a method and kit for diagnosing neoplastic transformation associated with infection by an oncogenic virus, or a potential for such neoplastic transformation. In general, the method includes the steps of: (a) obtaining from a patient a sample selected from the group consisting of a cell sample, a tissue sample, and a bodily fluid sample; (b) establishing a baseline level of expression or biological activity of at least one cyclooxygenase for the sample, the cyclooxygenase being COX-1 and/or COX-2; (c) detecting expression or biological activity of the cyclooxygenase in the sample; (d) comparing the expression or biological activity of the cyclooxygenase as determined in step (c) to the baseline level of cyclooxygenase expression or biological activity established in step (b); and, (e) making a diagnosis of the patient. In the general method, detection of increased cyclooxygenase expression or biological activity as compared to the baseline level of cyclooxygenase expression or biological activity, indicates a positive diagnosis of neoplastic transformation or a potential therefor in the patient. According to the present invention, the phrase "neoplastic transformation" refers to a change of a cell or population of cells from a normal to malignant state, involving cellular proliferation at a rate which is more rapid than normal and which is typically characterized by one or more of the following traits : continued growth even after the instigating factor (e.g., xenobiotic, virus) is no longer present; a lack of structural organization and/or coordination with normal tissue, and typically, a formation of a mass of tissue, or tumor. A neoplasia, therefore, is a proliferation of cells (e.g., a tumor, growth, polyp) resulting from neoplastic growth and includes both benign and malignant tumors. In the case of neoplastic transformation, a neoplasia is malignant or is predisposed to become malignant. Malignant tumors are typically characterized as being anaplastic (primitive cellular growth characterized by a lack of differentiation), invasive (moves into and destroys surrounding tissues) and/or metastatic (spreads to other parts of the body). As used herein, reference to a "potential for neoplastic transformation" refers to an expectation or likelihood that, at some point in the future, a cell or population of cells will display characteristics of neoplastic transformation, including rapid cellular proliferation characterized by anaplastic, invasive and metastatic growth. In the present invention, the expectation or likelihood of neoplastic transformation is determined based on a positive diagnosis of increased cyclooxygenase expression or biological activity, particularly in the presence of an oncogenic virus, as discussed in detail below.

The terms "diagnosis", "diagnosing" and variants thereof refer to the identification of a disease or condition on the basis of its signs and symptoms. As used herein, a "positive diagnosis" indicates that the disease or condition, or a potential for developing the disease or condition, has been identified, hi contrast, a "negative diagnosis" indicates that the disease or condition, or a potential for developing the disease or condition, has not been identified. Therefore, in the present invention, a positive diagnosis of neoplastic transformation or a potential therefor, means that the indicators (e.g., signs, symptoms) of neoplastic transformation or a likelihood of developing neoplastic transformation (described in detail below) have been identified in the sample obtained from the patient. Such a patient can then be prescribed treatment to reduce or eliminate the neoplastic transformation. A negative diagnosis of neoplastic transformation or a potential therefor means that the indicators of neoplastic transformation or a likelihood of developing neoplastic transformation described herein have not been identified in the sample obtained from the patient, h this instance, the patient is typically not prescribed any treatment, but may be reevaluated at one or more timepoints in the future for neoplastic transformation.

According to the present invention, the method for diagnosing neoplastic transformation is suitable for use in a patient that is a member of the Vertebrate class, Mammalia, including, without limitation, primates, livestock and domestic pets (e.g., a companion animal). Most typically, a patient will be a human patient or a companion animal (e.g., dog or cat).

The method of the present invention is useful for diagnosing neoplastic transformation, or the potential therefor, which is symptomatic of a variety of cancers including, but not limited to: prostate cancer, cervical cancer, uterine cancer, brain cancer, bone cancer, epithelial cell-derived neoplasia (epithelial carcinoma, e.g., basal cell carcinoma, adenocarcinoma, gastrointestinal cancer (e.g., lip cancer, mouth cancer, esophageal cancer, small bowel cancer and stomach cancer), colon cancer, liver cancer, bladder cancer, pancreas cancer, ovary cancer, lung cancer, breast cancer and skin cancer (e.g., squamous cell and basal cell cancer), renal cell carcinoma, and any cancers that effect epithelial cells throughout the body. The method is particularly effective for diagnosis of neoplastic transformation or a potential therefor associated with cancers that are induced or associated with infection by oncogenic viruses. Such cancers include, but are not limited to prostate cancer, cervical cancer and uterine cancer. According to the present invention, an oncogenic virus can include any DNA or RNA virus that can directly or indirectly (i.e., through events associated with viral infection) cause cells to undergo uncontrolled proliferation resulting in neoplastic transformation. Oncogenic DNA viruses include, but are not limited to Papovaviruses, Herpesviruses, Hepadnaviruses, and Poxviruses. Oncogenic DNA viruses which infect human cells include, but are not limited to human papilloma virus, polyoma virus and Simian virus 40 (SN40). Oncogenic RΝA viruses include, but are not limited to, human lymphotropic virus and human immunodeficiency virus. The method and assay kit of the present invention are particularly useful for diagnosing neoplastic transformation associated with infection by oncogenic DΝA viruses, and most particularly, human papilloma virus. Cancers that are known to be associated with Papovaviruses include, but are not limited to, cervical cancer (papilloma virus), uterine cancer (papilloma virus) and prostate cancer (papilloma virus); cancers that are known to be associated with Herpesviruses include, but are not limited to, nasopharyngeal carcinoma and Burkitt's lymphoma; a cancer that is known to be associated with Hepadnaviruses include, but is not limited to, hepatocellular carcinoma; and cancers that are known to be associated with oncogenic RΝA viruses include, but are not limited to, human immunodeficiency virus-associated cancers (Kaposi's sarcoma, Hodgkin's Disease, non-Hodgkin's lymphoma) and human T-cell leukemia. The method of the present invention is useful for diagnosing any of the above-identified cancers, although the invention is not limited to the diagnosis of these cancers.

The first step of the method of the present invention involves obtaining a sample from a patient. The sample can be a cell sample, a tissue sample and/or a bodily fluid sample. According to the present invention, a cell sample is a specimen of cells, typically in suspension or separated from connective tissue which may have connected the cells within a tissue in vivo, which have been collected from an organ, tissue or fluid by any suitable method which results in the collection of a suitable number of cells for evaluation by at least one of the methods of the present invention. Cells can be obtained, for example, by scraping of a tissue, processing of a tissue sample to release individual cells, or isolation from a bodily fluid. A tissue sample, although similar to a cell sample, is defined herein as a section of an organ or tissue of the body which typically includes several cell types and or cytoskeletal structure which holds the cells together. One of skill in the art will appreciate that the term "tissue sample" may be used, in some instances, interchangeably with a "cell sample", although it is preferably used to designate a more complex structure than a cell sample. A tissue sample can be obtained by a biopsy, for example, including by cutting, slicing, or a punch. A bodily fluid sample is a fluid excreted or secreted by a tissue or organ to be evaluated. A bodily fluid is obtained by any method suitable for the particular bodily fluid to be sampled. Bodily fluids suitable for sampling include, but are not limited to, mucous, seminal fluid, saliva, breast milk, bile and urine.

In general, the sample type (i.e., cell, tissue or bodily fluid) is selected based on the accessibility and structure of the organ or tissue to be evaluated for neoplastic transformation and/or on whether the evaluation is for a particular type of cancer. For example, if the organ tissue to be evaluated is the cervix, the sample can be a sample of epithelial cells scraped from the cervix (i.e., a cell sample), a cervical tissue biopsy (a tissue sample), or a sample of cervical mucous (i.e., a bodily fluid). If the organ/tissue to be tested is the prostate, the most preferable sample is likely to be a urine or seminal fluid sample, although if necessary, cell or tissue samples may be obtained through more invasive procedures in the clinic or hospital. Additionally, the sample type to be evaluated can be selected based on whether the sampling is done in a medical clinic or hospital, or in the home of the patient to be evaluated. For example, in some embodiments of the present invention, the patient to be evaluated may obtain (e.g., collect) a sample of bodily fluid, such as urine, mucous or seminal fluid to be used in a home assay kit for neoplastic transformation.

The second step of the method of the present invention is establishing a baseline level of cyclooxygenase expression or biological activity for the sample type to be evaluated. As used herein, the terms "cyclooxygenase expression", "COX-1 expression", and/or "COX-2 expression" can refer to transcription of cyclooxygenase, Cox-1, and/or Cox-2 mRNA, respectively, or to the translation of cyclooxygenase, COX-1 and/or COX-2 protein, respectively. The term, "cyclooxygenase biological activity", "COX-1 biological activity" or "COX-2 biological activity" refers to any biological action of the referenced cyclooxygenase protein, including, but not limited to, catalyzing the synthesis of prostaglandin (e.g., prostaglandin E2), prostacyclin and/or thromboxane A₂. As used herein, the term "cyclooxygenase" can be used to generically refer to COX-1 and/or COX-2. According to the present invention, a "baseline level" is a control, or normal, level of cyclooxygenase expression or activity against which a level of cyclooxygenase expression or biological activity to be evaluated for correlation with neoplastic transformation can be compared. Therefore, it can be determined, based on the control or baseline level of cyclooxygenase expression or biological activity, whether a sample to be evaluated for neoplastic transformation has a measurable increase, decrease, or no change in cyclooxygenase expression or biological activity. Methods for detecting cyclooxygenase expression or biological activity are described in detail below.

The method for establishing a baseline level of cyclooxygenase expression or activity is selected based on the sample type, the tissue or organ from which the sample is obtained, and the status of the patient to be evaluated. In one embodiment, the baseline level of cyclooxygenase expression or biological activity is established in an autologous control sample obtained from the patient. The autologous control sample can be a cell sample, a tissue sample or a bodily fluid sample, and is preferably a cell sample or tissue sample. According to the present invention, and as used in the art, the term "autologous" means that the sample is obtained from the same patient from which the sample to be evaluated is obtained. Preferably, the control sample is obtained from the same organ or tissue as the sample to be evaluated, such that the control sample serves as the best possible baseline for the sample to be evaluated. In this embodiment, it is desirable to take the control sample from a population of cells or a tissue which is believed to represent a "normal" cell or tissue, or at a minimum, a cell or tissue which is least likely to be undergoing or potentially be predisposed to develop neoplastic transformation. For example, if the sample to be evaluated is an area of apparently abnormal cell growth, such as a tumorous mass, the control sample is preferably obtained from a section of apparently normal tissue (i.e., an area other than and preferably a reasonable distance from the tumorous mass) in the tissue or organ where the tumorous mass is growing. For example, if a mass is to be evaluated in the uterus, the sample to be evaluated would be obtained from the mass and the control sample would be obtained from a different section of the uterus which is separate from the area where the mass is located and which does not show signs of uncontrolled cellular proliferation. In this embodiment, the sample type can be any type, although an autologous control sample is most readily obtained if the sample type is a cell or tissue. It will be clear to those of skill in the art that some samples to be evaluated will not readily provide an obvious autologous control sample, hi these instances, an alternate method of establishing a baseline level of cyclooxygenase expression or biological activity should be used, examples of which are described below.

A second method for establishing a baseline level of cyclooxygenase expression or biological activity is to establish a baseline level of cyclooxygenase expression or biological activity that is an average from at least two previous detections of cyclooxygenase expression or biological activity in a previous sample from the same patient. Preferably, each of the previous samples were of a same cell type, tissue type or bodily fluid type as the sample to be presently evaluated, and each of the previous evaluations resulted in a negative diagnosis (i.e., no neoplastic transformation, or potential therefor, was identified). Accordingly, a new sample is evaluated periodically (e.g., at annual physicals), and as long as the patient is determined to be negative for neoplastic transformation, an average or other suitable statistically appropriate baseline of the previous samples can be used as a "negative control" for subsequent evaluations. For the first evaluation, an alternate control can be used, as described below, or additional testing may be performed to confirm an initial negative diagnosis, if desired, and the value for cyclooxygenase expression or biological activity can be used thereafter. This type of baseline control is frequently used in other clinical diagnosis procedures where a "normal" level may differ from patient to patient and/or where obtaining an autologous control sample is either not possible or not practical. For example, for a patient who has periodic mammograms, the previous mammograms serve as baseline controls for the mammary tissue of the individual patient. Similarly, for a patient who is regularly screened for prostate cancer by evaluation of levels of prostate cancer antigen (PCA), previous PCA levels are frequently used as a baseline for evaluating whether the individual patient experiences a change.

A third method for establishing a baseline level of cyclooxygenase expression or biological activity is to establish a baseline level of cyclooxygenase expression or biological activity from control samples that were obtained from a population of matched individuals. Preferably, the control samples are of the same sample type as the sample type to be evaluated for neoplastic transformation. According to the present invention, the phrase "matched individuals" refers to a matching of the control individuals on the basis of one or more characteristics which are suitable for the type of neoplastic transformation to be evaluated. For example, control individuals can be matched with the patient to be evaluated on the basis of gender, age, race, or any relevant biological or sociological factor that may affect the baseline of the control individuals and the patient (e.g., preexisting conditions, consumption of particular substances, levels of other biological or physiological factors). For example, levels of cyclooxygenase expression in prostate of a normal individual (i.e., having prostate that is not neoplastically transformed or predisposed to such transformation) may be higher in individuals of a given classification (e.g., elderly vs. teenagers, men vs. women, smokers vs. non-smokers). To establish a control or baseline level of cyclooxygenase expression or biological activity, samples from a number of matched individuals are obtained and evaluated for cyclooxygenase expression or biological activity. The sample type is preferably of the same sample type and obtained from the same organ, tissue or bodily fluid as the sample type to be evaluated in the test patient. The number of matched individuals from whom control samples must be obtained to establish a suitable control level (e.g., a population) can be determined by those of skill in the art, but should be statistically appropriate to establish a suitable baseline for comparison with the patient to be evaluated (i.e., the test patient). The values obtained from the control samples are statistically processed to establish a suitable baseline level using methods standard in the art for establishing such values.

A fourth method of "establishing a baseline" can also be a step of referring to a form of stored information regarding a previously determined baseline level of cyclooxygenase expression, such as a baseline level established by any of the above-described methods . Such a form of stored information can include, for example, but is not limited to, a reference chart, listing or electronic file of population or individual data regarding "normal" or baseline cyclooxygenase expression (such as data that could be established by using the third method above); a medical chart for the patient recording data from previous evaluations; or any other source of data regarding baseline cyclooxygenase expression that is useful for the patient to be diagnosed.

After establishing a baseline level of cyclooxygenase expression or biological activity, the method of the present invention includes a step of detecting cyclooxygenase expression or biological activity in the sample which is obtained from the patient to be evaluated for neoplastic transformation. As discussed above, cyclooxygenase expression can generally refer to Cyclooxygenase mRNA transcription or cyclooxygenase protein translation. Preferably, the method of detecting cyclooxygenase expression or biological activity in the test patient is the same or qualitatively equivalent to the method used for detection of cyclooxygenase expression or biological activity in the sample used to establish the baseline level of cyclooxygenase expression or biological activity.

Methods suitable for detecting transcription of Cox-1 or Cox-2 include any suitable method for detecting and/or measuring mRNA levels from a cell or cell extract. Such methods include, but are not limited to reverse transcriptase PCR (RT-PCR), in situ hybridization, Northern blot, sequence analysis, and detection of a reporter gene. Such methods for detection of transcription levels are well known in the art, and many of such methods, including oligonucleotide primers useful in such methods, are described in detail below as well as in Sambrook et ah, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, 1989 and/or in Glick et al., Molecular Biotechnology: Principles and Applications of Recombinant DNA, ASM Press, 1998; Sambrook et al., ibid., and Glick et al . , ibid, are incorporated by reference herein in their entireties . In addition, U.S. Patent No . 5,543,297, which is incorporated herein by reference in its entirety, describes methods of measuring Cox-2 mRNA, including RT-PCR, Northern blot analysis, sequence analysis. Such methods can be applied to measuring Cox-1. U.S. Patent No. 5,543,297 also provides the nucleotide and amino acid sequence for human COX-2, as well as PCR primers useful in Cox-2 mRNA or cDNA detection methods. The nucleotide and amino acid sequence for COX- 1 and COX-2 in a variety of mammalian species can be found in public databases, such as GenBank in the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov). For example, the nucleotide and amino acid sequence for human COX-1 can be found in GenBank under the Primary Accession No. NM_000962, and the complete nucleotide and amino acid sequence for human COX-2 can be found in GenBank under Primary Accession No. U04636. Measurement of Cox-1 or Cox-2 transcription is most suitable when the sample is a cell or tissue sample, although when the sample is a bodily fluid sample containing cells or cellular extracts, measurement of Cox-1 or Cox-2 transcription can be used.

Cyclooxygenase expression can also be detected by detection of cyclooxygenase translation. Methods suitable for the detection of cyclooxygenase protein include any suitable method for detecting and/or measuring proteins from a cell or cell extract. Such methods include, but are not limited to, immunoblot (e.g., Western blot), enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA) and immunoprecipitation. Such methods are well known in the art, and antibodies against cyclooxygenases, both COX-1 and COX-2, are available, for example, from Cayman Chemical Corporation (Ann Arbor, Michigan). The Examples section demonstrates the detection of COX-1 and COX-2 in a cell sample using Western blot.

Methods to detect cyclooxygenase biological activity are also well known in the art and include assays for the detection of the synthesis or biological activity of biochemical endproducts of cyclooxygenase enzyme activity. Suchbiochemical endproducts include, but are not limited to, prostaglandins (PGE₂, PGF₂, PGD₂), prostacyclin and thromboxane A₂. Cyclooxygenases (i.e., both cyclooxygenase-1 and cyclooxygenase-2) are enzymes which catalyzes the conversion of arachidonic acid to these and other presently unknown biochemical endproducts which are involved in inflammation, pain, fever and blood clotting. Therefore, one can detect the enzyme activity of a cyclooxygenase using assays for the synthesis and/or activity of suchbiochemical endproducts. For example, assays for detection of the activity of prostaglandin E2 (PGE2) or for 15-R-hydroxyeicosatetraenoic acid (15-R- HETE) as an indicator of COX-2 activity are described in Mancini et al., 1994, FEBS Letters 342:33-37. Briefly, protein extracts are prepared from cells and the reaction commenced by the addition of arachidonic acid. After 30 minutes of incubation, samples are extracted using chloroform and the extracts analyzed using reverse-phase HPLC. Identity of reaction products is analyzed using mass specfrometry (15-R-HETE). PGE₂ is analyzed and identified using a radioimmunoassay. h addition, U.S. Patent No. 5,543,297, incorporated herein by reference in its entirety, describes assays for the detection of COX-2 biological activity, including microsomal and whole cell cyclooxygenase assays which use detection of prostaglandin E₂ (PGE₂) as a readout. Direct detection of biochemical endproducts which are indicative of cyclooxygenase biological activity can be accomplished using any of the cyclooxygenase protein detection methods described above and identification tools (e.g., antibodies, primers, probes) specific for the endproduct to be identified. The nucleic acid and amino acid sequences for these biochemical endproducts are known in the art, as are antibodies which specifically bind to such endproducts (See, for example, U.S. Patent No. 5,543,297). In the event that antibodies are not available, or as an alternative, endproducts can be identified using other means, such as mass specfrometry, as described above for 15-R- HETE.

After the level of cyclooxygenase expression or biological activity is detected in the sample to be evaluated for neoplastic transformation, such level is compared to the established baseline level of cyclooxygenase expression or biological activity, determined as described above. Preferably, the method of detecting used for the sample to be evaluated is the same or qualitatively and or quantitatively equivalent to the method of detecting used to establish the baseline level, such that the levels can be directly compared. In comparing the test sample to the baseline confrol, it is determined whether the test sample has a measurable increase in cyclooxygenase expression or biological activity over the baseline level. After comparing the levels of cyclooxygenase expression or biological activity in the samples, the final step of making a diagnosis of said patient can be performed. Detection of an increased level of cyclooxygenase expression or biological activity in the sample to be evaluated (i.e., the test sample) as compared to the baseline level indicates a positive diagnosis of neoplastic fransformation or potential for neoplastic transformation in the patient. As discussed above, a positive diagnosis indicates that neoplastic transformation is has occurred, is occurring, or is statistically likely to occur in the cells or tissue from which the sample was obtained. In order to establish a positive diagnosis, the level of cyclooxygenase activity is increased over the established baseline by an amount that is statistically significant (i.e., with at least a 95% confidence level, or p<0.05). Preferably, detection of at least about a 1.5 fold increase in cyclooxygenase expression or biological activity in the sample as compared to the baseline level results in a positive diagnosis for said sample. More preferably, detection of at least about a 3 fold increase, and more preferably at least about a 6 fold increase, and even more preferably, at least about a 12 fold increase, and even more preferably, at least about a 24 fold increase in cyclooxygenase expression or biological activity as compared to the baseline level, results in a positive diagnosis for said sample. If the level of cyclooxygenase expression or biological activity in the test sample is less than or statistically substantially the same as the baseline level of cyclooxygenase expression or biological activity, then a negative diagnosis is indicated. As discussed above, a negative diagnosis refers to a determination that neoplastic transformation has not occurred in the cells or tissue from which the sample was obtained and that there is no indication that neoplastic transformation is or will occur in such cells as of the time the evaluation is performed. A negative diagnosis may be used in future evaluations to establish a baseline for the patient and/or be used to assist with the establishment of a population control level when combined with results from other patients considered to be normal. A positive diagnosis of neoplastic transformation in a sample obtained from a patient is indicative of the development, or potential for development, of neoplastic fransformation of the cell type, tissue and/or organ from which the sample was obtained. For example, a positive diagnosis in a sample obtained from cervix, uterus or prostate (or associated fluids) is indicative of cervical cancer, uterine cancer or prostate cancer, respectively, in the patient. Once a positive diagnosis is made using the present method, the diagnosis can be substantiated, if desired, using any suitable alternate method of detection of neoplastic transformation, including biopsy and pathology screening. In one embodiment of the present invention, the method can include an additional step of confirming the diagnosis of neoplastic fransformation using such an alternate form of detection of neoplastic transformation such as biopsy and/or pathology/histology. A positive diagnosis of neoplastic transformation in an individual allows for the commencement of appropriate treatment protocols. Since the method of the present invention is useful for the early detection of neoplastic transformation, treatment protocols are expected to be more effective and result in prolonged survival rates.

In another embodiment of the present invention, the method can include an additional step, typically prior to a final step of making a diagnosis, of detecting alternate causes of elevated cyclooxygenase expression or biological activity in the patient. Although such a step is not required by the method of the present invention, such a step is useful to rule out such alternate causes of elevated cyclooxygenase expression or biological activity and assists in the confirmation of a diagnosis of neoplastic transformation in the patient. It is known in the art that increases in cyclooxygenase expression or biological activity as compared to a normal level of cyclooxygenase expression or biological activity can be caused by events which include tumor promoters, growth factors, cytokines and other inflammatory modulators, hormones, bacterial endotoxins and chemical carcinogens. Therefore, an additional step of detecting alternate causes of elevated cyclooxygenase expression or biological activity can include any method of detecting one or more indicators of one or more of such alternate causes of elevated cyclooxygenase expression or biological activity. For example, a medical history and/or other suitable diagnostic tests can be established for the patient, in order to confirm a decreased likelihood that any observed increase in cyclooxygenase expression or biological activity in the sample obtained from the patient is associated with inflammation, pregnancy, hormone imbalance, bacterial infections, and/or exposure to chemical carcinogens or other tumor promoters. Assays useful for the detection of inflammation include, but are not limited to, detection of inflammatory modulators (e.g., cytokines) in bodily fluids and/or tissues of a patient and detection of biological effects associated with inflammation (e.g., increased hypersensitivity reactions, fever, etc.). Pregnancy tests, assays for hormone levels, tests for bacterial infection and methods for the evaluation of potential exposures to various xenobiotics, including carcinogens, are well known in the art.

In one embodiment, the method of the present invention is used to diagnose neoplastic transformation, or a potential therefor, which is associated with infection by an oncogenic virus. As discussed above, the present inventor is believed to be the first to discover a positive correlation between viral carcinogenesis and increased cyclooxygenase expression and biological activity (i.e., COX-1 and/or COX-2), wherein neoplastic transformation occurs in the absence of other cancer inducers such as tumor promoters (e.g., chemical carcinogens, mutagens, and other xenobiotics). According to the present invention, viral carcinogenesis, or oncogenic virus-associated neoplastic transformation, is neoplastic fransformation that is caused either directly or indirectly as a result of an active infection by an oncogenic DNA or RNA virus. The events leading to the neoplastic transformation can be either directly or indirectly caused by the oncogenic virus (e.g., directly caused by viral oncogenes or indirectly by the action of the virus on a cellular protein which, through a cyclooxygenase-associated mechanism, allows the cell to become transformed). Oncogenic DNA and RNA viruses which can be indirectly or directly causative of neoplastic transformation detectable by the method of the present invention are described in detail above.

Although other investigators have noted that COX-2 is elevated in a variety of spontaneous and chemically induced cancers, the present inventor is believed to be the first to discover that COX-2 is elevated as a direct result of viral carcinogenesis (i.e., in the absence of oncogenic induction by tumor promoters). Moreover, the present inventor is believed to be the first to discover that COX-1 is elevated as a direct result of viral carcinogenesis. The advantage of this discovery is that the method of the present invention can be used to diagnose oncogenic virus-associated neoplastic transformation by detecting a combination of the presence of an oncogenic virus and elevated cyclooxygenase in a sample. A positive diagnosis of both oncogenic viral infection and cyclooxygenase elevation (i.e., COX-1 and or COX-2) is indicative of neoplastic transformation or a potential for development of neoplastic transformation, and can be valuable for early diagnosis of such cancers. In contrast, neoplastic fransformation associated with other causative factors (i.e., spontaneous, hereditary, chemical carcinogens) lack a correlative marker which easily enables early detection of the neoplastic fransformation. Moreover, detection of cyclooxygenase expression or biological activity alone in the absence of establishing a clear baseline level of activity and/or ruling out other possible causes of cyclooxygenase expression or activity, as is described above for the general method of diagnosis, would be expected to produce a high rate of false positive or meaningless results. This embodiment of the method of the present invention provides a reliable means for early detection of oncogenic virus-associated neoplastic transformation, or a potential for development of such neoplastic transformation. Prior to the present invention, viral carcinogenesis was difficult to detect at an early stage, even though the virus was detected in a tissue, because: (1) such cancers can be caused as a rare effect by ubiquitous viruses, which can often be considered to be innocuous bystanders (i.e., even though a virus can be detected, an association with neoplastic fransformation is missed or not suspected); (2) some oncogenic viruses have heterogeneous viral particles and infect cells without inducing cancer (i.e., the presence of the virus alone can not be positively correlated with increased risk of cancer); (3) the disease may not overtly develop until long after viral infection (i.e., there is no suggestion to look at a viral correlation); and, (4) the cancers may not seem related to a contagious factor because the method of transmission of the virus is not apparent (i.e., again, no suggestion to look at a viral correlation).

In this embodiment, the method includes an additional step, performed after step (a) of obtaining a sample from the patient and before step (e) of making a diagnosis, of detecting whether the sample carries an oncogenic RNA or DNA virus. Detection o f an oncogenic RNA or DNA virus in the test sample, in combination with detection of increased cyclooxygenase expression or biological activity in the sample as compared to the baseline level of cyclooxygenase expression or biological activity, indicates a positive diagnosis of virus-associated neoplastic transformation or a potential therefor. A positive detection of an oncogenic virus in this step is any measurable (detectable) evidence of the presence of such a virus in the sample, and therefore, there is no baseline control for this step. According to the present invention, evidence of the presence of a virus in the sample, or detection of a virus in the sample, includes any measurement or detection of a viral gene, viral protein, viral-specific marker (e.g., enhanced phosphorylation of particular kinases), which can be identified as being associated with the presence of an oncogenic DNA or RNA virus, and which indicates that the cells or tissues from which the sample was obtained have been infected by, or otherwise in contact with the virus.

Many oncogenic DNA and RNA viruses are known in the art, and viral genes and proteins associated with such viruses have been identified, isolated and sequenced. Therefore, the method of the present invention can include a step of detecting any oncogenic DNA or RNA virus, including by initially detecting a viral gene or protein that is common to two or more such viruses, followed by additional detection, if necessary, to detect the presence of specific viruses. The step of detection can also be designed to detect oncogenic viruses by family or by specific viral strain by selection of detection means suitable for the desired level of detection, as discussed below. Suitable oncogenic DNA viruses to detect using the method of the present invention include viruses from families which include, but are not limited to, Papovaviruses (e.g., papilloma virus, polyoma virus, Simian virus 40 (SN40)), Herpesviruses (e.g., Epstein Barr Virus) Hepadnaviruses (e.g., hepatitis B virus (HBN)), and Poxviruses. Preferred viruses to detect in the method of the present invention include, but are not limited to, human papilloma virus, polyoma virus, and/or Simian virus 40 (SN40). In a preferred embodiment, the method of the present invention includes a step of detecting an oncogenic virus from the Papovavirus family, with detection of human papilloma virus being most preferred. Suitable oncogenic RΝA viruses to detect using the method of the present invention include, but are not limited to, human lymphofropic virus and human immunodeficiency virus. The step of detecting the presence of an oncogenic DΝA or RΝA virus can be performed by any suitable method of detecting a virus in a cell, tissue, or bodily fluid sample. Such methods are known in the art. As discussed above, viral genes and proteins for most oncogenic DΝA and RΝA viruses have been identified, isolated and sequenced. Therefore, information necessary to generate oligonucleotide primers, hybridization probes and/or antibodies useful in standard detection methods are known and readily available in the art. For example, the step of detecting can include, but is not limited to, a molecular detection technique such as PCR, RT-PCR, Northern blot, Southern blot, sequence analysis, and in situ hybridization. The Examples section describes the use of RT-PCR and PCR to detect bovine papilloma virus in a cell sample, for example. Similar methods can be used to detect human papilloma virus and other viruses in a cell sample. Alternatively, for viral proteins for which antibodies or other ligands are available, the virus may be detected by a method such as immunoblot, enzyme-linked immunosorbant assay (ELIS A), radioimmunoassay (RLA), and immunoprecipitation.

One embodiment of the present invention relates to a method for diagnosing neoplastic transformation or a potential for neoplastic transformation that is associated with an oncogenic virus (i.e., viral carcinogenesis). This method includes the steps of: (a) obtaining from a patient, who has been identified as having been infected with an oncogenic virus, a sample to be evaluated for neoplastic transformation, wherein the sample is selected from the group of a cell sample, a tissue sample, and a bodily fluid sample; and (b) detecting expression or biological activity of at least one cyclooxygenase in the sample, the cyclooxygenase being COX-1 and/or COX-2. h this method, detection of increased cyclooxygenase expression or biological activity over a baseline cyclooxygenase expression or biological activity indicates a positive diagnosis of virus-associated neoplastic fransformation. In this embodiment of the present invention, the patient to be evaluated has been diagnosed as having been infected with an oncogenic virus prior to the performance of the present method. The establishment of viral infection in the patient can be determined by any suitable method in the art, including by performing any of the molecular techniques and/or viral protein detection techniques described for viral detection previously herein. Jn this embodiment, the method is used to determine whether cells or tissues of the patient to be evaluated are undergoing or are likely to undergo neoplastic fransformation as a result of infection by the virus .

In this embodiment of the present invention, a sample to be evaluated for neoplastic transformation is first obtained from the patient. Methods for obtaining the sample are described above. Preferably, the cells, tissue or organ from which the sample is obtained is known to be a target of infection of the particular oncogenic virus identified in the patient. For example, if it is known that the patient has been infected with human papilloma virus, it is desirable to obtain the sample from the cervix, particularly if the previous identification of viral infection was made through analysis of cervical tissue (e.g., by biopsy of a wart).

The second step in this embodiment of the present invention is to detect cyclooxygenase expression or biological activity in the sample obtained from the patient. Methods for detecting cyclooxygenase expression or biological activity in a sample have been described in detail above. In this step, the level of cyclooxygenase expression or biological activity, if any is detected, can be compared to a baseline level of cyclooxygenase expression or biological activity (i.e., a control level) as discussed above. Detection of increased cyclooxygenase expression or biological activity in the sample over such a baseline level indicates a positive diagnosis of oncogenic virus-associated neoplastic transformation. As set forth above for the general method for diagnosis of neoplastic fransformation, in order to establish a positive diagnosis, the level of cyclooxygenase activity is increased over the established baseline by an amount that is statistically significant (i.e., with at least a 95% confidence level, or p<0.05). Preferably, detection of at least about a 1.5 fold increase in cyclooxygenase expression or biological activity in the sample as compared to the baseline level results in a positive diagnosis for said sample. More preferably, detection of at least about a 3 fold increase, and more preferably at least about a 6 fold increase, and even more preferably, at least about a 12 fold increase, and even more preferably, at least about a 24 fold increase in cyclooxygenase expression or biological activity as compared to the baseline level, results in a positive diagnosis for said sample. If the level of cyclooxygenase expression or biological activity in the test sample is less than or statistically substantially the same as the baseline level of cyclooxygenase expression or biological activity, then a negative diagnosis is indicated.

Yet another embodiment of the method of the present invention is a method for diagnosing neoplastic transformation or a potential for neoplastic fransformation associated with an oncogenic virus. This method includes the steps of: (a) obtaining from a patient, wherein the sample can be a cell sample, a tissue sample, and/or a bodily fluid sample; (b) detecting whether said sample carries an oncogenic RNA or DNA virus; and, (c) detecting the expression or biological activity of at least one cyclooxygenase in the sample, the cyclooxygenase being COX-1 and or COX-2. In this embodiment, detection of: (1) an oncogenic virus in the sample, and (2) increased cyclooxygenase expression or biological activity, over a baseline cyclooxygenase expression or biological activity, indicates a positive diagnosis of virus-associated neoplastic transformation or a potential therefor. Step (a) of obtaining in this embodiment of the method is performed as described above for the other embodiments of the method. As for the other embodiments, the sample type and location from which the sample is obtained may be determined by' those of skill in the art based on any reasonable factor associated with the patient and the reason for making an evaluation of the patient including, but not limited to, the medical history of the patient, a medical complaint by or observation of the patient which suggests early neoplastic transformation or potential therefor, a routine screening of a particular tissue or organ, and/or screening based on hereditary risk. Steps (b) and (c) of detecting are performed as described above for the general method for diagnosing neoplastic transformation in a patient. This embodiment of the method is particularly useful when it is not known whether a patient has been or is infected with an oncogenic virus. Detection of both the presence of an oncogenic virus and elevated cyclooxygenase expression in a patient can be extremely valuable for early diagnosis of potentially deadly tumors resulting from viral carcinogenesis. Moreover, the detection of an oncogenic virus concomitantly with detection of elevated cyclooxygenase increases the likelihood that the positive diagnosis of neoplastic fransformation or a potential therefor will be confirmed by subsequent analysis, since other potential causes of cyclooxygenase elevation may be less suspect. Following a positive diagnosis by this method, however, the patient maybe screened by other known assays for neoplastic fransformation and/or screened for the presence of other inducers of cyclooxygenase elevation, as discussed above, in order to confirm the diagnosis and provide additional information for establishing a treatment protocol.

Another embodiment of the present invention is a method for diagnosing cervical, uterine or prostate cancer or a potential for development of cervical, uterine or prostate cancer in a patient. This method includes the steps of: (a) obtaining a sample from a patient, wherein the sample is a cell sample, a tissue sample or a bodily fluid sample taken from cervix, uterus or prostate, such tissue being evaluated for neoplastic fransformation; and (b) detecting the expression or biological activity of at least one cyclooxygenase in said sample, the cyclooxygenase being COX-1 and or COX-2. h this embodiment, detection of increased cyclooxygenase expression or biological activity over a baseline level of cyclooxygenase expression or biological activity in the cervix, uterine or prostate sample indicates a positive diagnosis of cervical, uterine or prostate cancer, respectively, or a potential for development of cervical, uterine or prostate cancer, respectively. Each of steps (a) of obtaining a sample and (b) of detecting have been described in detail above. This embodiment of the present invention relates to the use of the method of the present invention to diagnose specific cancers that are associated with specific organs and tissues of the body. Each of these cancers is further characterized as being associated, at least a portion of the time, with viral carcinogenesis. In particular, each of cervical, uterine and prostate cancer can be directly or indirectly caused by infection with a papilloma virus. Therefore, in one embodiment of this method, the method includes an additional step of detecting whether said sample carries papilloma virus. Such methods are described in detail above and are exemplified in the Examples section below for papilloma virus. Detection of both papilloma virus and elevated cyclooxygenase expression or biological activity indicates a positive diagnosis of papilloma virus-associated neoplastic transformation in the tissue or organ from which the sample was obtained. Cervical, uterine and prostate cancer indicators are routinely screened in women (cervical and uterine) and men (prostate), and the method of the present invention provides a new method of early detection of postive indicators of neoplastic transformation. This method is particularly valuable, because the sample can be collected in a non-invasive manner (i.e., cervical mucous or cells from a woman and urine or seminal fluid from a man), and method can be readily foπnulated into a simple assay kit which may even be modified for home use (discussed in detail below).

Yet another embodiment of the present invention relates to an assay kit for diagnosing neoplastic transformation or a potential for neoplastic fransformation in a patient. The assay kit includes: (a) a means for detecting the presence of an oncogenic virus in a sample obtained from a patient, wherein the sample is selected from the group consisting of a cell sample, a tissue sample and a bodily fluid sample; and, (b) a means for detecting cyclooxygenase expression or biological activity in said sample. In a preferred embodiment, the assay kit is configured to test a bodily fluid sample such as, but not limited to, mucous, seminal fluid, saliva, breast milk, bile and urine. Such an assay kit is therefore provided in a format suitable for receiving a bodily fluid sample such that the bodily fluid can be contacted with the means for detecting in parts (a) and (b) of the kit and such that an oncogenic virus and cyclooxygenase expression or biological activity, respectively, can be detected, if present, in the bodily fluid sample.

The means for detecting the presence of an oncogenic virus can be any reagent that is used in a method of detecting the presence of a virus in a sample, such as by a method for detecting the presence of a virus described previously herein. Such means for detecting include, but are not limited to: a probe that hybridizes under stringent hybridization conditions to a nucleic acid molecule from the virus; PCR primers which amplify a nucleic acid molecule from the virus; an antibody that selectively binds to an oncogenic viral protein in the sample and a viral antigen that can be bound by anti-virus antibodies in the sample. As discussed above, nucleic acid and amino acid sequences for many viral genes and proteins, respectively, are known in the art and can be used to produce such reagents for detection.

According to the present invention, a probe is a nucleic acid molecule which typically ranges in size from about 8 nucleotides to several hundred nucleotides in length. Such a molecule is typically used to identify a target nucleic acid sequence in a sample by hybridizing to such target nucleic acid sequence under stringent hybridization conditions. As used herein, stringent hybridization conditions refer to standard hybridization conditions under which nucleic acid molecules are used to identify similar nucleic acid molecules. Such standard conditions are disclosed, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, 1989. Sambrook et al., ibid., is incorporated by reference herein in its entirety (see specifically, pages 9.31-9.62). hi addition, formulae to calculate the appropriate hybridization and wash conditions to achieve hybridization permitting varying degrees of mismatch of nucleotides are disclosed, for example, in Meinkoth et al, 1984, Anal. Biochem. 138, 267-284; Meinkoth et al., ibid., is incorporated by reference herein in its entirety.

More particularly, stringent hybridization and washing conditions, as referred to herein, refer to conditions which permit isolation of nucleic acid molecules having at least about 70% nucleic acid sequence identity with the nucleic acid molecule being used to probe in the hybridization reaction, more particularly at least about 80%>, and more particularly at least about 85%>, and most particularly at least about 90%. Such conditions will vary, depending on whether DNA:RNA or DNA:DNA hybrids are being formed. Calculated melting temperatures for DNA:DNA hybrids are 10 C less than for DNA:RNA hybrids, h particular embodiments, stringent hybridization conditions for DNA:DNA hybrids include hybridization at an ionic strength of 6X SSC (0.9 MNa⁺) at a temperature of between about 20 C and about 35 C, more preferably, between about 28 C and about 40 C, and even more preferably, between about 35 C and about 45 C. In particular embodiments, stringent hybridization conditions for DNA:RNA hybrids include hybridization at an ionic strength of 6X SSC (0.9 M Na⁺) at a temperature of between about 30 C and about 45 C, more preferably, between about 38 C and about 50 C, and even more preferably, between about 45 C and about 55 C. These values are based on calculations of a melting temperature for molecules larger than about 100 nucleotides, 0% formamide and a G + C content of about 40%). Alternatively, T_m can be calculated empirically as set forth in Sambrook et al., supra, pages 9.31 TO 9.62.

PCR primers are also nucleic acid sequences, although PCR primers are typically oligonucleotides of fairly short length which are used in polymerase chain reactions. PCR primers and hybridization probes can readily be developed and produced by those of skill in the art, using sequence information from the target sequence. (See, for example, Sambrook et al., supra or Glick et al., supra).

Antibodies that selectively bind to an oncogenic viral protein in the sample and a viral antigen that can be bound by anti- virus antibodies in the sample can be produced using viral protein information available in the art. As usedherein, the term "selectively binds to" refers to the ability of such an antibody to preferentially bind to a specific viral protein. Antibodies useful in the assay kit and methods of the present invention can be either polyclonal or monoclonal antibodies. Such antibodies include functional equivalents such as antibody fragments and genetically-engineered antibodies, including single chain antibodies, that are capable of selectively binding to at least one of the epitopes of the protein used to obtain the antibodies. Such antibodies can include chimeric antibodies in which at least a portion of the heavy chain and or light chain of an antibody is replaced with a corresponding portion from a different antibody. For example, a chimeric antibody of the present invention can include an antibody having an altered heavy chain constant region (e.g., altered isotype), an antibody having protein sequences derived from two or more different species of animal, and an antibody having altered heavy and/or light chain variable regions (e.g., altered affinity or specificity). Preferred antibodies are raised in response to viral proteins or peptides from the virus to be detected.

Generally, in the production of an antibody, a suitable experimental animal, such as a rabbit, hamster, guinea pig or mouse, is exposed to an antigen against which an antibody is desired. Typically, an animal is immunized with an effective amount of antigen that is injected into the animal. An effective amount of antigen refers to an amount needed to induce antibody production by the animal. The animal's immune system is then allowed to respond over a pre-determined period of time. The immunization process can be repeated until the immune system is found to be producing antibodies to the antigen, hi order to obtain polyclonal antibodies specific for the antigen, serum is collected from the animal that contains the desired antibodies. Such serum is useful as a reagent. Polyclonal antibodies can be further purified from the serum by, for example, treating the serum with ammonium sulfate. In order to obtain monoclonal antibodies, the immunized animal is sacrificed and B lymphocytes are recovered from the spleen. The B lymphocytes are then fused with myeloma cells to obtain a population of hybridoma cells capable of continual growth in suitable culture medium. Hybridomas producing a desired antibody are selected by testing the ability of an antibody produced by a hybridoma to bind to the antigen.

According to the present invention, a means for detecting cyclooxygenase expression or biological activity can be any suitable reagent which can be used in a method for detection of cyclooxygenase expression or biological activity as described previously herein. Such reagents include, but are not limited to: a probe that hybridizes under stringent hybridization conditions to a nucleic acid molecule encoding the cyclooxygenase or a fragment thereof; RT-PCR primers for amplification of mRNA encoding the cyclooxygenase or a fragment thereof; an antibody that selectively binds to the cyclooxygenase; a probe that hybridizes under stringent hybridization conditions to a nucleic acid molecule encoding a cyclooxygenase biochemical endproduct or a fragment thereof; RT-PCR primers for amplification of mRNA encoding a cyclooxygenase biochemical endproduct or a fragment thereof; and/or an antibody that selectively binds to a cyclooxygenase biochemical endproduct. Hybridization probes, PCR primers and antibodies have been described above in the discussion related to detection of viruses, and such discussion can be readily applied to the detection of cyclooxygenase expression or biological activity. Similarly, cyclooxygenase biochemical endproducts have been previously discussed herein and the technology used to develop probes, primers and/or antibodies can be similarly applied. Preferably, such a cyclooxygenase biological endproduct is selected from the group of a prostaglandin, prostacyclin and thromboxane A₂.

The means for detecting of part (a) and or part (b) of the assay kit of the present invention can be conjugated to a detectable marker. Such a marker can be any suitable marker which allows for detection of the means of part (a) or (b) and includes, but is not limited to, a fluorescent marker, a chemiluminescent marker, a radioactive tag, a colorimefric tag, an enzyme, or other such detectable markers which are commonly used in detection assays such as those described herein. In addition, the means for detecting of part (a) and or part (b) of the assay kit of the present invention can be immobilized on a substrate. Such a substrate can include any suitable substrate for immobilization of a detection reagent such as would be used in any of the previously described methods of detection. Briefly, a substrate suitable for immobilization of a means for detecting includs any solid support, such as any solid organic, biopolymer or inorganic support that can form a bond with the means for detecting without significantly effecting the activity and/or ability of the detection means to detect the desired targefr molecule. Exemplary organic solid supports include polymers such as polystyrene, nylon, phenol-formaldehyde resins, acrylic copolymers (e.g., polyacrylamide), stabilized intact whole cells, and stabilized crude whole cell/membrane homogenates. Exemplary biopolymer supports include cellulose, polydextrans (e.g., Sephadex®), agarose, collagen and chitin. Exemplary inorganic supports include glass beads (porous and nonporous), stainless steel, metal oxides (e.g., porous ceramics such as ZrO₂, TiO₂, Al₂O₃, andNiO) and sand.

Yet another embodiment of the present invention relates to a method for evaluating the carcinogenicity of a compound. The method includes the steps of: (a) contacting with the compound being tested for carcinogenicity a test cell; and, (b) detecting the expression or biological activity of at least one cyclooxygenase by the test cell, wherein the cyclooxygenase is COX-1 and or COX-2, and wherein an increase in cyclooxygenase expression in the test cell after contact with the compound as compared to before contact with the compound indicates that the compound is carcinogenic. The test cell can be any suitable cell which can be used to detect a change in cyclooxygenase expression or biological activity.

In a preferred embodiment, the test cell is transfected with a recombinant nucleic acid molecule from a DNA or RNA virus encoding a fransforming protein, wherein said test cell maintains a copy number of said recombinant nucleic acid molecule of within at least about 50%) over 5 passages of said test cell. The DNA or RNA virus encoding a transforming protein can be any of the oncogenic DNA or RNA viruses previously described herein. In a preferred embodiment, the recombinant nucleic acid molecule is from a papilloma virus, including bovine papilloma virus (BPN) and human papilloma virus. In one embodiment, the recombinant nucleic acid molecule is selected from the group of the BPN E5, E6 and/or E7 open reading frames, h another embodiment, the recombinant nucleic acid molecule is selected from group of the BPN E6 open reading frame and/or the BPN E7 open reading frame. In yet another embodiment, the recombinant nucleic acid molecule is a BPN genome in which a viral gene selected from the El and/or E2 open reading frames has been removed or made non-functional. Preferably, the test cell is mammalian and can include, but is not limited to, mouse C127 cells, C3H/IOTV2 cells, human newborn foreskin keratinocytes and primary fibroblast cultures of C57BL/6J mice. Such a test cell has been described in detail in U.S. Patent No. 5,821,049, issued October 13, 1998, to Kowalski, incorporated herein by reference in its entirety. U.S. Patent No. 5,821,049 did not, however, disclose the detection of COX-1 or COX-2 expression or biological activity by the test cell as a method for evaluating carcinogenicity of a compound. This method of the present invention includes contacting a test cell with a compound being tested for carcinogenicity. For example, test cells can be grown in liquid culture medium or grown on solid medium in which the liquid medium or the solid medium contains the compound to be tested. In addition, the liquid or solid medium contains components necessary for cell growth, such as assimilable carbon, nitrogen and micro-nutrients. The assay involves contacting cells with the compound being tested for a sufficient time to allow for a detectable change (e.g., an increase, elevation or upregulation) in cyclooxygenase expression or biological activity in the test cells in the presence of carcinogenic compounds. It may be that for more toxic substances a shorter time of contact with the substance being tested is suitable. As used herein, the term "contact period" refers to the time period during which cells are in contact with the compound being tested. The term "incubation period" refers to the entire time during which cells are allowed to grow prior to detection of cyclooxygenase expression or biological activity. Thus, the incubation period includes all of the contact period and may include a further time period during which the compound being tested is not present but during which growth is continuing prior to scoring. After the period of incubation, step (b) of detecting is performed. As described for the methods of above, step (b) of detecting can include detecting Cyclooxygenase (i.e., Cox-1 or Cox-2) mRNA transcription, detecting cyclooxygenase translation, and/or detecting cyclooxygenase biological activity, which can include detection of a biochemical endproduct of the cyclooxygenase activity. Suitable methods for detection by any of such means of detection have been described in detail previously herein. This method of the present invention can additionally include further evaluation of phenotypic fransformation characteristics of the cell after contact with the putative carcinogen as compared to before contact with the carcinogen. Such phenotypic transformation characteristics include, but are not limited to, formation of foci, loss of growth factor requirements, loss of serum requirements, tumorigenicity in nude mice and anchorage independence. Methods for evaluating such phenotypic transformation characteristics in a test cell are described in detail in U.S. Patent No. 5,821,049, which is incorporated herein by reference in its entirety.

The following examples are provided for the purposes of illustration and are not intended to limit the scope of the present invention.

EXAMPLES

Example 1

This example demonstrates that CSH/IOT¹ - fibroblasts containing bovine papilloma virus genes and having a partially transformed phenotype, constitutively express higher levels of COX-2 than wild-type C3H/10T% cells. The present inventor has investigated the expression of COX-1 and COX-2 in

C3H/10T'/2 mouse embryo fibroblasts which had been transfected with pdBPN- 1(142-6), a plasmid which carries the full-length bovine papilloma virus genome cloned into pML2d at the unique Bam HI site (Sarver Ν, Rabson MS, Byrne JC, Howley PM. 1982 Transformation and replication in mouse cells of a bovine papilloma virus pML2d plasmid vector that can be rescued in bacteria. Proc. Natl. Acad. Set (USA) 79:7147-7151). Aresulting cell line, TI, produced as described below, developed more foci in co-cultures with C3H/10T¹ ! when exposed to a wide range of carcinogens and tumor promoters than the TI cell line which had not been exposed to such chemicals (Kowalski, L.A., Laitinen, A.M., Mortazavi-Asl, B., Wee, R. K.-H., Erb,H.E., Assi, K.P., Madden, Z. (2000) In Vitro Determination of Carcinogenicity of Sixty-four Compounds Using a Bovine Papilloma virus DΝA-carrying C3H/10T¹ ! Cell Line. Env. Mol Mutag., accepted for publication). In contrast, the parent C3H/10T¹ : cell line produced very few foci in the presence or absence of such chemicals. The present inventor found that the TI cell line responded with increased numbers of foci to a very wide range of chemicals such as nitrosamines, polyaromatic hydrocarbons, nitrogen heterocycles and amides. These chemicals normally must be metabolized by the P450 monooxygenases to active carcinogens in order to act as mutagens. Conversely, C3H/10T/2, is known to express only two active P450 enzymes: CYP 1B1 and CYP E/F an isozyme unique to the cell line (Shen, Z, Liu, J, Wells, RL, Elkind, MM (1994) DNA and Cell Biology 13: 763-769 and Shen, Z, Wells, RL, Elkind, MM (1994) Cancer Research 54: 4052-4056; Shen, Z, Wells, RL, , Liu, J, Elkind, MM (1993) Proc. Natl Acad, Sci (USA) 90: 11483-11487; and Christou, M, Stewart, P, Pottenger, LH, Fahl, WE, Jefcoate, CR (1990) Carcinogenesis 11: 1691-1698). Therefore, the present inventor questioned how such a variety of xenobiotics could be metabolized by such a small variety of functional P450 enzymes.

COX- 1 and COX-2 possess both oxygenase and peroxidase activity. They can oxidize a wide variety of xenobiotics, such as aromatic amines, phenacetin, 5-nitrofurans, phenols, hydroquinones and polycyclic hydrocarbons to electrophilic free radicals (Parkinson, A.(1996) Biotransformation of xenobiotics, In: CD. Klaassen, ed., Casarett & Doull's Toxicology, Fifth Edition, McGraw Hill Inc. They can also generate a variety of active oxygen free radicals such as peroxyl radicals in the process of oxidizing arachidonic acid ibid. Without being bound by theory, the present inventor believed that COX-1 or 2 was abnormally active in the TI cell line and that it facilitated metabolism of xenobiotics to free radical intermediates. Using Western blot as a method of detection of COX-2, the present inventor has demonstrated herein that COX-2, which is normally almost undetectable in C3H/10T/2 cells unexposed to tumor inducing agents, was present in almost 3-fold higher amounts in TI cells, also unexposed to tumor inducing agents. Conversely, levels of COX-1 were the same in C3H/10T/2 cells and TI cells.

More specifically, to produce the TI cell line, C3H/10T/2 fibroblasts, publicly available from the American Type Culture Collection (ATCC No. CCL-226), were grown in Dulbecco's modified Eagle's medium (Gibco)containing 20 mM HEPES [N2hydroxyethylpiperazineN'2ethanesulfonic acid] (Sigma), 60 μg/ml of penicillin G (Sigma) and 100 μg/ml of streptomycin sulfate (Sigma) and supplemented with 10%> fetal calf serum (Gibco). Cells were grown initially in 25 cm² tissue culture flasks (Nunc), then passaged to 75 cm² tissue culture flasks at the first passage. Cultures were grown at 37°C in 3% CO₂/95%_> air. Medium was changed twice weekly. Passage 19 ofa culture of C2>ΗJ\0TVι cells was transfected with the pdBPNl (1426) plasmid (publicly available through the ATCC; ATCC No.37134), prepared by CsCl₂ density gradient cenfrifugation. The plasmid pdBPNl (1426) contains the complete BPN1 genome cloned into the plasmid pML2d at the Bam HI site (Sarver, A., et al., ibid.). For each 100 mm² plate (Νunc), 1.25 x 10⁵ CSH/IOT¹ . cells were transfected with pdBPNl (1426) by the calcium phosphate precipitation method (Graham, E. L and Nander Erb, A J. (1973) "A new technique for the assay of infectivity of human adenovirus 5 DΝA." Virol 52: 456-467, incorporated herein by reference in its entirety) using 1.05 μg of plasmid DΝA and 27.5 μg of sheared calf thymus DΝA (as carrier DΝA) for 4 hours followed by glycerol shock for three minutes. Cultures were then incubated overnight in normal medium.

On the following day, transfected cells on 100 mm plates were subcultured at a ratio of 1:17 into 60 mm² plates (Νunc). Mezerein (Sigma), a non-genotoxic carcinogen, was diluted in dimethylsulfoxide (BDH) to a stock of 0.2 mg/ml, then diluted in medium to the desired concentrations. Mezerein (Sigma) was added at to a final concentration of 0.5 ng/ml to 60 mm² plates containing transfected cells. Cultures were processed in dim light following addition of mezerein and evaluated for the formation of foci. Cloned cell lines were established from foci in transfected cells exposed to mezerein, as follows. Cultures were incubated as described above in the constant presence of 0.5 ng/ml of mezerein with twice weekly changes of medium. Fifteen days after transfection, when foci were clearly visible (1-2 mm in diameter), cultures were rinsed twice with 0.25%> trypsin containing 0.02% EDTA, and the centers of individual foci, well isolated from other foci, were extirpated into about 10 μl of 0.25% trypsin containing 0.02% EDTA in 25 mm² dishes (Νunc) using an Eppendorf Pipettman. Foci were vigorously agitated by pipetting for 5 minutes to separate cells, then 2 ml of medium (without mezerein) added. Ten foci were isolated separately in this way.

Cells obtained from trypsinized foci on 25 mm plates were incubated until they reached confluence (3 days), then transferred to 60 mm plates and grown to confluence (7 days). Cells from each clone were harvested and frozen in medium containing 10% DMSO for later testing and subcloning. Frozen clones were thawed and grown to subconfluence in 25 cm² culture flasks (Νunc). To determine response to mezerein of each clone, 2000 untransfected C3H/1 OT¹/- cells, passage 20, were coincubated with 200 cells from the cloned cell lines in medium containing 0.5 ng/ml of mezerein. Control co-cultures of each clone were prepared containing no mezerein. Cultures were prepared in duplicate. Medium was changed twice weekly.

After 21 days of incubation, the cultures were stained with 0.025%> methylene blue in 50:50 methanohwater, dried, and the foci were counted. Several clones were identified which produced a higher number of foci in the presence of mezerein than in control cultures. The culture with the greatest difference was labeled as S 1 and produced an average of 60 foci per 60 mm² plate in the presence of mezerein and an average of 21 in the absence of mezerein.

To subclone SI, 50 cells were plated, using serial dilutions, on 100 mm² plates in triplicate. The plates were incubated in normal medium for 8 days, at which time colonies were 2-3 mm in diameter but not in contact with each other. Cultures were rinsed twice with 0.25%) trypsin containing 0.02%> EDTA, and the centers of individual colonies, wellisolated from other colonies, were extirpated into about 10 μl of 0.25% trypsin containing 0.02% EDTA in 25 mm² dishes (Nunc) using an Eppendorf Pipettman. Colonies were vigorously agitated by pipetting for 5 minutes to separate cells, then 2 ml of medium was added. Ten colonies were isolated separately in this way.

The subclones were grown and passaged into larger flasks, then tested in co-culture assays in the presence and absence of mezerein in the same way as the foci were tested above. The original S 1 clone resulted in ten subclones, one of which produced an average of 70 foci in the presence of mezerein and an average of 9 foci in the absence of mezerein. This subclone was designated TI.

Western blot analysis of protein isolated from TI cells unexposed to xenobiotics and of protein isolated from CθH/lOT¹. cells, also unexposed to xenobiotics, was performed using COX-2 or COX-1 antibodies (Cayman Chemical Company). Briefly, cell lines were seeded in 100 mm dishes (Corning) at 1 X 10⁶ cells / dish and grown until confluence as described above. Cells were serum deprived for 18 hrs and treated for 12 hrs with or without mezerin (5 and 50 ng/ml) in the presence or absence of platelet derived growth factor (PDGF) (50 ng/ml). Cells were then washed with ice cold PBS, scraped into cold PBS, and pelleted by cenfrifugation. Proteins were extracted from cells with 100 ml of 50 mM Tris-HCl (pH 7.4) containing 0.1 % SDS, 0.5 % NP-40, 1 mM PMSF, 20 mg/ml Aprotinin, μg/ml Leupeptin, and 2 mM Na3 VO₄ for 30 min. on ice. The crude protein extracts were centrifuged at 5,000 rpm for 5 min. at 4 C°. Protein concentrations of each sample were measured by the Biorad DC protein assay. After mixing and boiling with sample buffer, 50 μg of protein samples were applied on 10%> SDS-PAGE and transferred to a Hybond ECL membrane (Amersham). Membranes were incubated with 5 % nonfat dry milk in washing buffer (0.1%

Tween-20 in PBS, pH 7.5) overnight at 4 C°. After washing, blots were incubated either an anti Cox-1 monoclonal antibody or an anti Cox-2 polyclonal antibody (Cayman Chemical Co.) in 5 % nonfat dry milk in washing buffer at room temperature for 2 hrs. Following washing with washing buffer, blots were incubated with either peroxidase conjugated anti-mouse or anti-rabbit IgG second antibody (Amersham) in 5 % nonfat dry milk in washing buffer at room temperature for 1 hr. Chemiluminescent detection was performed using ECL western blotting detection reagent (Amersham) and were visualized after exposure to Hyperfilm ECL (Amersham).

The results of the Western blot demonstrated that COX-2 protein levels were three times higher in TI cells as compared to the parent line, CSH/IOT¹ - (data not shown). COX-1 protein levels were similar in the two cell lines (data not shown). The only known difference between the parent C3H/10T/2 cell line and the TI cell line is the presence of the transfected pdBPN- 1(142-6) genome. Therefore, it was concluded that BPV, a papilloma virus, either directly upregulates COX-2 expression or initiates cellular events which result in upregulation of COX-2.

Example 2

The following example demonstrates that a COX-2 inhibitor is capable of decreasing chemically-induced transformation of cells containing bovine papilloma virus genes. The present inventor has also demonstrated that the COX-2 inhibitor, ΝS398, decreased numbers of foci induced by benzo[a]pyrene in TI cells. Briefly, TI cells lines were seeded in 100 mm dishes (Corning) at 200 cells / dish together with C3H/10T1/2 cells 50,000 cells/dish and grown for 21 days in the above medium with twice weekly changes of medium. From the time of setting up co-cultures, benzo[a]pyrene (Sigma) at either 0.011 μM or 0.033 μM was added to each dish. Some dishes contained NS-398 (Sigma) at 1 μg/ml.or 2 μg/ml. Control cultures were exposed to no chemicals or only 0.011 μM or 0.033 μM benzo[a]pyrene or NS-398 at 1 μg/ml or 2 μg/ml. All co-cultures were set up in triplicate. After 21 days of incubation, the cultures were stained with 0.025% methylene blue in 50:50 methanokwater, dried, and the foci were counted. Cultures exposed to benzo[a]pyrene at 0.011 μM developed on average 36 foci as compared with 26 foci on average in cultures exposed only to medium. Cultures exposed to 0.011 μM of benzo[a]pyrene and 1 μg/ml of NS-398 developed on average 20 foci (p<0.05) and cultures exposed to 0.011 μM of benzo[a]pyrene and 2 μg/ml of NS-398 developed on average 19 foci (p<0.05). The results demonsfrated that benzo[a]pyrene-exposed cultures of TI cells co-cultured with C3H/10T1/2 cells which were freated with the COX-2 inhibitor, NS-398, formed significantly fewer numbers of foci than benzo[a]pyrene-exposed cultures of TI cells which were not treated with the inhibitor. These data indicate that the formation of foci in TI cells in response to exposure to a chemical tumor promoter is at least partially associated with COX-2 expression by said cells.

Example 3

The following example demonstrates that exposure of a cell line which has been partially transformed by papilloma virus genes to a tumor promoter results in further elevation of COX-2 expression by the cell line. The phorbol ester tumor promoter, mezerein (Sigma), a non-genotoxic carcinogen, was diluted in dimethylsulfoxide (BDH) to a stock of 0.2 mg/ml, then diluted in medium to the desired concentrations. Mezerein (Sigma) was added to a final concentration of 50 ng/ml to 100 mm² plates containing TI cells at 80% confluence. Cultures were processed in dim light for 12 hours following addition of mezerein and evaluated for expression of COX-2 by Western blot as described in Example 1. The results demonstrated that exposure to mezerein increased COX-2 protein levels in TI cells 5 fold as compared to the parent C3H/10T/2 cell line, but mezerein had no effect on levels of COX-1. Example 4

This example demonstrates that cells which express wild type BP V-E5 have a higher constitutive level of COX-2 than C3H/10T ^lΛ cells, whereas cells expressing a nonfunctional E5 mutant have a similar level of COX-2 as C3H/10T ^lA cells. C3H/1 OT/2 mouse embryo fibroblast cells were transfected with one of the following using Ca PO₄ precipitation: pJS63, a derivative of pSG5 (Sfratagene), which contained the wild-type E5 ORF; pJS66, which contained the E5 ORF mutated to substitute glycine for the wild-type glutamine; or pJW6/7 which contained the BPV-1 URR, E6 and E7 ORF's substituted for the E5 ORF in pJS63. All pJS (not pJW6/7) vectors were obtained as a gift from Dr. J. Sparkowski, Georgetown University. Clones were developed from foci induced in cultures exposed to 0.5 ng/ml of mezerein according to the above protocol. Of the resulting cell lines, 6/7UM-1 carried the BPV URR and E6 andE7 ORF's; M63.1 carried the wild type E5 ORF; and M66.2 carried the mutant E5 ORF. Transfection of this mutant E5 ORF into C127 cells eliminates the ability of the cells to form foci. Western blot analysis was used to detect the level of COX-2 protein expressed by the various cell lines as described above. RT-PCR was used to verify that M63.1 and M66.2 expressed only BPV-1 E5, and PCR was used to verify that: (1) M63.1 and M66.2 carried only BPV-1 E5 sequences; and (2) 6/7UM-1 carried only BPV-1 E6 and E7 sequences.

Briefly, for the RT-PCR procedure, cultured cells were aliquoted into 0.5x10⁶ cells per tube and washed twice with ice cold PBS. The cells were then cenfrifuged at 4 C at 5,000 rpm for 10 min. Cell pellets were vortexed and 1 μl of Rnase inhibitor was added. 200 μl of lysis buffer was added to the cell pellet and vortexed. The mixture was passed through a 21 gauge needle fitted to 3ml syringe 4 times. Samples were cenfrifuged at 1 l,000rpm for 30sec, at 4 C and the supernatant was transferred to fresh 1.5ml microfuge tubes. For hybridization, biotin-labeled Oligo(dT)₂₀ (lμl + 19μl nuclease free H₂O) was diluted and added to the lysate. The mixture was hybridized at 37 C for 15min. 50 μl of the biotin-labeled PolyA+ mRNA was added to Streptavidin-coated PCR tubes and incubated at 37 C for 15min. Primers were prepared and added to appropriate samples with the following master mixes. Master Mix 1 : Sterile nuclease free H₂O (14.5μl); PCR nucleotide mix (l.Oμl); DDT (100 mM solution)(2.5μl); Rnase Inhibitor (3.0μl). 23μl of the master mix was added to each polyA-mRNA tube. Master Mix 2 was then added to each tube (Master Mix 2: nuclease free water (12 μl); 5x RT-PCR (10 μl); MgCl 25mM (2 μl); and polymerase (lul). Reverse transcription was carried out at 55 C for 30min, followed by 1 cycle at 94 C for 2 minutes; 10 cycle repeats (30 sec at 94 C; 30 sec at 41 C and 30 sec at 55 C); 45 sec at 72 C; 20 cycle repeats (15 sec at 94 C; 30 sec at 41 C; and 55 sec at 72 C); 2 min. 50 sec at 72 C and a final elongation for 7 min. at 72 C. Quantitation of amplified product was performed by Southern blot.

For both RT-PCR and PCR procedures described in the Examples section herein, upsfream and downstream primer sequences were used as follows: BPV E5: (DD914WT) 5' CTGACT GGT GTA CTA TGC CAA 3' (SEQ ID NO:l)

BPV E5: (DD915WT)

5' GGCATT AAA AGG GCA GAC C 3' (SEQ ID NO.2)

BPV E6:

5 ' GCT GAA TTA TTG CAT GGC AAA A 3 ' (SEQ ID NO:3) 5' CTATGG GTA TTG GGA CCT TGA A 3' (SEQ ID NO.4)

BPV E7:

5' TTC AAG GTC CAA ATA CCC 3' (SEQ ID NO:5)

5' CAC AGC AAA AGT CAG CTC 3' (SEQ ID NO:6)

COX 2: COXFOR 5 ' TTA CTG CTG AAG CCC ACC CCA AAC 3 ' (SEQ ID NO:7)

COXREV5' CCA GGT CCT CGC TTA TGA TCT G 3' (SEQ ID NO:8)

SEQ ID NOs: 1 and 2 were kindly provided by D. DiMaio, Yale University. SEQ ID NOs:3-6 were kindly provided by L. Nasir, University of Glasgow. SEQ ID NOs:7 and 8 were produced by Canadian Life Technologies (Gibco; Burlington, Ontario) at the direction of the present inventor. The primers were diluted according to the manufacturer's instructions and the following master mixes were used. Master Mix 1 : nuclease free water (45.0 μl); PCR lOmM nucleotides (2.0 μl); template DNA eg 142. (61.0 μl). Control tubes contained the master mix in the absence of either primer, nucleotides, DNA or enzyme. Primers were added to appropriate samples at the following final concentrations: Upstream Primer eg for E5 (DD914) was added at 1.0 μl (200nM final concentration); Downstream Primer eg for E5 (DD915) was added at 1.0 μl (200nM final concentration). 50 μl of Master Mix 2 was added to each tube (Nuclease free water (36.25 μl); 10 X Expand buffer with MgCl₂ (Roche Diagnostic)(10.00 μl (1.5mM MgCl)); 25mM MgCl₂ ( 2.00 μl (0.5mM MgCl)); Rnase inhibitor (Roche Diagnostic)(l .0 μl); Expand High Fidelity Enzyme (Roche Diagnostic)(added last) (0.75 μl). The PCR cycles were as follows: 2 min. at 94 C; 10 cycle repeats (15 sec at 94 C; 30 sec at 50 C; and 45 sec at 72 C); 20 cycle repeats (15 sec at 94 C; 30 sec at 50 C; 1 min 5 sec (45 sec + 20 sec increment) cycle 11 at 72 C; 7 min 25 sec at 72 C and 15 min. at 72 C.

The results of the detection of COX-2 protein and BPV genes in the various cell lines was as follows. M63.1 which carried the wild type BPV-1 E5 ORF showed approximately a 2-fold increase in COX-2 protein (unexposed to mezerein) as compared with C3W10ΥY2. M66.2, which carried the mutant (nonfunctional) BPV-1 E5 ORF, showed a very low level of COX-2 expression, similar to C3H/10T/2 cells. 6/7UM-1, which carried the BPV-1 E6 and E7 ORF's showed a three to four fold increase in level of COX-2 protein expression (same as TI). RT-PCR conducted on mRNA isolated from the same cell lines verified that M63.1 and M66.2 expressed only BPV-1 E5 (no E6 or E7 mRNA was detected). 6/7UM-1 mRNA contained no BPV-1 E5 transcripts. PCR verified that M63.1 and M66.2 carried only BPV-1 E5 sequences and that 6/7UM-1 carried only BPV-1 E6 and E7 sequences.

Since mutation of the E5 gene into a nonfunctional form by substitution of glycine at codon 17 abolishes upregulation of COX-2, the mechanism must involve the E5 ORF. In addition, the combined effect of BPV E6 and E7 enhances COX-2 expression even further, hi summary, the results demonstrated that: (1) clones which express wild type BPV-E5 have a higher constitutive level of COX-2 than C3H/10T Vi cells but clones expressing a nonfunctional E5 mutant have a similar level of COX-2 as C3H/10T Vi cells; and, (2) Clones which express BPV-E6 and E7 express a higher constitutive level of COX-2 than clones expressing only wild type BPV E5. Therefore, without being bound by theory, the present inventor believes that for maximum upregulation of COX-2, E6 and or E7 is essential but E5 can also induce some upregulation; and (3) the presence of the papilloma virus genome is clearly essential for upregulation of COX-2. Example 5

In this example, upregulation of COX-1 in a human cell line expressing and carrying human papillomavirus type 18 (HP V- 18) is demonsfrated, thereby illustrating the usefulness of COX-1 as a marker for HPV-18 which is a high risk vims associated with cervical carcinoma.

An immortalized human keratinocyte cell line carrying and expressing HPV-18 was obtained from J. McDougall, Fred Hutchinson Cancer Research Centre, Seattle, WA (cell line 1811). As a control, newborn human foreskin keratinocytes (NFK cells) were purchased from the American Type Culture Collection, Manassus, VA. NFK cells were grown in either 100 mm plates (Coming, Ithaca, NY) for protein isolation for Western blotting or grown in 75 cm² tissue culture flasks (Coming, Ithaca, NY) for isolation of RNA for RT-PCR. Cells were cultured in Keratinocyte Serum Free Medium (KSFM) (Canadian Life Technologies, Burlington, ON), with a final bovine pituitary extract concentration of 22.118 mg/ml and a final recombinant epidermal growth factor concentration of 37.2 ng/μl. Cells were incubated in 97%) air/3 %> CO₂ with medium changes twice weekly. For 1811 cells, cells were cultured in the same way as for NFK cells. For isolation of protein for Western blotting, 4 x 100 mm plates of 80% confluent NFK cells, passage 3 and 4 x 100 mm plates of 80% confluent 1811 cells passage 26 were harvested and collected by scraping into PBS and processed exactly in the same fashion as in Example 1 with the exception that human COX- 1 and human COX- 2 antibodies (Cayman Chemicals, Ann Arbor, MI) were used instead of mouse antibodies and the cells were neither serum deprived nor exposed to mezerein prior to harvest.

To evaluate expression of HPV-18 using RT-PCR, 2 x 75 cm² flasks of 80% confluent passage 26 of 1811 cells and 2 75 cm² flasks of 80%> confluent passage 4 of NFK cells were harvested. RNA isolation and RT-PCR were conducted in the same manner as in Example 4 using the following primers:

Upsfream: ATG GCG CGC TTT GAG GAT CC (SEQ ID NO:9) Downstream: TTA CTG CTG GGA TGC ACA CC (SEQ ID NO: 10)

Primers were constructed by Canadian Life Technologies (Burlington, ON) on the design of the present inventor. It was determined, using Western blots, that the level of COX- 1 protein in the 1811 cells carrying HPV-18 was 3-fold higher than the amount of COX-1 protein in the confrol NFK cells carrying no HPV-18. The COX-2 protein level was 4-fold lower in the HPV-18 carrying cell line than in the control. It was also verified that the HPV-18 E6 ORF (a fransforming gene of HPV-18) was expressed.

Example 6

In this example, upregulation of COX-1 in a human cell line expressing and carrying human papillomavirus type 16 (HPV- 16) is demonsfrated, thereby illustrating the usefulness of COX-1 as a marker for HPV-16 which is a high risk virus associated with cervical carcinoma. An immortalized human keratinocyte cell line carrying and expressing HPV- 16 was obtained from M. Nassari, Wyeth Ayerst, Pearl River, NY (cell line 2i2). As a control, newborn human foreskin keratinocytes (NFK cells) were purchased from the American Type Culture Collection, Manassus, VA and grown as in example 5. 2i2 cells were cultured as described in Example 5 for 1811 cells in KSFM. Isolation of protein for Western blotting, and blotting using human COX-1 and COX-2 antibodies was done as in Example 5 using 4 x 100 mm plates of 80% confluent NFK cells, passage 3 and 3 x 100 mm plates of 80% confluent 2i2 cells passage 3. RT-PCR was not conducted for the 2i2 cells.

It was determined, using Western blots, that the level of COX-1 protein in the 2i2 cells carrying HPV-16 was 2.5-fold higher than the amount of COX-1 protein in the confrol NFK cells carrying no HPV-16. COX-2 protein level was 2.5-fold lower in the HPV-16 carrying cell line than in the confrol.

Example 7

In this example, lack of upregulation of COX-1 in a human cell line expressing and carrying human papillomavirus type 6 (HPV-6) is demonstrated. This illustrates the ability of COX-1 to be useful as a marker for HPV-18 and HPV-16 which are high risk viruses associated with cervical carcinoma but not to be altered in a cell line carrying a low risk virus

(HPV-6) which is very similar but is not associated with cervical carcinoma.

A human fibroblast cell line expressing HPV-6 E6 (one of the two fransforming genes) and a second human fibroblast cell line expressing HPV-6 E7 (the second transforming gene) were obtained from D. Galloway, Fred Hutchinson Cancer Research Centre, Seattle, WA (cell lines HF6E6 and HF6E7). As a confrol, primary human fibroblasts (hs 27 cells) were purchased from the American Type Culture Collection, Manassus, VA. hs 27, HF6E6 and HF6E7 cells were cultured in Dulbecco's modified Eagle's medium (Canadian Life Technologies, Burlington, ON ) supplemented with 10% fetal calf serum (Canadian Life Technologies, Burlington, ON), 9.96 U/ml of penicillin G (Sigma Aldrich Canada, Oakville, ON), 72.5 μg/ml of streptomycin sulfate (Sigma Aldrich Canada, Oakville, ON), and 20mM HEPES [N-2 hydroxyethylpiperazine-N'-2-ethanesulfonic acid] (Sigma Aldrich Canada, Oakville, ON) in 3% CO₂ in air at 37 C. Medium was changed twice weekly. Isolation of protein for Western blotting, and blotting using human COX- 1 and COX-

2 antibodies was done as in example 5 using 4 x 100 mm plates of 80% confluent hs27 cells, passage 19 and 4 x 100 mm plates of 80% confluent HF6E6 or HF6E7 cells passage 8. RT- PCR was not conducted for these cells.

The present inventor found that, using Western blots, the level of COX-1 protein in the HF6E6 or HF6E7 cells was the same as in the control hs27 cells. In the HF6E6 cells, the level of COX-2 protein was 2-fold less than in the control hs27 cells. In the HF6E7 cells, the level of COX-2 protein was 0.5 -fold lower than in the control cells.

Example 8 In this example, lack of change of level of expression of COX-1 or COX-2 in a human cell line expressing and carrying simian virus 40 (SV40) compared with uninfected controls is demonstrated. This illustrates the ability of COX-1 change of expression to be useful as a marker to distinguish between cells infected with high risk HPV strains which is a high risk virus associated with cervical carcinoma from human cells infected a related viruses which is not associated with cervical carcinoma.

Two immortalized human keratinocyte cell lines, 98 and 130, carrying and expressing SV40 were obtained from M. Steinberg, City College of New York, NY, NY. As a control, newborn human foreskin keratinocytes (NFK cells) were purchased from the American Type Culture Collection, Manassus, VA. The control NFK cells and the cell lines 98 and 130 were cultured in Dulbecco ' s modified Eagle' s medium (Canadian Life Technologies, Burlington, ON ) supplemented with 10% fetal calf serum (Canadian Life Technologies, Burlington, ON), hydrocortisone at a final concentration of 0.5 μg/ml (Sigma Aldrich Canada, Oakville, ON), cholera toxin (Sigma Aldrich Canada, Oakville, ON) at a final concentration of 20 ng/ml, 9.96 U/ml of penicillin G (Sigma Aldrich Canada, Oakville, ON), 72.5 μg/ml of streptomycin sulfate (Sigma Aldrich Canada, Oakville, ON), and 20mM HEPES [N-2 hydroxyethylpiperazine-N'-2-ethanesulfonic acid] (Sigma Aldrich Canada, Oakville, ON) in 3% CO₂ in air at 37 C. Medium was changed twice weekly.

Isolation of protein for Western blotting, and blotting using human COX- 1 and COX- 2 antibodies was done as in example 5 using 4 100 mm plates of 80% confluent NFK cells, passage 3 and 4 x 100 mm plates of 80%> confluent 98 cells, passage 143 or 130 cells passage 143.

To evaluate expression of SV40 using RT-PCR, 2 x 75 cm² flasks of 80% confluent passage 143 of either 98 or 130 cells and 2 x 75 cm² flasks of 80%> confluent passage 3 of NFK cells were harvested. RNA isolation and RT-PCR were conducted in the same manner as in example 4 using the following primers: Upstream: GAT TAA AAT CAT CC (SEQ ID NO: 11)

Downstream: CTG AGG CGG AAA GAA CCA GCT GTG GAA TGT GT (SEQ ID NO: 12)

Primers were constructed by Canadian Life Technologies (Burlington, ON) on the design of M. Steinberg.

The present inventor found that, using Western blots, the levels of COX-1 protein or COX-2 protein in either 98 or 130 cells was barely detectable. It was also verified that SV40 was expressed in both cell lines but not in the confrol NFK cells.

While various embodiments of the present invention have been described in detail, it is apparent that modifications and adaptations of those embodiments will occur to those skilled in the art. It is to be expressly understood, however, that such modifications and adaptations are within the scope of the present invention, as set forth in the following claims:

Claims

What is claimed is:

1. A method for diagnosing neoplastic transformation or a potential for neoplastic transformation in a patient, comprising: a. obtaining from a patient a sample selected from the group consisting of a cell sample, a tissue sample, and a bodily fluid sample; b. establishing a baseline level of expression or biological activity of at least one cyclooxygenase for said sample, said cyclooxygenase being selected from the group consisting of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2); c. detecting expression or biological activity of said cyclooxygenase in said sample; d. comparing said expression or biological activity of said cyclooxygenase as determined in step (c) to said baseline level of expression or biological activity of said cyclooxygenase established in step (b); and, e. making a diagnosis of said patient, wherein detection of increased cyclooxygenase expression or biological activity as compared to said baseline level of expression or biological activity of said cyclooxygenase, indicates a positive diagnosis of neoplastic transformation or a potential therefor in said patient.

2. The method of Claim 1 , wherein said step (c) of detecting comprises detecting mRNA transcription of said cyclooxygenase.

3. The method of Claim 2, wherein said step (c) of detecting is by a method selected from the group consisting of reverse transcriptase-PCR (RT-PCR), in situ hybridization, Northern blot, sequence analysis and detection of a reporter gene.

4. The method of Claim 1 , wherein said step (c) of detecting comprises detecting translation of said cyclooxygenase.

5. The method of Claim 4, wherein said step (c) of detecting is by a method selected from the group consisting of immunoblot, enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA), and immunoprecipitation.

6. The method of Claim 1 , wherein said step (c) of detecting comprises detecting biological activity of said cyclooxygenase.

7. The method of Claim 6, wherein said step (c) of detecting is by a method selected from the group consisting of an assay for detection of prostaglandin E2 (PGE2) activity and an assay for detection of 15-R-hydroxyeicosatefraenoic acid (15-R-HETE) activity.

8. The method of Claim 1 , wherein said step (c) of detecting comprises detecting production of a cyclooxygenase biochemical endproduct.

9. The method of Claim 8, wherein said biological endproduct is selected from the group consisting of a prostaglandin, prostacyclin and thromboxane A₂.

10. The method of Claim 1 , further comprising, after said step (a) of obtaining and prior to step (e) of making a diagnosis, a step of detecting whether said sample carries an oncogenic RNA or DNA virus; wherein detection of said oncogenic RNA or DNA vims, in combination with detection of increased cyclooxygenase expression or biological activity as compared to said baseline level of cyclooxygenase expression or biological activity, indicates a positive diagnosis of vims-associated neoplastic fransformation or a potential therefor.

11. The method of Claim 10, wherein said oncogenic DNA vims is selected from the group consisting of Papovavimses, Herpesviruses, Hepadnaviruses and Poxvimses.

12. The method of Claim 10, wherein said oncogenic DNA vims is selected from the group consisting of human papilloma vims, polyoma vims and Simian viras 40 (SV40).

13. The method of Claim 10, wherein said oncogenic DNA vims is human papilloma virus.

14. The method of Claim 10, wherein said oncogenic RNA vims is selected from the group consisting of human lymphotropic viras and human immunodeficiency viras.

15. The method of Claim 10, wherein said step of detecting whether said sample carries an oncogenic RNA or DNA viras is selected from the group consisting of PCR, RT- PCR, Northern blot, Southern blot, sequence analysis, and in situ hybridization.

16. The method of Claim 10, wherein said step of detecting whether said sample carries an oncogenic RNA or DNA viras is selected from the group consisting of immunoblot, enzyme-linked immunosorbant assay (ELIS A), radioimmunoassay (RIA), and immunoprecipitation.

17. The method of Claim 1, wherein said bodily fluid is selected from the group consisting of mucous, seminal fluid, saliva, breast milk, bile and urine.

18. The method of Claim 1 , wherein detection of at least about a 1.5-fold increase in cyclooxygenase expression or biological activity in said sample as compared to said baseline level, indicates a positive diagnosis for said sample.

19. The method of Claim 1 , wherein detection of at least about a 3 -fold increase in cyclooxygenase expression or biological activity in said sample as compared to said baseline level, indicates a positive diagnosis for said sample.

20. The method of Claim 1, wherein said sample is from a tissue selected from the group consisting of cervix, uterus and prostate, and wherein said positive diagnosis is indicative of cervical cancer, uterine cancer or prostate cancer, respectively.

21. The method of Claim 1 , wherein said baseline level is established by a method selected from the group consisting of: a. establishing a baseline level of expression or biological activity of said cyclooxygenase in an autologous control sample from said patient, wherein said autologous sample is of a same cell type, tissue type or bodily fluid type as said sample of step (a); b. establishing a baseline level of expression or biological activity of said cyclooxygenase that is an average from at least two previous detections of expression or biological activity of said cyclooxygenase in a previous sample from said patient, wherein each of said previous samples were of a same cell type, tissue type or bodily fluid type as said sample of step (a), and wherein said previous evaluations resulted in a negative diagnosis; and, c. establishing a baseline level of expression or biological activity of said cyclooxygenase from control samples of a same cell type, tissue type or bodily fluid type as said sample of step (a), said control samples having been obtained from a population of matched individuals.

22. A method for diagnosing neoplastic fransformation or a potential for neoplastic fransformation associated with an oncogenic virus, said method comprising: a. obtaining from a patient, who has been identified as having been infected with an oncogenic vims, a sample to be evaluated for neoplastic transformation, said sample selected from the group consisting of a cell sample, a tissue sample, and a bodily fluid sample; b. detecting expression or biological activity of at least one cyclooxygenase in said sample, said cyclooxygenase being selected from the group consisting of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2); wherein detection of increased expression or biological activity of said cyclooxygenase over a baseline expression or biological activity of said cyclooxygenase indicates a positive diagnosis of vims-associated neoplastic fransformation.

23. A method for diagnosing neoplastic fransformation or a potential for neoplastic transformation associated with an oncogenic vims, said method comprising: a. obtaining from a patient a sample selected from the group consisting of a cell sample, a tissue sample, and a bodily fluid sample; b. detecting whether said sample carries an oncogenic RNA or DNA vims; and, c. detecting expression or biological activity of at least one cyclooxygenase in said sample, said cyclooxygenase being selected from the group consisting of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2); wherein detection of: (1) said oncogenic vims in said sample, and (2) increased expression or biological activity of said cyclooxygenase, over a baseline expression or biological activity of said cyclooxygenase, indicates a positive diagnosis of vims-associated neoplastic transformation or a potential therefor.

24. A method for diagnosing cervical, uterine or prostate cancer or a potential for development of cervical, uterine or prostate cancer, said method comprising: a. obtaining from a patient a sample from a tissue selected from the group consisting of cervix, uteras and prostate tissue to be evaluated for neoplastic transformation, said sample selected from the group consisting of a cell sample, a tissue sample, and a bodily fluid sample; b. detecting expression or biological activity of at least one cyclooxygenase in said sample, said cyclooxygenase being selected from the group consisting of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2); wherein detection of increased expression or biological activity of said cyclooxygenase over a baseline level of expression or biological activity of said cyclooxygenase in said cervix, uterine or prostate sample indicates a positive diagnosis of cervical, uterine or prostate cancer, respectively, or a potential for development of cervical, uterine or prostate cancer, respectively.

25. The method of Claim 24, wherein said method further comprises a step of detecting whether said sample carries papilloma viras.

26. An assay kit for diagnosing neoplastic transformation or a potential for neoplastic fransformation in a patient, comprising: a. a means for detecting the presence of an oncogenic viras in a sample obtained from a patient, wherein said sample is selected from the group consisting of a cell sample, a tissue sample and a bodily fluid sample; and, b. a means for detecting expression or biological activity of at least one cyclooxygenase in said sample, said cyclooxygenase being selected from the group consisting of cyclooxygenase- 1 (COX-1) and cyclooxygenase-2 (COX-2).

27. The assay kit of Claim 26, wherein said assay kit is configured to test a bodily fluid selected from the group consisting of mucous, seminal fluid, saliva, breast milk, bile and urine.

28. The assay kit of Claim 26, wherein said means for detecting the presence of an oncogenic viras is selected from the group consisting of: a probe that hybridizes under stringent hybridization conditions to a nucleic acid molecule from said viras; PCR primers which amplify a nucleic acid molecule from said viras; an antibody that selectively binds to an oncogenic viral protein in said sample and a viral antigen that can be bound by anti- viras antibodies in said sample.

29. The assay kit of Claim 26, wherein said means for detecting expression or biological activity of said cyclooxygenase is selected from the group consisting of: a probe that hybridizes under stringent hybridization conditions to a nucleic acid molecule encoding said cyclooxygenase or a fragment thereof; RT-PCR primers for amplification of mRNA encoding said cyclooxygenase or a fragment thereof; an antibody that selectively binds to said cyclooxygenase; a probe that hybridizes under stringent hybridization conditions to a nucleic acid molecule encoding a biochemical endproduct of said cyclooxygenase or a fragment thereof; RT-PCR primers for amplification of mRNA encoding a biochemical endproduct of said cyclooxygenase or a fragment thereof; and an antibody that selectively binds to a biochemical endproduct of said cyclooxygenase.

30. The method of Claim 29, wherein said biological endproduct of said cyclooxygenase is selected from the group consisting of a prostaglandin, prostacyclin and thromboxane A₂.

31. The method of Claim 26, wherein said means for detecting in part (a) is conjugated to a detectable marker.

32. The method of Claim 26, wherein said means for detecting in part (b) is conjugated to a detectable marker.

33. The method of Claim 26, wherein said means for detecting of part (a) is immobilized on a substrate.

34. The method of Claim 26, wherein said means for detecting of part (b) is immobilized on a substrate.

35. A method for evaluating the carcinogenicity of a compound, said method comprising: a. contacting with said compound being tested for carcinogenicity a test cell; and, b. detecting expression or biological activity of at least one cyclooxygenase by said test cell, said cyclooxygenase being selected from the group consisting of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), wherein an increase in expression of said cyclooxygenase in said test cell after contact with said compound as compared to before contact with said compound indicates that said compound is carcinogenic.

36. The method of Claim 35, wherein said test cell is transfected with a recombinant nucleic acid molecule from a DNA or RNA viras encoding a transforming protein, wherein said test cell maintains a copy number of said recombinant nucleic acid molecule of within at least about 50% over 5 passages of said test cell

37. The method of Claim 36, wherein said recombinant nucleic acid molecule is from a DNA viras .

38. The method of Claim 36, wherein said recombinant nucleic acid molecule is from an RNA virus.

39. The method of Claim 36, wherein said recombinant nucleic acid molecule is from a papilloma viras.

40. The method of Claim 36, wherein said recombinant nucleic acid molecule is from bovine papilloma vims (BPV).

41. The method of Claim 36, wherein said recombinant nucleic acid molecule is from the group consisting of the BPV E5, E6 and E7 open reading frames.

42. The method of Claim 36, wherein said recombinant nucleic acid molecule is selected from group consisting of the BPV E6 open reading frame and the BPV E7 open reading frame.

43. The method of Claim 36, wherein said recombinant nucleic acid molecule is a BPV genome in which a viral gene selected from the El and E2 open reading frames has been removed or made non-functional.