DETERMINATION OF THE PRESENCE OF ABNORMAL

CELLULAR PROLIFERATION THROUGH THE DETECTION OF

ONE OR MORE CYCLIN DEPENDENT KINASES

This is a continuation-in-part of PCT Patent Application PCT/US94/00961 filed

January 21, 1994 which claims priority for U.S. Patent Application Serial No. 08/075,744 filed January 21, 1993 which in turn is a continuation-in-part application of 08/007,636 filed June 11 , 1993.

FIELD OF THE INVENTION

This invention relates to in vitro and in vivo assays for measuring cell growth propensity, and for measuring the concentration of at least one cyclin dependent kinase in tlie blood, plasma or serum of an animal or human and correlating that concentration to the presence of abnormal cellular proliferation or tumor in any tissue of the organism from which the sample was obtained.

BACKGROUND OF INVENTION

Cell proliferation is the most fundamental phenotypic property of cancer. The stimulus for cellular proliferation is central not only at the late steps in carcinogenesis, the cancer, but also at the earliest known step, initiation (1,2) and Figure 1. In fact, cell proliferation exerts an influence in the initiation of carcinogenesis in that cells in the S phase are more sensitive toward many initiators than at other times in the cell cycle (3). A myriad of short-term tests exist for the assessment of the carcinogenic potential of chemicals. These tests detect only carcinogens that interact with nucleic acids, or induce DNA repair synthesis or mutations in bacterial or mammalian cells (4-8).

As testing of the genotoxicity and carcinogenicity of chemicals has become routine, a growing number of compounds have been found that induce tumors in chronic bioassays while exhibiting negative results in genotoxicity tests (9). Significant examples of these classes of compounds include the dioxins, chlorinated biphenyls and peroxisome proliferators. These chemicals are often active as tumor promoters in two-stage experiments and exhibit biological activities as hormones (ethinylestradiol), peroxisome proliferators (pirnixic acid) or enzyme inducers (phenobarbital) (10).

At the present time only the initiation- promotion assay is employed routinely. In this assay the test compounds are examined for their ability to promote hepatic tumors or foci formation after initiation with a known genotoxic agent (11,12). Currently this assay utilizes animals, requires several months to perform, and produces histological endpoints that are difficult to quantify and do not lend to rigorous dose- response calculations for the purposes of risk assessment (13).

Stimulation of DNA synthesis has been proposed as an assay for short-term assessment of nongenotoxic carcinogens and tumor promoters in vivo (14,15). This methodology has potential for application to routine testing. So far, only one result has been detected that is inconsistent with carcinogenicity bioassay data. The different carcinogenicity of di(2-ethylhexyl)adipate (negative in rats) and di(2-ethylhexyl)phthalate (positive) was not detectable by DNA stimulation index using ³ H-thymidine. Both plasticizers were positive in this short-term system with doubling doses of 0.7 mmol/kg for di(2-ethylhexyl)adipate and 0.5 mmol/kg for di(2-ethylhexyl)phthalate. Other disadvantages of this system include the use of radioactivity and the high coefficient of variation in the endpoint.

Several in vitro models have been utilized for the assessment of nongenotoxic carcinogens. Chida et al. (16) modeled the activation of protein kinase C and specific phosphorylation of a 90,000 kDa membrane protein of promotable BALB/3T3 and C3H/IOT1/2 cells by tumor promoters. Smith and Colburn also utilized protein kinase C and its substrates in tumor promoter-sensitive and tumor-resistant cells as a biochemical marker for the response of cells to tumor promoters (17). However, these systems were flawed by both false positive and false negative values. The false positive values may be

due to the fact that the activation of protein kinase C represents a biochemical signal far upstream from the final proliferative signal, while the false negatives may result from the fact that protein kinase C represents only a single receptor-mediated response. At least four other receptor responses, which are independent of protein kinase C, are known for tumor promotion and activity of nongenotoxic carcinogens (e.g. dioxin receptor, peroxisome proliferator receptor, phenobarbital receptor and estrogen receptor) (14,18).

Protein tyrosine phosphorylation

Protein-tyrosine kinases (PTK) constitute a class of enzymes that catalyze the transfer of the γ- phosphate of either ATP or GTP to specific tyrosine residues in certain protein substrates. Evidence suggests that these enzymes are important mediators of normal cellular signal transduction (19-21), with PTK being the intracellular effectors for many growth hormone receptors (22-24). PTKs are also frequently the products of proto- oncogenes (25) and their aberrant expression has been associated with a variety of human cancers (26).

The cascade of protein tyrosine phosphorylation following the activation of protein tyrosine kinases appears to regulate the proliferative response (27, 28). Specifically, protein tyrosylphosphorylations are common to a wide variety of nongenotoxic carcinogens independent of associated receptors or known mechanism of action. The present invention demonstrates the xenobiotic alterations in protein tyrosine phosphorylation at a fundamental point in the control of cellular proliferation and on an assay protocol that characterizes the ability of a xenobiotic test chemical to initiate cellular proliferation.

Cyclin proteins and Cyclin-dependent kinases (CDK)

Intrinsic defects in the cell cycle machinery may themselves help cause cancer. Recent research has indicated that tumor cells tend to overexpress the cyclin dependent kinases (CDKs) as wells as the cyclins. It is becoming increasingly apparent that the transeriptional regulation of the CDK is important in the control differentiation and cellular proliferation. It must be noted that the assays of the invention do not measure of the kinase activity associated with the p34 ^cdc2 enzyme as described by others and in other microtiter assays (52). In particular, the assay taught by Ducommen & Beach (52) measures kinase activity - usually with histone HI

as the substrate - which is maximal at the G ₂ /M phase transition and is associated with the p34/cyclin B complex. Ducommen and Beach also suggest an immunoassay, however, they provide no teachings with regard to how such an assay would work with the p34/cyclin B complex nor do they teach an antibody that is capable of recognizing the complex. Furthermore, they do not teach that the concentration of CDK is correlated to cellular proliferation.

The teachings of the prior art falsely assumed that the levels of CDK remained the same from the G ₀ to G* phase and throughout the cell cycle. Although once in the cell cycle, levels of CDK remain relatively stable, when the cell progresses from G ₀ to G- phase the level of CDK expression or concentration increases, contrary to what was previously assumed by prior art.

If the cyclins are overproduced in a cell or made at the wrong time, they would be expected to stimulate inappropriate cell division by keeping their partner kinases "on" when they should be turned off — a malfunction that could lead to cancer. That is just what appears to be happening with cyclin Dl (29, 30 ). The gene encoding one component of the cell cycle machinery, a protein called cyclin Dl, is apparently an oncogene itself and several others are oncogene candidates. Still other genes, which code for a group of newly discovered cell cycle inhibitors, including the one made in response to the p53 protein, have the potential to be tumor suppressors.

Some of the most compelling evidence that points to the direct involvement of cell cycle components in cancer comes from studies of cyclin Dl. This protein is one of a group of eight or so cyclins so far discovered in mammalian cells. The name cyclin comes from the fact that their concentrations rise and fall in a regular pattern during the cell cycle, a pattern that enables them to do their critical job: turning on, at the appropriate moment, enzymes called cyclin dependent kinases (CDKs), whose activity is needed to propel cells through the cell cycle.

The D cyclins are active at a particularly critical time in the cell cycle: during the Gl phase when cells grow and decide whether to begin replicating their DNA in preparation for cell division (Figure 2). Overexpression of the gene for cyclin Dl may contribute to more

common cancers, including those of the breast and esophagus. The cyclin Dl gene is both amplified and producing greater than normal amounts of protein in about 15% of breast cancers and approximately one-third of esophageal cancers examined (29). In some circumstances the cyclin Dl gene can cooperate with the ras oncogene in transforming cells (31).

While cyclin Dl has been the focus of most research on cell transformation, it is not the only cyclin whose expression is altered in cancer cells. The gene for cyclin E, which also becomes active during the Gl phase of the cell cycle, is overexpressed in cultured breast cancer cell lines and in primary breast tumors.

Cyclin A may also be involved in oncogenesis. Like cyclins D and E, cyclin A is important for the passage into the DNA-synthesizing stage of the cell cycle. In addition, overexpression of the cyclin A gene may lead to another classic feature of cancer cells: the ability to grow without being anchored to a surface.

The partners of the cyclins in the control of the cell cycle, the kinases, may also be significant in terms of the transformation of a normal cell into a cancer cell. Cyclin dependent kinases were originally believed to have an apparent molecular weight between 32 and 34 kD, however, as new CDKs are discovered, this range has expanded to between 30-37 kD. Among the nine or so kinases that have been identified, CDK4 is perhaps most likely to serve as an oncogenic protein. Cells that have been altered so that they produce CDK4 all the time cannot be inhibited by TGF-beta. Since many cancer cells lose their responsiveness to TGF-beta's growth suppressing effects, these results raise the possibility that CDK4 production may contribute to the failure of growth controls in cancer cells.

CDKSs as tumor markers

If the cyclins and CDKs contribute to cancer, they would presumably act positively, like the oncogenes. However, cell cycle inhibitors might be more like the negatively acting tumor suppressors whose loss or inactivation leads to cancer. Currently there are three such inhibitors. One is made in response to p53 and apparently mediates its growth suppressive effects by blocking the activity of CDK2 and other CDKs. The second cell cycle inhibitor is more specific in its action, apparently blocking only CDK4. The third helps to mediate TGF-

beta's inhibitory effects and also the growth inhibition brought about when the cells come into contact with each other.

Diagnosing cancer by detecting the presence of elevated amounts of cancer markers offers inherent advantages over other types of cancer diagnostics. Markers can be measured in blood, serum or urine tests. However, the disadvantage is that markers are not considered as accurate as the other diagnostic methods. Therefore, research into developing more accurate cancer markers continues.

Currently about five cancer markers are FDA approved for diagnosing cancer and for monitoring disease progression or therapeutic effectiveness. No markers have been approved for screening healthy people for cancer. Some of the available markers and the cancers they are used to detect or monitor are:

A) Prostate specific antigen (PSA) for prostate cancer. PSA is probably considered the most accurate of all tumor markers.

B) Prostatic acid phosphatase (PAP), which is also used for prostate cancer. PAP is not considered as sensitive as PSA in therapeutic drug monitoring or in the early detection of new and recurring cases of prostate cancer.

C) Carcinoembryonic antigen (CEA), which is used for lung, breast, liver, pancreas, stomach and colon cancers. CEA levels are monitored periodically to detect cancer recurrence and are often monitored in surgery patients. However, CEA levels also may be increased in benign tumors and can vary widely among people. Consequently, CEA tests have been found to have a high rate of false positives.

D) CAl 25 for ovarian, pancreatic, breast and lung cancers.

E) Alpha fetoprotein (AFP), which is used primarily for diagnosing liver cancer and to a lesser extent, for ovarian and testicular cancers. Elevated

AFP levels are also sometimes found in cancers of the lungs and digestive system.

F) Estrogen and progesterone receptors, which have their presence monitored to indicate prognosis of hormone therapy.

With the possible exception of PSA, most of the currently approved tumor markers have not received widespread acceptance among practicing oncologists. Physicians contend that most markers are best at detecting tumors that have reached advanced stages, but that markers are not as accurate in detecting cancers at their earliest stages when they are most treatable. Oncologists also maintain that the results of tumor marker tests must too often be confirmed through further testing. Given the shortcomings of some markers, an extensive amount of research is being conducted into developing new tumor markers, yet no single marker yet described appears useful in detecting a wide range of cancers.

Examples of cancer markers in development include:

C-reactive peptide (CRP) Hodgkin's disease

Interleukin- 10 (IL-10) Non-Hodgkin's lymphoma

CA125 in combination with immunosuppressive acetic Ovarian cancer protein, tissue polypeptide antigen, amylase and alkaline phosphatase

CA125 with M-CSF and OVXl Ovarian cancer

CA125 and lipid-associated sialic acid Ovarian cancer

Combination of CA27-29, CA15-3 mucin-like antigen, Breast and gastrointestinal and CA19-9 cancers

CA15-3, TPS Breast cancer

CA15-3 Breast cancer

CA549 and CEA Breast cancer

CA15-3, CEA, TPA Breast cancer

Tumor-associated glycoprotein antigen Breast cancer

Tissue polypeptide-specific antigen Breast cancer

Soluble interleukin-2(sIL-2) and sIL-2 Breast cancer receptors

HER-2/neu-related protein NRP Breast cancer

DES-R-CARBOXY PROTHROMBIN Hepatocellular carcinoma

Insulin-like growth factor (IGF-l) Neuroendocrine neoplasms

Progastrin-releasing peptide fragment Lung cancer

CYFRAN21-1 Lung cancer

Neuron-specific enolase Lung cancer

Ring-shaped particle Lung cancer

OVXl Endometrial cancer

PTH RP1-34, N-term, PTH RP1-86 Lymphoma

Pyridinoline Bone cancer

Soluble interleukin-6 receptor Melanoma

Ki-67 Several rapidly dividing tumors

The gene p53 and the gene's protein product, also called p53, were discovered in 1979 during research into cells infected with the simian virus 40. For the next 10 years, research into p53 proceeded slowly, and the p53 gene was largely dismissed as one of the less important oncogenes. The medical significance of p53 was not realized until 1989 when two important discoveries occurred:

A) Researcher investigating possible genetic causes of colon cancer identified a point mutation in p53 that appeared to be linked to the onset of colon cancer.

B) Separate research found that only mutated p53 fosters the abnormal cell growth occurring in cancer. The wild type of p53 gene was found to suppress tumors. Further, the role of p53 in halting normal cell division and promoting the normal self-destruction of cells was elucidated. In short, when p53 functions normally, it is involved in halting cell growth and thus helps to prevent cancer. When mutated, p53 contributes to abnormal cell growth and suppresses the healthful function of the wild gene.

The ubiquity of p53 is spurring research into its use as a diagnostic marker. Initial indications are that p53 markers may be most useful in the prognosis of cancers because mutated forms of p53 have been correlated with metastatic cancer. Preliminary research has focused on the prognosis of breast, colon, lung, cervical, bladder, kidney and prostate cancers and melanoma. Most of the reported research has been conducted at universities and research institutions rather than in corporations, although it is widely expected that the prevalence of p53 will lead to extensive corporate involvement. The diagnostic technologies reported to have been used most frequently in p53-related research are flow cytometry and polymerase chain reaction.

Recent experimental evidence suggests that the cell cycle of all eukaryotic cells is controlled at several checkpoints by different members of a novel class of protein kinase,

the cyclin-dependent kinases (32, 34, 39, 49). The most well known of these kinases is the 34 kD product of the cdc2 gene in the fission yeast, p34 ^cdc2 ; however, several putative cyclin-dependent kinases (CDK) have now been cloned or identified. Some of these clones resemble p34 cdc2

At least nine CDKs have been described in the literature; these all have a common

PSTAIR epitope. Therefore anti-PSTAIR would be expected to cross react with the entire complement of CDKs showing up in the 32 to 34 kD region. (Apparently some cyclins also cross react with the anti-PSTAIR antibody and this explains the banding at approximately 60 kD observed in some of the immunoblots with anti-PSTAIR.)

The antibody to the C-terminus region is more specific for p34 ^cdc2 kinase, since the

C-terminus region is more variable than the highly conserved PSTAIR region. However, it is obviously not species-specific since it was generated against human cdc2 and it cross reacts with mouse, rat and dog p34 ^cdc2 kinase.

SUMMARY OF THE INVENTION

The invention provides a method for measuring cyclin dependent kinase concentration in tissue, blood, plasma, serum or cell samples. The invention also includes a kit measures cyclin dependent kinase concentration in the sample.

The invention provides a diagnostic method for determining whether a tissue has undergone transformation to a cancerous phenotype, said method comprising measuring a parameter that indicates the concentration of at least one cyclin dependent kinase in tissue, blood, plasma, serum or cell line sample and indicating a likelihood of transformation in relation to the level of CDK.

The invention provides a diagnostic method for determining cell growth propensity comprising measuring a parameter indicative on concentration of at least one cyclin dependent kinase in tissue, blood, plasma, serum or cell line sample and indicating a cell growth propensity in relation to the level of CDK.

The invention includes method of measuring carcinogenicity of a test substance comprising:

a) selecting a test substance to be tested for carcinogenicity;

b) selecting an assay system selected from the group comprising: an animal, cell culture, cell lines, or a panel of tissue cells or tissues capable of expressing cyclin dependent kinase;

c) contacting said test substance to said assay system

d) measuring a parameter that indicates concentration of at least one cyclin dependent kinase;

e) indicating a relative carcinogenicity for said test substance in relation to said parameter that indicates said concentration of at least one cyclin dependent kinase.

The invention also provides a method for determining efficacy of a regimen for reducing or enhancing cell growth, said method comprising the steps of measuring at parameter indicative of concentration levels of at least one cyclin dependent kinase following treatment of animals or humans with said regimen and determining efficacy in relation to cyclin dependent kinase concentration.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Schematic of the multistage nature of carcinogenesis. Nongenotoxic carcinogens and tumor promoters affect, respectively, defects in terminal differentiation and selective clonal expansion of initiated cells.

Figure 2. Schematic of the role of cyclins and cyclin dependent kinases in the progression of a cell through the four phases of the cell cycle. The CDKs, complexed with an appropriate phase cyclin, coordinate the movement of cells through the cycle until they divide in M phase (mitosis).

Figure 3. The cell cycle. A cell can either be quiescent or continue to grow. The decision point is early in the G* phase when a cell either passes START - and then is committed to growing, finishing the rest of the cycle and dividing (G-, S, G ₂ and M) - or the cell enter the G ₀ state in which it continues to metabolize but does not grow.

Figure 4. Immunoblot using anti-PSTAIR antibody. An anti-phosphotyrosine immunoprecipitate of the murine hepatic S-9 protein is separated using an 11 % SDS- PAGE gel. The separated proteins are transferred to a blotting membrane and probed with the anti-PSTAIR antibody.

Figure 5. Scanning densitometry of anti-PSTAIR immunoblots for hepatic S-9 fraction of 2,3,7,8- tetrachlorodibenzo-p-dioxin treated female, C57BL/6J mice.

Figure 6. Bar graph depicting the quantification of the results of the scanning densitometry. The cyclin dependent kinase (CDK) quantified from the anti-PSTAIR immunoblot was at 32 kDa. The administration of a single dose of 2,3,7,8- tetrachlorodibenzo-p-dioxin results in enhanced tyrosylphosphorylation of the CDK compared to control animals, which exhibit no tyrosylphosphorylation of CDK. Each group on the graph represents the single result of scanning an anti-PSTAIR immunoblot produced from the pooled hepatic S-9 of three animals. Error bars represent the 10 percent coefficient of variation in the quantification of density.

Figure 7. A typical BIAcore* sensorgram produced on immobilization of anti-cdc2 kinase C- terminus.

Figure 8. Anti-phosphotyrosine immunoblots of rat hepatic S-9 protein separated using 11 % SDS-PAGE gels for pirnixic acid-treated (lanes 1,2) and control (3,4) rats. Each lane represents a single rat.

Figure 9. Scanning densitometry of anti- phosphotyrosine immunoblots for pirnixic acid-treated rats [A and B] and paired vehicle controls [C and D, respectively]. Bolding of peaks indicates difference of greater than 40 percent between treatment and control.

Figure 10. Bar graph depicting the quantification of the results of the scanning densitometry. The phosphotyrosyl protein quantified from the anti-phosphotyrosine

immunoblot was at 33 kDa. Results indicate that the administration of five, twice- daily doses of pirnixic acid (50 mg/kg each dose) produces enhanced tyrosylphosphorylation of p33 compared to control animals, which exhibit no tyrosylphosphorylation at 33 kDa. Each group on the graph represents the average of two rats. Error bars represent the 10 percent coefficient of variation in the quantification of density.

Figure 11. BIAcore* sensorgram displaying binding of pirnixic acid-treated S-9 protein and control S-9 protein over immobilized anti-cdc2 PSTAIR monoclonal antibody.

Figure 12. Summary bar graph depicting BIAcore* quantification of the interaction of tyrosylphosphorylated cyclin dependent kinases (CDK) with anti-CDK monoclonal antibodies (PSTAIR and C-terminus) from control and pirnixic acid-treated rats. Error bars represent standard deviations of n = 6 (anti-PSTAIR) and n = 8 (anti-C Terminus) control rats. RU (response units) value for pirnixic acid-treated rats represents the mean of 2 animals. The treatment of rats with 50 mg pirnixic acid/kg twice a day for 5 days results in enhanced tyrosylphosphorylation of CDK (p34 ^cdc2 kinase) compared to control rats.

Figure 13. Anti-phosphotyrosine immunoblots of rat hepatic S-9 protein separated using 11 % SDS-PAGE gels for diethylhexylphthalate-treated (lanes 1 ,2) and control (3,4) rats. Each lane represents a single rat.

Figure 14. Scanning densitometry of anti- phosphotyrosine immunoblots for diethylhexylphthalate- treated rats [A and B] and paired vehicle controls [C and D, respectively]. Bolding of peaks indicates difference of greater than 40 percent between treatment and control.

Figure 15. Bar graph depicting the quantification of the results of the scanning densitometry. The phosphotyrosyl protein quantified from the anti-phosphotyrosine immunoblot was at 34 kDa. Results indicate that the administration of five, twice- daily doses of diethylhexylphthalate (500 mg/kg each dose) produces enhanced tyrosylphosphorylation of the p34 compared to control animals, which exhibit no tyrosylphosphorylation at 34 kDa. Each group on the graph represents the average of two rats. Error bars represent the 10 percent coefficient of variation in the quantification of density.

Figure 16. Summary bar graph depicting BIAcore* quantification of the interaction of tyrosylphosphorylated cyclin dependent kinases (CDK) with anti-CDK monoclonal antibodies (PSTAIR and C-terminus) from control and diethylhexylphthalate-treated rats. Error bars represent standard deviations of n = 6 (anti- PSTAIR) and n = 8 (anti-C Terminus) control rats. RU value for diethylhexylphthalate-treated rats represents the mean of 2 animals. The treatment of rats with 500 mg diethylhexylphthalate/kg twice a day for 5 days produces enhanced tyrosylphosphorylation of CDK (p34 ^cdc2 kinase) compared to control rats.

Figure 17. Anti-phosphotyrosine immunoblots of rat hepatic S-9 protein separated using 11 % SDS-PAGE gels for diethylnitrosamine-treated (lanes 3,4) and control (lanes 1,2) rats.

Figure 18. Scanning densitometry of anti-phosphotyrosine immunoblots for diethylnitrosamine-treated rats [A and B] and paired vehicle controls [C and D, respectively]. Bolding of peaks indicates difference of greater than 40 percent between treatment and control.

Figure 19. Bar graph depicting the quantification of the results of the scanning densitometry. The phosphotyrosyl protein quantified from the anti-phosphotyrosine immunoblot was at 34 kDa. Results indicate that the administration of five, twice-daily doses of diethylnitrosamine (500 mg/kg each dose) produces no enhanced tyrosylphosphorylation of p34compared to control animals. Each group on the graph represents the average of two rats. Error bars represent the 10 percent coefficient of variation in the quantification of density.

Figure 20. Summary bar graph depicting BIAcore ^® quantification of the interaction of tyrosylphosphorylated cyclin dependent kinases (CDK) with anti-CDK polyclonal antibodies (PSTAIR and C-terminus)from control and diethylnitrosamine-treated rats. Error bars represent standard deviations of n = 6 (anti-PSTAIR)and n = 8 (anti-C- terminus) control rats. RU value for diethylnitrosamine-treated rats represents the mean of 2 animals. Results indicate that the treatment of rats with 500 mg diethylnitrosamine/kg

twice a day for 5 days produces no enhanced tyrosylphosphorylation of CDK (p34 ^cdc2 kinase) compared to control rats.

Figure 21. Anti-phosphotyrosine immunoblot of dog hepatic S-9 protein separated using 11 % SDS-PAGE gels for Aroclor*- treated dogs. Lanes 1, 2, 3, 4 and 5 are control, 0.6, 0.8, 4 - 8, and 5 - 10 mg Aroclor*/kg-day, respectively.

Figure 22. Scanning densitometry of anti- phosphotyrosine immunoblots at 34 kDa for Aroclor*- treated dogs. From top to bottom the figures represent 0.6, 0.8, 4 - 8, and 5 - 10 mg Aroclor*/kg-day, respectively.

Figure 23. Bar graph depicting the quantification of the scanning densitometry of the putative cyclin dependent kinase (p34) from the anti-phosphotyrosine immunoblot. The daily administration of Aroclor* for a period of 11.5 weeks results in enhanced tyrosylphosphorylation of the p34 at all doses compared to the control dog. Each bar on the graph represents the result of scanning an immunoblot produced from the hepatic S-9 of a single dog. Error bars represent the 10 percent coefficient of variation in the quantification of density.

Figure 24. Anti-phosphotyrosine immunoblots of 3T3 cell lysate protein separated using 11 % SDS-PAGE gels for 3T3 cells exposed to 10 nM 2,3,7,8- tetrachlorodibenzo-p- dioxin (lane 3B) or DMSO vehicle (lane IB) for 24 h in 0.5% serum supplemented media.

Figure 25. Scanning densitometry of anti- phosphotyrosine immunoblots for 3T3 cells treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or DMSO vehicle (Control) for 24 h in 0.5% serum media. Bolded peaks indicate p34 and p33 tyrosylphosphoproteins.

Figure 26. Bar graph depicting the quantification of the scanning densitometry of the putative cyclin dependent kinases (p34/p33) from the anti-phosphotyrosine immunoblot. Exposure of 3T3 cells to 10 nM 2,3,7,8-tetracholordibenzo-p-dioxin for 24 h results in an increase in tyrosylphosphorylation of p34 and p33 of 67 and 32%, respectively, compared to the vehicle control. Each bar on the graph represents the result of scanning an immunoblot produced from the pooled whole cell lysates of four plates per

treatment. Error bars represent the 10 percent coefficient of variation in the quantification of density.

Figure 27. Anti-phosphotyrosine immunoblots of 3T3 cell lysate protein separated using 11 % SDS-PAGE gels for 3T3 cells exposed to 160 nM 12-0-tetra- decanoylphorbol- 13-acetate (TPA; lane 4B) or DMSO vehicle (Control; lane IB) for 24 h in 0.5% serum supplemented media.

Figure 28. Scanning densitometry of anti- phosphotyrosine immunoblots for 3T3 cells treated with 160 nM 12-0-tetra-decanoylphorbol- 13-acetate (TPA) or DMSO vehicle for 24 h in 0.5% serum media. Bolded peaks indicate p34 and p33 tyrosylphosphoproteins.

Figure 29. Bar graph depicting the quantification of the scanning densitometry of the putative cyclin dependent kinases (p34/p33) from the anti-phosphotyrosine immunoblot. Exposure of 3T3 cells to 160 nM 12-0-tetra-decanoylphorbol- 13-acetate (TPA) for 24 h results in an increase in tyrosylphosphorylation of p34 and p33 of 54% and 95%, respectively, compared to the vehicle control. Each bar on the graph represents the result of scanning an immunoblot produced from the pooled whole cell lysates of four plates per treatment. Error bars represent t e 10 percent coefficient of variation in the quantification of density.

Figure 30. Anti-phosphotyrosine immunoblots of BNL CL.2 cell lysate protein separated using l it SDS-PAGE gels for BNL CL.2 cells exposed to 0.1 , 1, 10, or 100 nM2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; lanes 3, 4, 5 and 6, respectively) or DMSO vehicle (lane 1) for 24 h in 0.5% serum supplemented media. Lane 2 is the 20% serum-supplemented control.

Figure 31. Scanning densitometry of anti-phosphotyrosine immunoblots in the 35 to 30 kDa molecular weight range for BNL CL.2 cells treated with 0.1, 1, lOor 100 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or DMSO vehicle (Control) for 24 h in 0.5% serum supplemented media. p34 and p33 tyrosylphosphoproteins are indicated for the respective treatments. Top row (left to right) 0.5% and 20% serum supplementation; Middle row 0.1 and 1 nM TCDD; Bottom row 10 and 100 nM TCDD.

Figure 32. Bar graphs depicting the quantification of the scanning densitometry of the putative cyclin dependent kinases (p34-top/p33-bottom) from the anti-phosphotyrosine immunoblot. Exposure of BNL CL2 cells to 0.1, 1, 10 or 100 nM 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD) for 24 h results in a similar increase in tyrosylphosphorylation of p34, averaging 180% of the vehicle control over all concentrations of TCDD. Twenty percent serum supplementation results in an increase of tyrosylphosphorylation of p34 of 229% of the vehicle control. Vehicle controls at 0.5% serum supplementation exhibit no tyrosylphosphorylation at p33, while TCDD exposure at the four concentrations enhances tyrosylphosphorylation of this putative CDK to 0.9, 2.0, 2,0 and 1.9 density units, respectively. The increases in tyrosylphosphorylation of p33 by TCDD are 3.4 times the p33 tyrosine phosphorylation produced by 20% serum supplementation. Each bar on the graph represents the result of scanning an immunoblot produced from the pooled whole cell lysates of four plates per treatment. Error bars represent the 10 percent coefficient of variation in the quantification of density.

Figure 33. Anti-phosphotyrosine immunoblots of BNL CL.2 cell lysate protein separated using 11 % SDS-PAGE gels for BNL CL.2 cells exposed to 1, 10, 100, or 1000 nM pirnixic acid (lanes 7, 8, 9 and 10, respectively) or DMSO vehicle (lane 1) for 24 h in 0.5% serum supplemented media. Lane 2 is the 20% serum-supplemented control.

Figure 34. Scanning densitometry of anti- phosphotyrosine immunoblots in the 35 to 30 kDa molecular weight range for BNL CL.2 cells treated with 1 , 10, 100, or 1000 nM pirnixic acid or DMSO vehicle (Control) for 24 h in 0.5% serum media. p34 and p33 tyrosylphosphoproteins are indicated for the respective treatments. Top row (left to right) 0.5 % serum and 20% serum; Middle row 1 and 10 nM pirnixic acid; Bottom row 100 and 1000 nM pirnixic acid.

Figure 35. Bar graphs depicting the quantification of the scanning densitometry of the putative cyclin dependent kinases (p34-top/p33-bottom) from the anti-phosphotyrosine immunoblot. Exposure of BNL CL2 cells to pirnixic acid for 24 h results in increases in tyrosylphosphorylation of p34 relative to the vehicle control for the 1 and 10 nM concentrations, 96 and 58% increases, respectively. At 100 nM pirnixic acid, the tyrosylphosphorylation of p34 is similar to the vehicle control, while at 1000 nM tyrosine

phosphorylation of p34 is depressed 60% from the vehicle control. Twenty percent serum supplementation results in an increase of tyrosylphosphorylation of p34 of 229%, relative to the vehicle control. The 0.5% serum supplementation control exhibits no tyrosylphosphorylation at p33, while pirnixic acid exposure enhances tyrosylphosphorylation of this putative CDK to 2.0, 2.5 and 0.5 density units, respectively, at the 1, 10, and 100 nM concentrations. The increases in tyrosylphosphorylation of p33 by pirnixic acid at 1 and 10 nM are roughly 4 times the p33 tyrosine phosphorylation produced by 20% serum supplementation. Each bar on the graph represents the result of scanning an immunoblot produced from the pooled whole cell lysates of four plates per treatment. Error bars represent the 10 percent coefficient of variation in the quantification of density.

Figure 36. Bar graph depicting the microtiter methodology for quantification of tyrosylphosphorylation of tissue CDK. The capture antibody was anti-PSTAIR and the secondary antibody was anti-phosphotyrosine. Dosing of C57BL/6J female mice daily with 0, 0.25, 0.5, 1 or 2 ng TCDD/kg-day (A, B, C and D, respectively) results in enhanced tyrosylphosphorylation of hepatic CDK but not pulmonary or renal CDK. This identifies the target tissue for the cellular proliferative effects of TCDD as the liver. Maximal increase in tyrosylphosphorylation of hepatic CDK is observed at the 0.5 ng TCDD/kg-day dose regimen. The error bars represent the 95 percent confidence interval of the mean absorbance determined at 415 nm for each of the treatments (n= 10 mice per treatment).

Figure 37. Bar graph depicting the microtiter methodology for quantification of tyrosylphosphorylation of tissue p34 ^cdc2 kinase. The capture antibody was anti-C terminus and the secondary antibody was anti- phosphotyrosine. Dosing of C57BL/6J female mice daily with 0. 0.25, 0.5, 1 or 2 ng TCDD/kg-day (A, B, C and D, respectively) results in enhanced tyrosylphosphorylation of hepatic p34 ^cdc2 kinase but not pulmonary or renal p34 ^cdc3 kinase. This identifies the target tissue for the cellular proliferative effects of TCDD as the liver. Maximal increase in tyrosylphosphorylation of hepatic p34 ^cdc2 kinase is observed at the 0.5 ng TCDD/kg-day dose regimen. The error bars represent the 95 percent confidence interval of the mean absorbance determined at 415 nm for each of the treatments (n = 10 mice per treatment).

Figure 38. The anti-cdc2 C-terminus immunoblot of rat hepatic S9 proteins separated using 10 to 11 % SDS PAGE gels for control (lanes 1 and 3) and WY 14,643- treated rats (lanes 2 and 4). A single intensely-stained band was visible in the CDK region (32 to 35 kDa) in hepatic S9 samples obtained from rats three days after receiving a single does of 50 mg WY14,643/kg. This band is barely visible in hepatic S9 from control rats.

Figure 39. Bar graph depicting the microtiter methodology for quantification of CDK expression in rat liver S9. The treated rats receive a single does of 50 mg pirnixic acid/kg and are killed 1 , 2 or 3 days later; control rats are dosed with the vehicle alone. The mean absorbance developed at 415 nm over 10 min is presented on the y-axis. Error bars represent standard deviations of n = 4 (1 day) and n = 5 (2 and 3 day) rat per treatment. The extent of CDK expression the livers of young, male rats receiving a single does of 50 mg/kg of WY 14,643 increases steadily during the 3-day post dosing observation period. CDK expression in control animals remains constant over the same 3- day period.

Figure 40. Anti-cdc2 C-terminus immunoblot of BNL CL.2 cell lysate protein separated using 10 to 11 % SDS-Page gels for BNL CL.2 cells exposed to 0.1, 1, or lOnM 2,3,7,8-tetrachIorodibenzo-p-dioin (TCDD; lanes 8, 9,and 10, respectively) or DMSO vehicle (lane 6) for 48 hr 0.5% serum supplemented media. Lane 7 is the 20% serum- supplemented control. TCDD exposure results in increased expression of CDK relative to the DMSO control.

Figure 41. An anti-CDK2 immunoblot of serum from normal woodchucks (lanes 1, 3, 5, 8 and 11) and woodchucks with hepatocellular carcinoma (lanes 2, 4, 6, 7, 9, 10 and 12). Sera from animals with hepatocellular carcinoma exhibited darker staining bands at 33 kDa relative to healthy animals.

Figure 42. Bar graph depicting the results of the microtiter immunoassay of woodchuck plasma or sera concentrations of CDK2 from normal woodchucks and woodchucks with hepatocellular carcinoma. Values presented represent the means of six woodchucks per group; error bar on the control group represents one standard deviation. Serum CDK2 content was

increased an average of 4.3-fold in woodchucks with hepatocellular carcinoma relative to controls.

Figure 43. An anti-CDK2 immunoblot of serum from normal dogs (lanes 1, 2, 3, 4, 5 and 6) and dogs with a variety of cancers (lanes 7, 8, 9, 10, 11, 12, 13, 14 and 15). Sera from dogs with cancers exhibited dark staining bands at 33 kDa, while the 33 kDa staining bands were not visible for any of the six healthy dogs.

Figure 44 . Bar graph depicting the results of the microtiter immunoassay of canine plasma or sera concentrations of CDK2 from normal dogs and dogs diagnosed with a variety of cancers. Values presented represent the means ± SD of 32 normal dogs and five dogs diagnosed with cancer. Serum CDK2 content was increased an average of 20-fold in dogs diagnosed with cancer relative to normal dogs.

Figure 45 . An anti-CDK2 immunoblot of serum from normal cats (lanes 1, 2, 3, 4, 5 and 6) and dogs with a variety of cancers (lanes 7, 8, 9, 10, 11, 12, 13, 14 and 15). Sera from dogs with cancers exhibited dark staining bands at 33 kDa, while the 33 kDa staining bands were not visible for any of the six healthy dogs.

Figure 46 . Bar graph depicting the results of the microtiter immunoassay of feline plasma or sera concentrations of CDK2 from normal cats and cats diagnosed with a variety of cancers. Values presented represent the means of 32 normal cats and two cats diagnosed with cancer; error bar on the control group represents one standard deviation. Serum CDK2 content was increased an average of 9.3-fold in cats diagnosed with cancer relative to normal cats.

Figure 47. An anti-CDK2 immunoblot of serum from normal human males without cancer (lanes 1 through 9) and prostate cancer patients (lanes 10 through 18). Sera from patients with prostate cancer exhibited dark staining bands at 33 kDa (CDK2), while the 33 kDa staining bands were barely visible for any of the nine healthy human males.

Figure 48. Bar graph depicting the results of the microtiter assay of serum CDK2 content of normal males (controls) and males diagnosed with prostate cancer. Values presented represent means and standard deviations of four healthy males and four males diagnosed with

prostate cancer. Mean serum CDK2 concentration of the prostate cancer group was increased 3-fold relative to the noncancer control group.

Figure 49. An anti-CDK2 immunoblot of serum from normal human females without cancer (lanes 1 through 9) and breast cancer patients (lanes 10 through 18). Sera from patients with breast cancer exhibited dark staining bands at 33 kDa (CDK2), while the 33 kDa staining bands were barely visible for any of the nine healthy human females.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In accordance with the present invention, there is provided novel methods, and assay kits for performing the methods, for measuring parameters indicative of the concentration of at least one cyclin dependent kinase in human or animal tissues, blood, plasma, cell lines, cell lysates, tissue homogenates and the like. Applicants have discovered a relationship between cyclin dependent kinase concentration and cell growth (or propensity therefor). Because cell proliferation is the most fundamental phenotypic property of cancer, the present invention has broad application to, inter alia, determining whether cells or tissue have transformed to a cancerous phenotype, determining the likelihood of such transformation later occurring, detecting and quantifying carcinogenicity of test substances (even substances which are nongenotoxic and/or nonmutagenic), testing putative antineoplastic agents, etc.

The invention also has broader applications in determining cell growth in general, and in evaluating the effectiveness of regimens designed to increase or decrease cell growth. Without intending to be bound by theory, the concentration of cyclin dependent kinase is believed to be indicative of the proportion of cells which are out of the G ₀ phase of their cell cycle. Therefore, measuring cyclin dependent kinase concentration (or a related parameter) provides a very early indication of increased cell growth (or a propensity therefor) significantly sooner than cell growth or cell transformation can be observed utilizing most other techniques.

In accordance with the invention, cyclin dependent kinase may either be measured directly or, alternatively, by measuring other parameters which are indicative of cyclin dependent kinase concentration. These other parameters may vary in direct or inverse proportion with cyclin dependent kinase concentration. For example, in some embodiments of the invention, parameters are measured which are related to either the formation or later metabolic fate of cyclin dependent kinase. For example, mRNA for cyclin dependent kinase could be measured, as could proteases involved in the degradation of cyclin dependent kinase. In one embodiment, tyrosylphosphorylation of cyclin dependent kinase is measured. The foregoing measurements are preferably performed by ELISA or immunohistochemical techniques utilizing antibodies to at least one cyclin dependent kinase, or to other antigens the concentration of which is indicative of cyclin dependent kinase concentration (e.g., some of the related parameters discussed above).

After measurements are taken, it is preferred but not required that measurements be compared to a control which may be either a historical or concurrent control, standard curve, archival materials, or the like. However, for a given purpose, a user's own prior experience with the measurement, and with its implications may be sufficient for subjective evaluation of the measurement by the user. In some embodiments of the invention, "before" and "after" measurements are taken to determine the effect of an intervening regimen or of exposure to stimulus. In other embodiments, abnormal measurement levels based on historical or archival data or standard curves are determined. Depending upon the sensitivity desired, for example, a positive indication could be set at one, two or three standard deviations above the mean of a normal control. Those of skill in the art will recognize a wide range of uses for the present methods and kits, only a representative sample of which are discussed below.

For purposes of diagnostic evaluation of tissues, samples suspected of having undergone transformation to cancerous phenotype, e.g., breast, prostate, colon, lung, stomach or pancreas tissues, or lymphocytes, etc., may be subjected to the methods of the recent invention wherein abnormal measurements of parameters indicative of cyclin dependent kinase concentration will represent a positive signal for transformation to cancerous phenotype or likelihood of transformation. The ability to determine likelihood of

transformation is of particular value in biopsy, specially when a patient is to undergo surgery for removal of a tumor. The present invention provides an improved method of determining how radical such surgery should be, and how much tissue should be removed.

Other applications include the diagnostic evaluation of potential agents to induce cancer phenotype in any cell or tissues. Comparative testing could be done, for example, utilizing fish or other animals from waters polluted with certain pollutants (the same animals from cleaner water could be used as controls).

The invention also has research applications to laboratory animals, and to providing model in vitro systems for the potency of carcinogenic agents or potential antineoplastic agents.

It is also possible, for example, to test the potency of potential inhibitors of various biological responses. A cell's response to a mitogen can be measured in accordance with the present invention, and the response to a combination of mitogen and a test inhibitor (at increasing concentrations) can also be tested by the present invention. The decrease in proliferation induced by the mitogen at increasing concentrations of inhibitor can be shown by measuring cyclin dependent kinase concentration in accordance with the invention, thereby providing a test of the effectiveness of the inhibitor.

The test of the present invention could also be said to establish "no effect" thresholds for toxic effects of various compounds. The test of the invention an be utilized, for example, to determine a threshold concentration below which the test compound will not interfere, for example, with the function of liver cells tested in accordance with the invention.

The prdsent invention is able to provide statistically significant results after a very short period of time of cell incubation with a test compound. In preferred embodiments, in vivo tests involved administering a test compound to a animal and allowing about 24 hours before sampling tissue. In in vitro tests, 24-48 hours of cell exposure to a test compound is preferred.

In certain embodiments of an in vitro test for carcinogenicity of a test compound, cells are synchronized at operational G ₀ by deprivation of growth factors. The test compound is administered to some dishes, serum to a positive control, and nothing to a negative control. After about 24-48 hours, cells are harvested and lysed, the lysate being subjected to measurement of a parameter indicative of cyclin dependent kinase concentration in accordance with the invention. In one embodiment of this in vitro test, the cells to be tested are lysed (they should be kept cold through the procedure). Preferably, they are kept on ice and their temperature does not exceed 2-40°C. Both sample and standards are then bound to the plate, after diluting with a sample dilution buffer, e.g. a sodium borate buffer at pH 10.5 (about lOOmM). The protein concentration is preferably between 6 and 100 ) μg per ml of dilution buffer. In accordance with standard ELISA techniques, the above binding is preferably followed by blocking the remaining sites, adding the primary antibody (anti-PSTAIR by way of example only), adding the secondary antibody and color development (where the secondary antibody is detectable by color).

In a corresponding in vivo application, animal tissue is obtained about 24 hours after exposure to a test compound. The tissue is preferably slurried and then subjected to testing of a parameter indicative of cyclin dependent kinase concentration, one preferred embodiment proceeds like the in vitro test above. Protein concentration for the in vivo test being preferably between 12.5 and 50 μg protein per ml of dilution buffer.

Preferred methods of the invention provide a lysate buffer for an in vitro test, or homogenization buffer for an in vivo test and a dilution buffer for both.

Immunohistochemical analysis of cells suspected of having transformed to a cancerous phenotype, or suspected of having increased susceptibility to transformation, may proceed iii an analogous manner starting with a thin (e.g. 4-6 micron) sample immobilized on a slide that has preferably been microwaved for about 10 minutes. Positive and negative controls are preferably provided on the slide.

Naturally, it is preferred that the antibodies used are specific for the particular antigen being measured and that the antibody formulations are substantially free of contaminants and of other antibodies to avoid cross-reactivity. The antibodies may be, for

example, anti-cyclin dependent kinase (when cyclin dependent kinase concentration is being measured directly). Preferred anti- cyclin dependent kinase includes but is not limited to anti-PSTAIR, and antibodies to cyclin dependent kinases having an apparent molecular weight between about 30 and 37 kD, especially 33 kD and 34 kD, when measured on polyacrylamide gel. Cyclin dependent kinases were originally believed to have an apparent molecular weight between 32 and 34 kD, however, as new CDKs have been discovered, this range has expanded to between 30-37 kD.

Possible substances that may be tested in accordance with the invention include peroxisome proliferators, estrogens, estrogen receptor, testosterone, testosterone receptor. The invention may also measure carcinogenicity of compounds from the dioxin or PCB group.

As used herein, "cell samples" may include tissues that have the type of cells under discussion.

The invention also comprises a method and assay to determine whether a test compound or sample is a nongenotoxic carcinogen, wherein the compound or sample to be tested is added to a cyclin dependent kinase (CDK) assay system. The assay system can be, inter alia, a living organism, a cell culture or a cell lysate, as long as the assay system contains a cyclin dependent kinase (CDK). An increase in the tyrosylphosphorylation level of CDK (one indication of increased CDK concentration) indicates that the test compound is a nongenotoxic carcinogen, or that the test sample contains a nongenotoxic carcinogen.

This assay also detects nonmutagenic carcinogens and substances having a cell proliferation effect. The nongenotoxic carcinogens that can be identified through the assay include tumor promoters, chlorinated biphenyls, hormones, dioxins and peroxisome proliferators, among others. The assay system can be assembled in the form of a test kit for diagnostic and environmental testing.

The above assay could also be used to quantify the potency of a particular growth factor (peptide hormone). A peptide growth factor would be added to the assay system instead of a xenobiotic (foreign chemical) and otherwise the assay would proceed without modification.

The method and assay of the invention can also be used to determine the potential of a chemical as an antineoplastic agent by reversing the steps outlined above. Starting with a transformed cell or transformed cell lysate, a potential antineoplastic agent would be tested for the capacity of the chemical to put the cells into the G _c state. This capacity would be determined by quantifying the decrease in cyclin dependent kinase, e.g. by measuring tyrosylphosphorylation of the CDK. The only other modification necessary to convert the assay for nongenotoxic carcinogens to one for antineoplastic agents is to grow the neoplastic cells in vitro in a full serum complement (20% serum containing medium).

In Vivo Experiments

The following five examples show enhanced tyrophosphorylation of p34cdc2 kinase in in vivo preparations in response to different nongenotoxic carcinogens. Example 4, however, show how a genotoxic carcinogen does not enhance tyrosylphosphoylation.

EXAMPLE 1

Enhanced tyrosylphosphorylation of p34 ^c kinase in an hepatic cytosol (S-9) preparation from C57BL/6J female mice 24 hours following administration of the nongenotoxic carcinogen 2,3,7,8-tetrachlordibenzo-p-dioxin.

Summary

p34 ^cdc2 is the serine/threonine kinase subunit of M-phase promoting factor (MPF)

(32-34). The regulation of p34 1,2 tyrosine phosphorylation status is considered the control mechanism for entry into G- from G ₀ , the START signal, and also from G ₂ to M, the initiation of mitosis. It is demonstrated that a single dose of 2,3,7,8- tetrachlorodibenzo-p-dioxin administered at 0.25, 0.5, 1 , or 2 μg/kg to young, female mice increases the extent of tyrosylphosphorylation of hepatic p34 ^cdc2 kinase compared to corn oil treated controls. These results indicate that the proliferative stimulus of the nongenotoxic carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin may be quantified as an

increase in hepatic p34 ^cdc2 kinase tyrosylphosphorylation and therefore that stimulation of tyrosylphosphorylation of hepatic p34 ^cdc2 kinase can serve to indicate the capacity of a dioxin-like chemical to function in vivo as a nongenotoxic carcinogen.

Materials and Methods

Chemicals: 2,3,7,8-tetrachlorodibenzo-p- dioxin (TCDD) is purchased from

AccuStandard, Inc. (New Haven, CT).

Anti-phosphotyrosine monoclonal, anti-PSTAIR (CDK), and anti-p34 ^cdc2 kinase C- terminus polyclonal antibodies are obtained from UBI (Lake Placid, NY). The acronym PSTAIR is the abbreviation for the amino acid sequence used as the antigen for developing the anti- PSTAIR antibody. The two antibodies (PSTAIR and anti-C terminus) recognize two different epitopes. At least nine CDKs have been described in the literature; these all have a common PSTAIR epitope. Therefore anti-PSTAIR would be expected to cross react with the entire complement of CDKs showing up in the 32 to 34 kD region (Apparently some cyclins also cross react with the anti PSTAIR antibody and this explains the banding at approximately 60 kD observed in some of the immunoblots with anti-PSTAIR.)

The antibody to the C-terminus region is more specific for p34 ^cdc2 kinase, since the C-terminus region more variable than the highly conserved PSTAIR region. However, it is obviously not species-specific since it was generated against human cdc2 and it cross reacts with mouse, rat and dog p34 ^cdc2 kinase. One or the other antibody is used depending upon the specificity desired in the experiments.

Bicinchoninic acid is obtained from Pierce (Rockford, IL). Molecular weight standards are supplies through BioRad (Melville, NY). All other chemicals are purchased from Sigma (St. Louis, MO) and are of the highest purity available.

Animals and dosing: Four to six-wk old, female C57BL/6J mice are obtained from Harton Sprague Dawley (Indianapolis, IN). The mice are fed Prolab RMH 1000 (Agway, Cortland, NY) and receive tap water ad libitum. All mice are housed three per cage and maintained on a photoperiod of 12 h. Mice are killed 24 h following an intraperitoneal injection of TCDD in corn oil at 0, 0.25, 0.5, 1, or 2 μg/kg. Three mice

are treated at each dose and the volume of the injections ranges from 0.1 to 0.2 L per mouse. All preparation procedures are performed on pooled hepatic samples of the three mice per dose.

Preparation and -80°C storage of hepatic S-9 fractions is performed exactly as previously described in the scientific literature (35). This procedure involves killing the mouse by cervical dislocation, removing the liver and homogenizing the liver in three volumes of 0.15 M KCI. This hepatic homogenate is centrifuged at 9,000 x g for 20 min at 4°C. The resulting supematant fraction, termed the S-9, is decanted into 1.5 mL plastic, conical tubes, frozen in a dry ice/ethanol bath and stored at 80°C until immunoprecipitation of phosphotyrosyl proteins can be performed.

Immunoprecipitation of tyrosine phosphorylated hepatic S-9 proteins with anti-phosphotyrosine monoclonal antibody: The hepatic S-9 is solubilized in immunoprecipitation buffer containing 20 mM Tris HCl (pH 8-0), 137 mM NaCl, 10% glycerol, 1 % NP-40, 1 mM phenyl- methylsulphonyl fluoride (PMSF), 0.15 U/mL aprotinin, and 1 mM sodium vanidate, centrifuged at 13,000 x g for 15 min at 4°C. The solubilized hepatic S-9 proteins are then incubated with anti-phosphotyrosine monoclonal antibody (5 μg/mL) at 4°C for 4 h or ovemight. After the incubation period, add 25 μL of protein A-Sepharose for each 5 μg of antibody. One h later the immune complexes are collected by centrifugation at 13,000 x g, washed twice with immunoprecipitation buffer, solubilized in SDS gel sample buffer and heated at 100°C for 5 min in preparation of SDS PAGE and immunoblotting.

Gel electrophoresis and immunoblotting: SDS PAGE is carried out as described in the scientific literature (36) using 11 % polyacrylamide gels with the modification that hepatic S-9 (100 μg protein/well) are subjected to heat treatment (100°C) for 3 min. The immunoblotting assay is performed as described by Towbin et al. (39), however a

Milliblot SDE electroblot apparatus (Millipore, Bedford, MA), is used to transfer proteins from polyacrylamide gels to an Immobilon* membrane filter (Millipore,Bedford, MA). Complete transfers are accomplished in 25-30 min at 500 mA and are assessed by tracking pre-stained molecular weight standards on the membrane filter.

Membrane filters are blocked by incubating in TBS (Tris buffered saline) containing 5% commercial nonfat dry milk (any commercial brand is suitable) for 30 min at room temperature. The membranes are then washed in TBST (TBS with 0.05% Tween 20) and incubated for 2 h with anti-human CDK (PSTAIR) antibody (2 - 5 μg/mL) in 5 TBST or anti-mouse cdc2 kinase (C-terminus) polyclonal antibody in TBST. The antibody reaction is visualized by incubating the membranes for 2 h at room temperature with alkaline phosphatase-conjugated anti-mouse IgG diluted 1:1000 in TBST and developed for 15 min. Molecular weights are determined by adding molecular weight standards (Bio Rad, Melville, NY) to reference lanes and staining the membrane filters with amido black 10 10B. The resulting immunoblots are scanned into TIFF-formatted files (Macintosh*; Apple Computers, Cupertino, CA) with a Microtech 600GS scanner (Torrance, CA) and quantified using Scan Analysis (BIOSOFT, Cambridge, UK). Summary scans are then printed and peak heights are measured directly from the figure. One density unit (U) is defined as one mm of the resulting peak height.

15 Protein determination: Bicinchoninic acid is used for the spectrophotometric determination of protein concentration (38). Mix 100 μL of sample (standard or unknown) with 2 mL of working reagent in a test tube. Color development occurs by incubation at 37°C for 30 min. Absorbance is read at 562 nm. Working reagent is made by adding 100 volumes of Reagent A with 2 volumes Reagent B. Reagent A: is made by combining 1.0 g

20 bicinchoninic acid (Pierce Chemical, Rockford, IL); 2.0 g Na. ₂ CO ₃ *H ₂ 0; 0.16 g NaOH; and 0.95 g NaHCO ₃ with water to 100 mL and adjust the pH to 11.25 with 50% NaOH. Reagent B consists of 4.9 9 CuSO ₄ * 5H ₂ 0 to 100 mL in double distilled H ₂ O.

Results:

The anti-phosphotyrosine immunoprecipitate of the murine hepatic S-9 is run on an 25 11 % polyacrylamide gel as described above and immunoblotting is performed with the anti-PSTAIR monoclonal antibody. The resulting anti-PSTAIR immunoblot is depicted in Figure 4. Density scans of the immunoblot are presented in Figure 5 and the quantification of these bands is presented in Figure 6. The bands in Figure 4 at 34 and 32 kDa immunoreactive with anti-PSTAIR have been identified as cyclin dependent kinases and at 30 this time it is not known if they represent isoforms of a single pp34 ^cdc2 kinase or whether

they are two separate cyclin dependent kinases (39). The large anti-PSTAIR immunoreactive band at approximately 60 kDa has been identified as a cyclin protein (40,41).

The results demonstrate that the Cyrosylphosphorylated CDK (pp34 ^cdc2 ) does not exist in measurable quantities in the hepatic S-9 of com oil treated control mice. However, dosing of mice with TCDD enhanced the tyrosylphosphorylation of a p34 and p32 to a maximum at 0.5 μg TCDD/kg. At higher doses of TCDD the tyrosylphosphorylation of the kinase(s) becomes attenuated, perhaps due to overt toxicity of TCDD to the mice at these higher doses.

EXAMPLE 2

Enhanced tyrosylphosphorylation of p34 ^c kinase (CDK) in an hepatic cytosol preparation (S-9) from young male rats 24 hours following administration of the nongenotoxic carcinogen pirnixic acid.

Summary

p34 ^cdc2 is the serine/threonine kinase subunit of M-phase promoting factor (MPF) (29-31). The regulation of p34 ^cdc2 tyrosine phosphorylation status is considered the control mechanism for entry into G _! from G ₀ , the START signal, and also from G ₂ to Ml the initiation of mitosis. It is demonstrated that twice daily doses of 50 mg pirnixic acid/kg of body weight for 5 days to young male rats increases the extent of tyrosylphosphorylation of hepatic p34 ^cdc2 kinase compared to com oil treated controls. These results indicate that the proliferative stimulus of the nongenotoxic carcinogen pirnixic acid may be quantified as an increase in hepatic p34 ^{c c} kinase tyrosylphosphorylation and therefore that stimulation of tyrosylphosphorylation of hepatic p34 ^cdc2 kinase can serve to indicate the capacity of chemicals that are termed peroxisome proliferators to function in vivo as a nongenotoxic carcinogen.

Materials and Methods

Chemicals: Pirnixic acid (CAS 50892-23-4 [4- chloro-6-(2,3-xylidino)-2- pyrimidiylthiol acetic acid) is purchased from ChemSyn Science Labs (Lenexa, KY). Anti- phosphotyrosine monoclonal, anti-PSTAIR (CDK), and anti-p34 ^cdc2 kinase C-terminus polyclonal antibodies are obtained from UBI (Lake Placid, NY). Bicinchoninic acid is obtained from Pierce (Rockford, IL). Molecular weight standards are supplied through BioRad (Melville, NY). Sensor Chips CM5, Surfactant P20, and amine coupling kit (EDC, NHS, and ethanolamine hydrochloride) were purchased from Pharmacia Biosensor AB. All other chemicals are purchased from Sigma (St. Louis, MO) and are of the highest purity available.

Animals and dosing: Eight-wk old male Sprague-Dawley rats are purchased from

Charles River Laboratory (Charles River, MA) and housed four to a cage in polycarbonate cages (24 x 34 x 20 cm). Bedding consists of hardwood chips. Rats are allowed free access to tap water and fed Agway RMH 3000 (Cortland, NY) ad libitum. Photoperiod is maintained at 12 h of light and 12 h of darkness.

Treatments begin after a week of acclimation to new surroundings. Treatments consists of twice daily doses of the test compound administered by oral gavage. The pirnixic acid is dissolved in com oil. Sham-treated animals are given an equal volume of plain com oil. Doses are adjusted daily on the basis of weight. The volume of com oil is generally on the order of 2 mL/ rat throughout the treatment period. The second dose is given between the h of 13:00-16:00, approximately 6-h after the first dose given between the h of 7:00-10:00. The pirnixic acid is administered for 5 days at a dose of 50 mg/kg twice a day.

On the day of sacrifice the rats are anesthetized with ethyl ether and decapitated. Livers are removed, weighed and homogenized using a Potter- Elvehjem* tissue grinder with 3 mL of ice-cold 0.15 M KCI per g of wet weight of liver. This material is pooled for each rat and spun in a high speed centrifuge (Beckman J2-MI, Beckman Instruments, Fullerton, CA) for 10 min at 9000 x g at 40C. The supematant liquid is decanted, distributed as aliquot and frozen at -900C.

Gel electrophoresis and immunoblotting with anti-phosphotyrosine: These procedures are carried out essentially as described in Example 1 except that anti- phosphotyrosine antibody is used in place of anti-PSTAIR antibody.

Protein determination: This procedure is performed as described in Example 1.

Real-time quantification of total tyrosylphosphorylated p34 ^cdc3 kinase: Surface plasmon resonance (SPR) is used for the real time quantification of p34 ^cdc2 kinase that exists in the tyrosylphosphorylated form. SPR is sensitive to changes in the optical properties of a medium close to a metal surface (42). SPR is suitable for macromolecular interaction studies at solid/liquid interfaces with the use of a carboxymethylated dextran hydrogel placed upon a thin layer of gold (42,43).

The detection system of a SPR monitor consists of a light source emanating both monochromatic and plane-polarized light, a glass prism, a thin metal film in contact with the base of the prism, and a photodetector. An evanescent field forms from the prism into the metal film when obliquely incident light on the base of the prism will exhibit total intemal reflection for angles greater than the critical angle. This evanescent field can couple to an electromagnetic surface wave, a surface plasmon, at the metal/liquid interface. Coupling is achieved at a specific angle of incidence, the SPR angle (42).

The SPR angle is highly sensitive to changes in the reactive index of a thin layer adjacent to the metal surface which is sensed by the evanescent wave. Therefore, it is a volume close to the surface that is probed. For example, when a protein layer is adsorbed on the metal surface, keeping all other parameters constant, an increase in the surface concentration occurs and the SPR angle shifts to larger values (42). The magnitude of the shift, defined as the SPR response, depends on the mean refractive index change due to the adsorption in the probed volume (a function of mass).

Utilizing SPR, biospecific interaction analysis is performed in real time in conjunction with a flow injection system and is as sensitive as other methods such as radiolabeling, fluorometry, and chemiluminescence. In short, biospecific interaction analysis is a sensitive, non-labile way of examining interactions between macromolecules in real time (44-46).

SPR measurements are performed on a BIAcore* unit manufactured by Pharmacia Biosensor AB (Uppsala, Sweden). Sensor Chips CM5, Surfactant P20, and amine coupling kit (EDC, NHS, and ethanolamine hydrochloride) were purchased from Pharmacia Biosensor AB.

Immobilization of PSTAIR and C-terminus antibodies via amine coupling was performed according to the general procedure recommended by the manufacturer. Briefly, the instrument was equilibrated with HBS buffer (10 mM HEPES, 150 mM NaCl, 0.050% surfactant P20, pH 7.4, and filtered with a 0.22 micron filter), then the following series of injections were made using the autosampler incorporated into the BIAcore unit:

(1) equal volumes of EDC (0.1 M in water) and NHS (0.1 M in water) were mixed and 35 μL injected to activate the carboxymethylated surface;

(2) ligand (35 μL, 50μg/mL in 10 mM sodium acetate pH 4.5) was then injected;

(3) the remaining NHS-esters on the surface were then deactivated with ethanolamine (35 μL, 1 M in water, pH 8.5);

(4) noncovalently bound material was then washed from the surface with hydrochloric acid (15 μL. 20 mM)- Immobilizations were executed with a continuous flow of HBS at a flow rate of 5μL/min.

A typical sensorgram produced on immobilization of anti-cdc2 C-terminus is depicted in Figure 7. Time required for immobilization is approximately 30 min.

BIAcore assay for tyrosylphosphorylation of cyclin dependent kinase (CDK)-Each binding/regeneration cycle is performed with a constant flow of HBS of 3 μL/min. Hepatic S-9 fractions of rats dosed with pirnixic acid or vehicle alone are diluted to a concentration of 1.5 mg protein/mL into exhausted FB-2 tissue culture supematant liquid and incubated ovemight at 4°C with anti-phosphotyrosine antibody. This equilibrated mixture (40 μL) is then injected over the immobilized PSTAIR and C-terminus antibodies and binding is recorded in RU. Binding is directly proportional to the amount of

tyrosylphosphorylated protein interacting with the anti-PSTAIR or anti-C Terminus antibodies.

Inteφretation of results

Immunoblots - For scans of immunoblots, a change in phosphotyrosylprotein content of p34 ^cdc2 kinase greater than 40 percent was considered biologically meaningful.

BIAcore assay- Research on the cell cycle has shown that the concentration of cdc2 kinase remains constant and that tyrosine phosphorylation can be utilized as a marker of cells that are preparing to enter the M phase of the cell cycle (46-51). Therefore, increased binding indicate increased tyrosylphosphorylation of cdc2 kinase, thus more cells are in the process of preparing to enter mitosis. Treatment effects from BIAcore analyses are considered significant when the instrument response of the treatment group is outside the upper bounds of the population 95 percent confidence interval (t ₍₅ x ₀ . ₉₅₎ = 2.015 times the standard deviation of the RU response of the control animals).

Results

Immunoblotting analysis - As seen in Figure 8, seven proteins exhibited an increased tyrosine phosphorylation in response to the administration of pirnixic acid. A 6.24-fold increase was noted in pp69, while the greatest relative difference in peak height was seen with a 13.16-fold increase in pp33. Five phosphotyrosylproteins also decreases in quantity. These were pp84, pp61, pp43, pp34 and pp23. Figure 9 depicts the scanning results and Figure 10 shows the quantification of the CDK at 33 kDa. Results indicate that the administration of five, twice-daily doses of pirnixic acid (50 mg/kg each dose) produces enhanced tyrosylphosphorylation of the CDK compared to control animals, which exhibit rio tyrosylphosphorylation of CDK at 33 kDa. Each group on the graph represents the average of two rats. Error bars in this figure represent the 10 percent coefficient of variation in the quantification of density.

BIAcore (SPR) - Hepatic S-9 samples from rats treated with pirnixic acid produced greater binding to both anti-PSTAIR or anti-C-terminus antibodies than hepatic S-9 samples from vehicle control rats (Figure 11). This increased binding exhibited by the

hepatic S-9 of test animals is due to enhanced tyrosylphosphorylation of cdc2 kinase (CDK). Figure 12 is a summary bar graph depicting BIAcore quantification of the interaction of tyrosylphosphorylated cyclin dependent kinases (CDK) with anti-CDK polyclonal antibodies (PSTAIR and C-terminus) from control and pirnixic acid-treated rats. Error bars represent standard deviations of n = 6 (anti-PSTAIR) and n = 8 (anti-C- terminus) control rats. RU value for pirnixic acid-treated rats represents the mean of 2 animals. The treatment of rats with 50 mg pirnixic acid/kg twice a day for 5 days results in enhanced tyrosylphosphorylation of CDK (p34 ^cdc2 kinase) compared to control rats.

EXAMPLE 3

Enhanced tyrosylphosphorylation of p3 ^c kinase (CDK) in an hepatic cytosol preparation (S-9) from young male rats 24 hours following administration of the nongenotoxic carcinogen diethylhexylphthalate.

Summary

p34 ^cdc2 is the serine/threonine kinase subunit of M-phase promoting factor (MPF)

(33-35). The regulation of p34 ^cdc2 tyrosine phosphorylation status is considered the control mechanism for entry into G, f rom G„, the START signal, and also from G ₂ to M, the initiation of mitosis.

It is demonstrated that twice daily doses of 500 mg diethylhexylphthalate/kg of body weight for 5 days to young, male rats increases the extent of tyrosylphosphorylation of hepatic p34 ^cdc2 kinase compared to com oil treated controls. These results indicate that the proliferative stimulus of the nongenotoxic carcinogen diethylhexylphthalate may be quantified as an increase in hepatic p34 ^cdc2 kinase tyrosylphosphorylation and therefore that stimulation of tyrosylphosphorylation of hepatic p34 ^cdc2 kinase can serve to indicate the capacity of chemicals that are termed peroxisome proliferators to function in vivo as a nongenotoxic carcinogen.

Materials and Methods

Chemicals: Diethylhexylphthalate (DEHP) [CAS 117-81-71 was purchased from Fluka Chemicals (Ronkonkoma, NY). Anti-phosphotyrosine monoclonal, anti-PSTAIR (CDK), and anti-p34 ^cdc2 kinase C-terminus polyclonal antibodies were obtained from UBI (Lake Placid, NY). Bicinchoninic acid was obtained from Pierce (Rockford, IL). Molecular weight standards were supplied through BioRad (Melville, NY). All other chemicals were purchased from Sigma (St. Louis, MO) and were of the highest purity available.

Animals and dosing: Rats are purchased and handled as described in Example 2.

After a wk of acclimation to new surroundings, treatments are begun. The treatment consists of twice daily doses of DEHP administered by oral gavage. The DEHP is dissolved in co oil. Sham-treated animals are given an equal volume of plain com oil. Doses are adjusted daily on the basis of weight. The volume of co oil is generally on the order of 2 mL/rat throughout the treatment period. The second dose is given between the h of 13:00-16:00, approximately 6 h after the first dose given between the h of 7:00 - 10:00. The DEHP is administered for 5 days at a dose of 500 mg/kg twice a day. Rats are anesthetized and livers are prepared as described in Example 2.

Gel electrophoresis and immunoblotting with anti-phosphotyrosine: These procedures are carried out essentially as described in Example 1 except that anti- phosphotyrosine antibody is used in place of anti-PSTAIR antibody.

Protein determination: This procedure is performed as described in Example 2.

Real-time quantification of total tyrosylphosphorylated p34 ^cdc2 kinase and interpretation of the results: These procedures are performed as described in Example 2.

Results

Immunoblotting analysis - Six phosphotyrosylproteins are shown to increase with the administration of DEHP (Figures 13 and 14). The range of relative increase is 1.48 to 4.19-fold. A decrease in pp31 and pp28 is also observed. Figure 15 depicts the quantification of the results of the scanning densitometry. The cyclin dependent kinase (CDK) quantified from the anti-phosphotyrosine immunoblot is at 34 kDa. Results indicate

that the administration of five, twice-daily doses of DEHP (500 mg/kg each dose) produces enhanced tyrosylphosphorylation of the CDK compared to control animals, which exhibit no tyrosylphosphorylation of CDK at 34 kDa. Each group on the graph represents the average of two rats. Error bars represent the 10 percent coefficient of 5 variation in the quantification of density.

BIAcore (SPR) - Hepatic S-9 samples from rats treated with DEHP produce greater binding to both anti- PSTAIR or anti-C-terminus antibodies than hepatic S-9 samples from vehicle control rats (Figure 16). This increase in binding by the hepatic S-9 of DEHP- treated animals is due to enhanced tyrosylphosphorylation of cdc2 kinase (CDK). Figure

10 16 is a summary bar graph depicting BIAcore* quantification of the interaction of tyrosylphosphorylated cyclin dependent kinases (CDK) with anti-CDK polyclonal antibodies (PSTAIR and C-terminus) from control and DEHP-treated rats. Error bars represent standard deviations of n = 6 (anti-PSTAIR) and n = 8 (anti-C-terminus) control rats. RU value for DEHP- treated rats represents the mean of 2 animals. The treatment of 5 rats with 500 mg DEHP/kg twice a day for 5 days results in enhanced tyrosylphosphorylation of CDK (P34 ^cdc kinase) compared to control rats.

EXAMPLE 4

The genotoxic carcinogen diethylnitrosamine does not enhance tyrosylphosphorylation 0 of p34 ^c kinase in an hepatic : ccyyssttooll pprreeppaarraattiioonn ((SS--99)) ffirom young male rats 24 hours following administration.

Summary

p34 ^cdc2 is the serine/threonine kinase subunit of M-phase promoting factor (MPF) (32-34). The regulation of p34 ^cdc2 tyrosine phosphorylation status is considered the control 5 mechanism f or entry into G, from G ₀ , the START signal, and also from G ₂ to M, the initiation of mitosis. It is demonstrated that twice daily doses of 500 mg diethylnitrosa ine/kg of body weight for 5 days to young, male rats did not affect the extent of tyrosylphosphorylation of hepatic p34 ^cdc2 kinase compared to com oil treated

controls. These results indicate that the early in vivo effects of the genotoxic carcinogen diethylnitrosamine can not be quantified through a change in hepatic p34 ^cdc2 kinase tyrosylphosphorylation and therefore that stimulation of tyrosylphosphorylation of hepatic p34 ^cdc2 kinase is specific for nongenotoxic carcinogens.

Materials and Methods

Chemicals: Diethylnitrosamine (DEN) [CAS 55- 18-51 was purchased from Fluka Chemicals (Ronkonkoma, NY). Anti-phosphotyrosine monoclonal, anti-PSTAIR (CDK), and anti-p34 ^cdc2 kinase C-terminus polyclonal antibodies were obtained from UBI (Lake Placid, NY). Bicinchoninic acid was obtained from Pierce (Rockford, IL). Molecular weight standards were supplied through BioRad (Melville, NY). All other chemicals were purchased from Sigma (St. Louis, MO) and were of the highest purity available.

Animals and dosing: Animals are purchased and handled as described in Example 2. After a wk. of acclimation to new surroundings, treatments are begun. The treatment consists of twice daily doses of DEN administered by oral gavage. The DEN is dissolved in com oil. Sham-treated animals are given an equal volume of plain co oil. Doses are adjusted daily on the basis of weight. The volume of com oil is generally on the order of 2 mL/rat throughout the treatment period. The second dose is given between the h of 13:00- 16:00, approximately 6 h after the first dose given between the h of 7:00 - 10:00. The DEN is administered for 5 days at a dose of 500 mg/kg twice a day. Rats are anesthetized and livers are prepared as described in Example 2.

Gel electrophoresis and immunoblotting with anti-phosphotyrosine: These procedures are carried out as described in Example 1 except that anti- phosphotyrosine antibody is used in place of anti-PSTAIR antibody.

Protein determination: This procedure is performed as described in Example 1.

Real-time quantification of total tyrosylphosphorylated p34 ^cdc2 kinase and interpretation of the results: These procedures are performed as described in Example 2.

Results

Immunoblotting analysis - Administration of DEN to young male rats did not produce any increases in phosphotyrosylproteins (Figures 17 and 18). A 61 % decrease in pp22 is observed. Figure 19 is a bar graph depicting the quantification of the results of the scanning densitometry. The band quantified from the anti-phosphotyrosine immunoblot is at 34 kDa. Results indicate that the administration of five, twice-daily doses of DEN (500 mg/kg each dose) produces no enhanced tyrosylphosphorylation of the p34 compared to control animals. Each group on the graph represents the average of two rats. Error bars represent the 10 percent coefficient of variation in the quantification of density.

BIAcore (SPR) - Hepatic S-9 samples from rats treated with DEN produce no greater binding to anti- PSTAIR or anti-C-terminus antibodies than hepatic S-9 samples from vehicle control rats. Figure 20 is a summary bar graph depicting BIAcore* quantification of the interaction of tyrosylphosphorylated cyclin dependent kinases (CDK) with anti-CDK polyclonal antibodies (PSTAIR and C-terminus) from control and DEN- treated rats. Error bars represent standard deviations of n = 6 (anti-PSTAIR) and n = 8 (anti-C Terminus) control rats. RU value for DEN- treated rats represents the mean of 2 animals. Results indicate that the treatment of rats with 500 mg DEN/kg twice a day for 5 days produces no enhanced tyrosylphosphorylation of CDK (p34 ^cdc2 kinase) compared to control rats.

EXAMPLE 5

Enhanced tyrosylphosphorylation of p34 in an hepatic cytosol preparation (S-9) from female Beagle dogs following administration of the nongenotoxic carcinogen Aroclor* polychlorinated biphenyls for eleven and one-half weeks

Summarv

It is demonstrated that daily doses of 0.6, 0.8, 4-8, or 5-10 mg /kg of body weight for 11.5 weeks to 2-year old, female Beagle dogs enhances the tyrosine phosphorylation status of an hepatic p34 compared to com oil treated controls. These results indicate that the early in vivo effects of the nongenotoxic carcinogen Aroclor ^® polychlorinated

biphenyls can be quantified through a change in hepatic p34 tyrosylphosphorylation and therefore that stimulation of tyrosylphosphorylation of hepatic p34 is specific for nongenotoxic carcinogens.

Materials and Methods

Chemicals: Aroclor ^® 1254 polychlorinated biphenyls (PCBs) is purchased from

AccuStandard, Inc. (New Haven, CT). Anti-phosphotyrosine monoclonal antibody is obtained from UBI (Lake Placid, NY). Bicinchoninic acid is obtained from Pierce (Rockford, IL). Molecular weight standards are supplied through BioRad (Melville, NY). All other chemicals were purchased from Sigma (St. Louis, MO) or stated suppliers and were of the highest purity available.

Animals and dosing: Five, purebred, 2-year old, female beagle dogs, obtained from Norwich Pharmaceutical (Norwich, NY), are used in this study. All dogs were fully vaccinated, dewormed and specific pathogen free (SPF) for at least 30 days prior to the initiation of the experiment. They are maintained indoors and individually housed according to Public Health service guidelines (NIH publication No. 85-23). At the beginning of the study the dogs weigh between 8.7 and 12.2 kg. Physical observations of the dogs are made daily during the 11.5-wk dosing period of the study.

Each dog is administered either com oil (controls) or Aroclor ^® PCBs at 0.6, 0.8, 4 or 5 mg/kg-day for seven wk. From seven to 11.5 wk, the 4 mg/kg-day dose and the 5 mg/kg-day dose are increased to 8 and 10 mg/kg-day, respectively. The com oil, as well as test material, is administered in a cube of agarose concealed in a small ball of canned dog food. After consuming the meatball, the dogs are immediately fed their daily caloric requirement of canned food.

Dogs were sacrificed using 2mIJkg of Fatal Plus (Vortech Pharmaceutical Company, Dearbome, MI). Hepatic S-9 fractions were prepared as previously described in Example 1.

Gel electrophoresis and immunoblotting with anti-phosphotyrosine: These procedures are canied out as described in Example 1 with the exception that anti- phosphotyrosine antibody is used in place of anti-PSTAIR antibody.

Protein determination: This procedure is performed as described in Example 1.

Results

The daily administration of Aroclor ^® polychlorinated biphenyls for a period of 11.5 wk results in enhanced tyrosylphosphorylation of a protein migrating at 34 kDa at all doses compared to the control dog. Figure 21 depicts the anti-phosphotyrosine immunoblot of dog hepatic S-9 protein separated using lit SDS-PAGE gels for control and Aroclor* polychlorinated biphenyls- treated dogs. Lanes 1, 2, 3, 4, and 5 are control, 0.6, 0.8, 4-8, and 5-10 mg Aroclor*/kg-day, respectively. The scanning densitometry of a single band at p34 of the anti-phosphotyrosine immunoblot is presented in Figure 22. Quantification of the scanning densitometry of p34 is presented in Figure 23 as a bar graph. Each bar on the graph represents the single result of scanning an immunoblot produced from the hepatic S- 9 of one dog. Error bars represent the 10 percent coefficient of variation in the quantification of density.

In Vitro Experiments

Examples 6 through 9 show enhanced tyrosylphosphorylation of p34/p33 (putative CDK) in 3T3 or BNL CL.2 cell lysates following exposure to various nongenotoxic carcinogens.

EXAMPLE 6

Enhanced tyrosylphosphorylation of p34/p33 (putative CDK) in 3T3 cell lysates 24 hours following exposure to the nongenotoxic carcinogen 2,3,7,8-tetrachlorodibenzo- p- dioxin.

Summary

Exposure of 3T3 cells to 10 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin for 24 h in a low serum media enhances the tyrosine phosphorylation status of two cell lysate proteins, p34 and p33, compared to dimethylsulfoxide-treated controls. These results indicate that the early in vitro effects of the nongenotoxic carcinogen 2,3,7,8-tetrachlorodibenzo-p- dioxin can be quantified through a change in cellular p34/p33 tyrosylphosphorylation and therefore that stimulation of tyrosylphosphorylation of p34/p33 is specific for nongenotoxic carcinogens.

Materials and Methods

Chemicals: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is purchased from

AccuStandard, Inc. (New Haven, CT). Anti-phosphotyrosine monoclonal antibody is obtained from UBI (Lake Placid, NY). Bicinchoninic acid is obtained from Pierce (Rockford, IL). Molecular weight standards are supplied through BioRad (Melville, NY). All other chemicals were purchased from Sigma (St. Louis, MO) or stated suppliers and were of the highest purity available.

Tissue culture cells, culture conditions and dosing: 3T3 cells (ATCC CCL-92) are purchased from American Type Culture Collection (Bethesda, MD). These cells are maintained in Dulbecco's Modified Eagle's Medium (DMEN; Gibco cat. ft 430-2100) supplemented with 10% Fetal bovine serum-heat inactivated (FBS-HI) (Intergen, Purchase, NY). For experimental purposes, the cells are plated in 100 mm x 20 mm tissue culture dishes containing 10 mL of the above maintained medium. The plates are placed in an incubator set at 37° C, 5% CO ₂ , 95% humidity, until they reach confluence (contact inhibited). At this point all the plates are then washed 2x with 5ml of Dulbecco's calcium and magnesium-free phosphate buffered saline (CMF-PBS). Four plates are then fed 10 mL of DMEM + 10% FBS-HI and all the other plates are fed 10 L of DMDM + 10%FBS-HI and incubated for 48 h in the above environmental conditions.

After the 48 h incubation period, the medium from the low-serum group (0.5% FBS-HI) was aseptically harvested and allocated into separated tubes containing 40 mL each (to provided 10 mL/plate for 4 plates per treatment ). The following concentrations

and reagents are added to the appropriate tubes (4 plates per treatment). Dimethyl sulfoxide (DMSO) is used as the diluent for TCDD.

10 mL of DMEM +20% FBS-HI + 0.1 % DMSO (positive control)

10 mL of DMEM +0.5% FBS-HI + 0.1 % DMSO

10 mL of DMEM +0.5 % FBS-HI + 10 nM TCDD

All plates were returned to the incubator for 24 h at the environmental conditions listed above. After the 24 h incubation period, the cells are harvested using the harvesting procedure described.

Gel electrophoresis and immunoblotting with anti-phosphotyrosine: These procedures are carried out as described in Example 1 except that anti- phosphotyrosine antibody is used in place of anti-PSTAIR antibody.

Protein determination: This procedure is performed as described in Example 1.

Results

Exposure of 3T3 cells to 10 nM TCDD for 24 h results in an increase in tyrosylphosphorylation of p34 and p33 of 67 and 32%, respectively, compared to the vehicle control. The anti-phosphotyrosine immunoblot of 3T3 cell lysate protein separated using an 11 % SDS-PAGE gel for 3T3 cells exposed to 10 nM TCDD is presented in Figure 24. Results of scanning the control and TCDD- treated lanes are presented in Figure 25; bolded peaks indicate p34 and p33 tyrosylphosphosphoproteins. In Figure 26 the putative cyclin dependent kinases (p34/p33) are quantified from the anti- phosphotyrosiηe immunoblot. Results of semm supplementation (cf. immunoblot in Figure 24, scan results not depicted in Figure 25) indicate enhanced tyrosylphosphorylation of p34/p33. This result would be expected if the pp34/pp33 are cyclin dependent kinases, since the semm supplemented media provide growth factor that stimulate the cells to mitosis and this stimulus is mediated through the CDK.

EXAMPLE 7

Enhanced tyrosylphosphorylation of p34/p33 (putative CDK) in 3T3 cell lysates 24 hours following exposure to the tumor promoter 12-0-tetra-decanoylphorbol-13- acetate.

Summary

Exposure of 3T3 cells to 12-0- tetra-decanoylphorbol- 13-acetate for 24 h in a low- serum media enhances the tyrosine phosphorylation status of two cell lysate proteins, p34 and p33, compared to dimethylsulfoxide-treated controls. These results indicate that the early in vitro effects of the tumor promoter 12-0-tetra-decanoylphorbol- 13-acetate can be quantified through a change in cellular p34/p33 tyrosylphosphorylation and therefore that stimulation of tyrosylphosphorylation of p34/p33 is specific to a mechanism relating to the process of nongenotoxic carcinogenesis.

Materials and Methods

Chemicals: 2-0-Tetra-decanoylphorbol-13- acetate (TPA) is purchased from ChemSyn Science Labs (Lenexa, KY). Anti-phosphotyrosine monoclonal antibody is obtained from UBI (Lake Placid, NY). Bicinchoninic acid is obtained from Pierce (Rockford, IL). Molecular weight standards are supplied through BioRad (Melville, NY). All other chemicals were purchased from Sigma (St. Louis, MO) or stated suppliers and were of the highest purity available.

Tissue culture cells, culture conditions and dosing: These procedures are performed as described in Example 6. The following concentrations and reagents are added to the appropriate tubes (4 plates/treatment). Dimethyl sulfoxide (DMSO) is used as the diluent for TPA.

10 mL of DMEM + 20% FBS-HI + 0.1 % DMSO

10 mL of DMEM + 0.5% FBS-HI + 0.1 % DMSO

10 L of DMEM + 0.5% FBS-HI + 160 nM TPA

Gel electrophoresis and immunoblotting with anti-phosphotyrosine: These procedures are carried out as described in Example 1 except that anti- phosphotyrosine antibody is used in place of anti-PSTAIR antibody.

Protein determination: This procedure is performed as described in Example 1.

Results

Exposure of 3T3 cells to 160 nM TPA for 24 h results in an increase in tyrosylphosphorylation of p34 and p33 of 54% and 95%, respectively, compared to the vehicle control. The anti-phosphotyrosine immunoblot of 3T3 cell lysate protein separated using an 11 % SDS-PAGE gel for 3T3 cells exposed to 10 nM TCDD is presented in Figure 27. Results of scanning the control and TCDD- treated lanes are presented in Figure 28; bolded peaks indicate p34 and p33 tyrosylphosphosphoproteins. In Figure 29 the putative cyclin dependent kinases (p34/p33) are quantified from the anti- phosphotyrosine immunoblot. Results of semm supplementation (cf. immunoblot in Figure 27, scan results not depicted in Figure 28) indicate enhanced tyrosylphosphorylation of p34/p33. This result would be expected if the pp34/pp33 are cyclin dependent kinases, since the serum supplemented media provide growth factor that stimulate the cells to mitosis and this stimulus is be mediated through the CDK.

EXAMPLE 8

Enhanced tyrosylphosphorylation of p34/p33 in BNL CL.2 cell lysates 24 hours following exposure to the nongenotoxic carcinogen 2,3,7,8-tetrachlorodibenzo-p- dioxin.

Summary

Exposure of BNL CL.2 cells to 0.1, 1, 10 or 100 nM 2,3,7,8-tetrachlorodibenzo- p-dioxin for 24 h in a low semm media enhances the tyrosine phosphorylation status of two cell lysate proteins, p34 and p33, compared to dimethylsulfoxide- treated controls. These results indicate that the early in vitro effects of the nongenotoxic carcinogen 2,3,7,8- tetrachlorodibenzo-p-dioxin can be quantified through a change in cellular

p34/p33 tyrosylphosphorylation and therefore that stimulation of tyrosylphosphorylation of p34/p33 is specific for nongenotoxic carcinogens.

Materials and Me hods

Chemicals: This section is as previously described in Example 6.

Tissue culture cells, culture conditions and dosing: BNL CL.2 cells (ATCC TIB73) are purchased from American Type Culture Collection (Bethesda, MD). These cells are representative of normal mouse hepatocytes. All other procedures were performed as detailed in Example 6.

The following concentrations and reagents are added to the appropriate tubes (4 plates/treatment). Dimethyl sulfoxide (DMSO) is used as the diluent for TCDD.

10 mL of DMEM + 20% FBS-HI + 0.1 % DMSO (positive control)

10 mL of DMEM + 0.5 % FBS-HI + 0.1 % DMSO

10 m.L of DMEM + 0.5% FBS-HI + 0.1 nM TCDD

10 mL of DMEM + 0.5% FBS-HI + 1.0 nM TCDD

10 mL of DMEM + 0.5% FBS-HI + 10 nlM TCDD

10 mL of DMEM + 0.5% FBS-HI + 100 nM TCDD

All plates were returned to the incubator for 24 h at the environmental conditions listed above. After the 24 h incubation period, the cells are harvested using the harvesting procedure described.

Gel electrophoresis and immunoblotting with anti-phosphotyrosine: These procedures are carried out as described in Example 1 except that anti- phosphotyrosine antibody is used in place of anti-PSTAIR antibody.

Protein determination: This procedure is performed as described in Example 1.

Results

Exposure of BNL CL2 cells to 0.1, 1, 10 or 100 niM 2,3,7,8-tetrachlorodibenzo-p- dioxin (TCDD) for 24 h results in a similar increase in tyrosylphosphorylation of p34, averaging 180% of the vehicle control over all test concentrations of TCDD. Twenty percent serum supplementation results in an increase of tyrosylphosphorylation of p34 of 5 229% of the vehicle control. Vehicle controls at 0.5% semm supplementation exhibit no tyrosylphosphorylation at p33, while TCDD exposure at the four concentrations enhances tyrosylphosphorylation of this putative CDK to 0.9, 2.0, 2,0 and 1.9 density units, respectively. The increases in tyrosylphosphorylation of p33 by TCDD are 3.4 times the p33 tyrosine phosphorylation produced by 20% semm supplementation. The anti- 0 phosphotyrosine immunoblot of BNL CL.2 cell lysate protein separated using an 11 % SDS- PAGE gel for BNL CL.2 cells exposed to the four concentrations of TCDD is presented in Figure 30. Results of scanning the control and TCDD-treated lanes are presented in Figure 31; the represented peaks are p34 and p33 tyrosylphosphosphoproteins. In Figure 32 the putative cyclin dependent kinases (p34/p33) are quantified from the anti-phosphotyrosine immunoblot.

EXAMPLE 9

Enhanced tyrosylphosphorylation of p34/p33 in BNL CL.2 cell lysates 24 hours following exposure to the nongenotoxic carcinogen pirnixic acid.

Summary

It is demonstrated that exposure of BNL CL-2 cells to 1, 10 or 100 nM pirnixic acid for 24 h in a low semm media enhances the tyrosine phosphorylation status of two cell lysate proteins, p34 and p33, compared to dimethylsulfoxide-treated controls. These results indicate that the early in vitro effects of the nongenotoxic carcinogen pirnixic acid can be quantified through a change in cellular p34/p33 tyrosylphosphorylation and that stimulation of tyrosylphosphorylation of p34/p33 is specific for nongenotoxic carcinogens.

Materials and Methods

Chemicals: This section is as previously described in Example 7.

Tissue culture cells, culture conditions and dosing: BNL CL.2 cells (ATCC TIB73) are purchased from American Type Culture Collection (Bethesda, MD). These cells are representative of normal mouse hepatocytes. All other procedures were performed as detailed in Example 7.

The following concentrations and reagents are added to the appropriate tubes (4 plates treatment). Dimethyl sulfoxide (DMSO) is used as the diluent for TCDD.

10 mL of DMEM + 20% FBS-HI + 0.1 % DMSO (positive control)

10 mL of DMEM + 0.5% FBS-HI + 0.1 % DMSO

10 mL of DMEM + 0.5% FBS-HI + 1 nM pirnixic acid

10 mL of DMEM + 0.5% FBS-HI + 10 nM pirnixic acid

10 mL of DMEM + 0.5% FBS-HI + 100 nM pirnixic acid

10 mL of DMEM + 0.5% FBS-HI + 1000 nM pirnixic acid

All plates were returned to the incubator for24 h at the environmental conditions listed above. After the 24 h incubation period, the cells are harvested using the harvesting procedure described.

Gel electrophoresis and immunoblotting with anti-phosphotyrosine: These procedures are carried out as described in Example 1 except that anti-phosphotyrosine antibody is used in place of anti-PSTAIR antibody.

Protein determination: This procedure is performed as described in Example 1.

Results

Exposure of BNL CL2 cells to pirnixic acid for 24 h results in increases in tyrosylphosphorylation of p34 relative to the vehicle control for the 1 and 10 nM concentrations, 96 and 58% increases, respectively. At 100 nM pirnixic acid the tyrosylphosphorylation of p34 is similar to the vehicle control, while at 1000 nM tyrosylphosphorylation of p34 is depressed 60% from the vehicle control. Twenty percent

semm supplementation results in an increase of tyrosylphosphorylation of p34 of 229%, relative to the vehicle control. The 5% semm supplementation control exhibits no tyrosylphosphorylation at p33, while pi ixic acid exposure enhances tyrosylphosphorylation of this putative CDK to 2.0, 2.5 and 0.5 density units, respectively, at the 1, 10, and 100 nM concentrations. The increases in tyrosylphosphorylation of p33 by pimixic acid at 1 and 10 nM are roughly 4 times the p33 tyrosine phosphorylation produced by 20% semm supplementation.

The anti-phosphotyrosine immunoblot of BNL CL.2 cell lysate protein separated using an 11 % SDS-PAGE gel for BNL CL.2 cells exposed to the four concentrations of pimixic acid is presented in Figure 33. Results of scanning the control and TCDD-treated lanes are presented in Figure 34; the represented peaks are p34 and p33 tyrosylphosphosphoproteins. In Figure 35 the putative cyclin dependent kinases (p34/p33) are quantified from the anti-phosphotyrosine immunoblot.

Assay Examples

EXAMPLE 10

Use of a microtiter assay for the assessment of enhanced tyrosylphosphorylation of cyclin-dependent kinases (CDK) or p34 ^c kinase in hepatic, pulmonary and renal cytosol (S-9) preparations from C57BL/6J female mice administered 2,3,7, 8- tetrachlorodibenzo-p-dioxin for 90 days

Summary

The regulation of the tyrosylphosphorylation status of the cytosolic cyclin dependent kinases (CDK) has been considered the control mechanism for the entry into G, from G ₀ , the START signal, and also for the movement of the cell from G ₂ to Ml the initiation of mitosis. We have discovered that this control mechanism is related to the level of CDK. However, as the level of CDK increase, so does the total level of tyrosylphosphorylation. A microtiter kit is described that allows for the demonstration of enhanced tyrosylphosphorylation of hepatic CDK as well as p34 ^cdc2 kinase following the

daily administration of 0.25, 0.5, 1 or 2 ng 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)/kg to young, female mice for 90 days. In addition, the microtiter kit may be used to assay for enhanced tyrosylphosphorylation of CDK in extrahepatic tissues and thus allow for the identification of the most sensitive responding tissue.

Materials and Methods

Materials and Chemicals:

Reagents;

A. Sodium carbonate buffer; 0. IM, pH 9.6

a) Mix 71.3 ml of 1 M NaHCO ₃ and 28 ml of IM Na ₂ CO ₃ -

b) Add 800 ml ddH ₂ 0.

c) Adjust pH to 9.6 and Qs to 11.

B. 1OX Phosphate buffered saline; 0. 15M, pH 7.2

a) NaCl, 80.0 g/1.

b) KCI, 2.0 g/1.

c) Na ₂ HPO ₄ , 11.5 g/1.

d) NaH ₂ PO ₄ , 2.0 g./l.

C. Blocking buffer; PBS with 3% BSA

a) IX PBS with 3 g. BSA per 100 ml.

D. Washing buffer; PBS with 0.2% Triton X-100

a) IX PBS with 0.2 ml of Triton X-100 per ml.

E. Prep Buffer, 25mM Tris-HCl, pH 8.0 with 10 M MgCI ₂ , 15mM EGTA, 0. 1 % Triton

X- 100,0. ImM PMSF, O. l M Na fluoride, 6OmM β-glycerophosphate, 15mM paranitrophenylphosphate, 0.1 mM Na orthovanidate, lμg/ l leupeptin, 10,μg/ml soybean trypsin inhibitor, lμg/ml aprotinin, and lOμg/ml tosyl phenylalanine.

F. Assay buffer; 5OmM Tris-HCI, pH 7.4 with lOmM MgCl ₂ , ImM DTT, and all inhibitors of phosphatases and proteases contained in Prep buffer.

G. Citrate buffer;

a) Add 9.6 g Citric acid (MW 192.12) to 950 ml ddH ₂ O.

b) Adjust pH to 4.0 with 5M NaOH and store at 4° C.

H. ABTS stock solution;

a) 0.5487 g ABTS to 25 ml with double distilled H ₂ 0 and store at 4°C.

I. ABTS substrate; mM ABTS

a) 0.05 ml ABTS

b) 0.02 ml diluted H20, (0.5M)

c) 5.0 ml citrate buffer

Animals and dosing: Four to six-wk old, femaleC57BL/6J mice are obtained from

Harton Sprague Dawley (Indianapolis, IN). The mice are fed Prolab RMH 1000 (Agway, Cortland, NY) and receive tap water ad libitum. All mice are housed three per cage and maintained on a photoperiod of 12 h. Mice are administered TCDD in co oil at 0, 0.25, 0.5, 1, or 2 ng/kg by oral gavage daily for a period of 90 days. Ten mice are treated at each dose and the volume of the dose is approximately 0.1 mL per mouse.

Procedure;

Plate preparation:

1. 100 μl of anti-PSTAIR or anti-C-terminus antibody at a concentration of 10 Ag/mL in of 0. IM Na carbonate buffer pH 9.6 is added to the wells of a microtiter plate and incubated ovemight at 4°C. These are the capture antibodies and will retain all

CDK or p34 ^cdc2 kinase, respectively.

2. Wash plates 3x with washing buffer by filling the wells, allowing them to sit for two minutes, and inverting and shaking them.

3. Block plates for two hours at room temp by filling the wells with blocking buffer. The plates can be washed lx with washing buffer and stored for several weeks at 4°C.

4. Wash fresh plated 3x or stored plates 2x with washing buffer prior to use.

Sample preparation:

AH preparation procedures are performed on individual or pooled hepatic, pulmonary or renal samples. Preparation and -80°C storage of tissue S-9 fractions is performed exactly as previously described in the scientific literature (32). This procedure

involves killing the mouse by cervical dislocation, removing the liver, lung or kidney sample and homogenizing the tissue in three volumes of Prep buffer. This tissue homogenate is centrifuged at 9,000 x g for 20 min at 4°C. The resulting supematant fraction, termed the S-9, is decanted into 1.5 mL plastic, conical tubes, frozen in a dry ice/ethanol bath and stored at -80°C until the microtiter assay can be performed.

Assay;

1. 200 μg of sample tissue protein is diluted in Prep buffer and mixed 1: 1 with blocking buffer.

2. This is added to the wells of a prepared plate and incubated for 5 hr at 40C with slow,constant shaking.

3. Plates are washed 3x with washing buffer and lx with assay buffer.

4. 200 μl of primary(anti-phosphotyrosine) antibody at a dilution of 1: 1000 in blocking buffer is added to each well and incubated for 2 hr at 4°C.

5. Plates are washed 3x with washing buffer.

6. 200 μl of peroxidase-conjugated (anti-mouse) secondary antibody at a dilution of 1:3000 in blocking buffer is added to each well and incubated for 1 hr at 4°C.

7. Wash plates 3x with washing buffer.

8. Add 200 μl of ABTS solution and read once a minute for 10 min in kinetics mode

(Biotek EL312) at 415 nm.

Inteφretation of results

Microtiter assay - The anti-PSTAIR or anti-C- terminus antibody will, respectively, capture all CDK or p34 ^cdc2 kinase present in the tissue S-9 fraction in the microtiter well. The anti-phosphotyrosine antibody quantifies the extent of tyrosylphosphorylation of the total CDK or p34 ^cdc2 kinase. This quantification represents the extent to which the cells from the sampled tissue have been signaled to exit the G ₀ stage of the cell cycle (index of

proliferative signaling) by exposure to the test chemical. The current state of knowledge in the role of the cyclin dependent kinases in controlling the cell cycle (47-52) does not allow for an absolute determination as to the extent of CDK tyrosylphosphorylation relating to the strength of the proliferative signal. The fact that molecules other than peptide-like growth factors have the ability to enhance the tyrosylphosphorylation status of the CDK has not been reported in the literature. Therefore, inteφreting the capacity of a test chemical to direct the cell toward mitosis requires a comparison to a control group treated only with the vehicle. A test chemical is considered positive for the capacity to function as a nongenotoxic carcinogen when the extent of CDK or p34 ^cdc2 kinase tyrosylphosphorylation is statistically greater (p < 0.05) than a concurrent control.

Results

Microtiter assay - As seen in Figure 36, the dosing of C57BL/6J female mice with 0, 0.25. 0.5, 1 or 2 ng TCDD/kg-day (A, B, C and D, respectively) for 90 days results in enhanced tyrosylphosphorylation of hepatic CDK but not pulmonary or renal CDK. This identifies the target tissue for the cellular proliferative effects of TCDD as the liver. Maximal increase in tyrosylphosphorylation of hepatic CDK is observed at the 0.5 ng TCDD/kg-day dose regimen. Results for the tyrosylphosphorylation of p34 ^cdc2 kinase are similar (Figure 37), although the absolute increase observed is lower. This is due to the ffaacctt tthhaatt pp3344 ^ccdcc2 kkiinnaassee rreepprreesseennttss oonnly one of several possible CDK in the cytosol that function to regulate cell replication.

EXAMPLE 11

Use of a rhicrotiter assay for the assessment of enhanced expression of cyclin- dependent kinases (CDK) or p34cdc2 kinase in hepatic cytosol (S-9) preparations from young male rats 1, 2, or 3 days following the administration of the nongenotoxic carcinogen pirinixic acid (WY14,643)

Summary

This example demonstrates of the utility of the assay for the quantification of CDK response elicited by a test chemical in vivo following an exposure period of any length and a description of a kit to perform the assay.

The administration of the nongenotoxic carcinogen pirinixic acid to young, male rats results in the enhanced expression of total cytosolic cyclin-dependent kinases (CDK). A microtiter kit is described that allows for the demonstration of enhanced expression of hepatic CDK as well as p34 ₍ cdc ^c 2 kinase following a single dose of 50 mg pirinixic acid.

Materials nd Chemicals;

Reagents:

A. Sodium carbonate buffer; 0. IM, pH 9.6

a) Mix 71.3 ml of IM NaHCO ₃ and 28 ml of IM Na ₂ CO ₃

b) Add 800 ml ddH ₂ 0.

c) Adjust pH to 9.6 and Qs to 1 1.

B. 1OX Phosphate buffered saline; 0. 15M, pH 7.2

a) NaCl, 80.0 g/1.

b) KCI, 2.0 g/1.

c) Na ₂ HP0 ₄ , 11.5 g/1.

d) NaHIP0 ₄ , 2.0 g/1.

C. Blocking buffer; PBS with 3% BSA

a) IX PBS with 3 g BSA per 100 ml.

D. Washing buffer; PBS with 0.2% Triton X-100

a) IX PBS with 0.2 ml of Triton X-100 per ml.

E. Prep Buffer; 25mM Tris-HCI, pH 8.0 with 10MM MgCl ₂ , 15mM EGTA, 0. 1 %

Triton X- 1 00, 0. I mM PMSF, 0. 1 mM Na fluoride , 60mM β-glycerophosphate, 15mM paranitrophenylphosphate, 0. ImM Na orthovanidate, lμg/ml leupeptin, lOμg/ml soy bean trypsin inhibitor, Iμg/ml aprotinin, and lOμg/ml tosyl phenylalanine.

F. Assay buffer; 5OmM Tris-HCI, pH 7.4 with lOmM MgCl ₂ , ImM DTT, and all inhibitors of phosphatases and proteases contained in Prep buffer.

G. Citrate buffer;

a) Add 9.6 g Citric acid (MW 192.12) to 950 ml ddH ₂ 0.

b) Adjust pH to 4.0 with 5M NaOH and store at 4°C.

H. ABTS stock solution;

a) 0.5487 g ABTS to 25 ml with double distilled H20 and store at 4°C.

I. AM substrate; OAmM ABTS

a) 0.05 ml ABTS

b) 0.02 ad diluted H ₂ 0 ₂ (0.5M)

c) 5.0 ml citrate buffer

Animals, dosing and preparation of tissue S9: This procedure is performed as described in Example 2 except only a single 50 mg/kg dose of pirinixic acid is administered. Livers are removed from rats on post dosing days 1, 2 and 3.

Gel electrophoresis and immunoblotting with anti- cdc2 C-terminus: These procedures are carried out as described in Example 1 except that anti-cdc2 C-terminus is used in place of anti-PSTAIR antibody.

Protein determinations: This procedure is performed as described in Example 1.

Microtiter assay procedure:

Sample preparation:

All preparation procedures are performed on individual or pooled hepatic (tissue) samples. Preparation and -80°C storage of tissue S9 fractions is performed exactly as previously described in the scientific literature (35). This procedure involves killing the rat by cervical dislocation, removing and homogenizing the tissue in three volumes of Prep buffer. This tissue homogenate is centrifuged at 9,000 x g for 20 min at 4°C. The resulting supematant fraction, termed the S9, is decanted into 1.5 ml plastic, conical tubes, frozen in a dry ice/ethanol bath and stored at -80°C until the microtiter assay can be performed.

Assay:

1. 50 Ag of S9 tissue protein is diluted in Prep buffer and mixed 1 : 1 with blocking buffer.

2. This is added to the wells of a prepared plate and incubated for 5 hr at 40C with slow constant shaking.

3. Plates are washed 3x with washing, buffer and lx with assay buffer.

4. 200 Al of primary (anti-cdc2 C-terminus) antibody at a dilution of 1: 1000 in blocking buffer is added to each well and incubated for 2 hr at 4°C.

5. Plates are washed 3x with washing buffer.

6. 200 Al of peroxidase-conjugated (anti-mouse) secondary antibody at a dilution of

1:3000 in blocking buffer is added to each well and incubated for 1 hr at 4°C.

7. Wash plates 3x with washing buffer.

8. Add 200 Al of ABTS solution and read once a minute for 10 min in kinetics mode (Biotek EL312) at 415 nm.

Inteφretation of results

Microtiter assay:

Due to cross-reactivity with other, unidentified CDK, the anti-cdc2 C-terminus antibody will quantify the total CDK expression in the tissue. This quantification represents the extent to which the cells from the sampled tissue have been signaled to exit the G„ stage of the cell cycle (index of proliferative signaling) by exposure to the test chemical. The current state of knowledge in the role of the cyclin dependent kinases in controlling the cell cycle (46-51) does not allow for an explanation as to the strength of the proliferative signal. The fact that molecules other than peptide-like growth factors have the ability to enhance the expression of the CDK has not been reported in the literature. Therefore, inteφretation of the capacity of a test chemical to direct the cell toward replication relies on a comparison to a concurrent control group treated only with the vehicle used to administer the test chemical. A test chemical is considered positive for the capacity to function as a nongenotoxic carcinogen when the extent of CDK or p34 ^{c c2} kinase expression is statistically greater (p < 0.05) than a concurrent control.

Results

Immunoblotting with anti - cdc2 C- terminus - Figure 38 depicts the immunoblot of rat hepatic S9 protein separated using 10 to 11 % SDS-PAGE gels for control (lanes 1 and 3) and WY14,643-treated rats (lanes 2 and 4). A single intensely-stained band was visible in the CDK region (32 to 35 kDa) in hepatic S9 samples obtained from rats 3 days after receiving a single dose of 50 mg WY14,643/kg.

Microtiter assay - As seen in Figure 39, the extent of CDK expression in the livers of young, male rats receiving a single dose of 50 mg/kg of WY14,643 increases steadily during the 3-day post dosing observation period. CDK expression in control animals remains constant over the same 3-day period.

EXAMPLE 12

Enhanced expression of CDK in BNL CL.2 cell lysates 48 hours following exposure to the nongenotoxic carcinogen 2,3,7,8-tetrachIorodibenzo-p-dioxin.

Summary

This example demonstrates the utility of the assay for the quantification of CDK response elicited by a test chemical in vitro following an exposure period of 48 hours.

It is demonstrated that exposure of BNL CL.2 cells to 0.1 , 1 , or 10 nM 2,3,7,8- tetrachlorodibenzo-p- dioxin for 48 hours in a low semm media enhances the expression of two cell lysate proteins, p34 and p33 immunoreactive with anti -cdc2 C- terminus antibody, compared to dimethylsulfoxide-treated controls. These results indicate that the early in vitro effects of the nongenotoxic carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin can be quantified through a change in cellular CDK expression and therefore that stimulation of CDK is specific for nongenotoxic carcinogens.

Materials and Methods

Chemicals: This section is as previously described in Example 6.

Tissue culture cells, culture conditions and dosing: BNL CL.2 cells (ATCC TIB73) are purchased from American Type Culture Collection (Bethesda, MD). These cells are representative of normal mouse hepatocytes. All other procedures were performed as detailed in Example 6.

The following concentrations and reagents are added to the appropriate tubes (4 plates/treatment) .Dimethyl sulfoxide (DMSO) is used as the diluent for TCDD.

10 mL of DMEM + 2O% FBS-HI + 0.1 %; DMSO (positive control)

10 mL of DMEM + 0.5% FBS-HI + 0.1 % DMSO

10 mL of DMEM + 0.5% FBS-HI + 0.1 nM TCDD

10 mL of DMEM + 0.5% FBS-HI + 1.0 nM TCDD

10 mL of DMEM + 0.5% FBS-HI + 10 nM TCDD

All plates were returned to the incubator for 48h at the environmental conditions listed above. After the 48 h incubation period, the cells are harvested using the harvesting procedure described.

Gel electrophoresis and immunoblotting with anti -Cdc2 C - terminus: These procedures are carried out as described in Example 1 except that anti-cdc2 C-terminus antibody is used in place of anti-PSTAIR antibody.

Protein determination: This procedure is performed as described in Example 1.

Results

Exposure of BNL CL2 cells to 0.1, 1, or 10 nM 2,3,7,8-tetrachlorodibenzo-p- dioxin (TCDD) for 48 h results in an increase in expression of anti-cdc2 C-terminus immunoreactive proteins p34 and p33 compared to the semm deprived DMSO control (Figure 40, lanes 8, 9and 10 compared to lane 6). CDK protein expression at lOnM TCDD was similar to that observed with semm stimulation (lane 10 compared to lane 7).

EXAMPLE 13

Testing Chemical Compounds or Test Samples for Nongenotoxic Carcinogens

The methods and assays systems disclosed in Examples 1-12 can be used to test chemical compounds, human and animal semm, air, water, and soil environmental samples for the presence of nongenotoxic carcinogens. Many cytotoxic compounds have been identified as anti-tumor leads based on in vitro cytotoxicity tests. The actual primary screen is carried out at three preselected does, using a 96-well microtiter plate. When "significant cytotoxicity" is found it is confirmed using six doses with three replicates per dose to define the dose-response curve. A substance may also be tested for its antineoplastic effects.

The above reagents, including antibodies, with or without aliquots of the cell lines described in the Examples may be packaged in the form of kits for the testing of suspected nongenotoxic carcinogens. Equivalent reagents, antibodies or cell lines may be substituted for the ones described in the Examples. In one preferred embodiment, a panel of three cell lines are included in the test kits. The three cell lines are a murine cell line, a rat cell line and a human cell line. Cell lines which are suitable for this puφose include murine BNL- CL.2 cells, a primary rat hepatic cell line developed by Paracelsian, Inc., PRLN-RH1, and a human hepatic cell line such as Hep G2 (ATCC: HB-8065).

Tissue samples, cells, and cell lysates from an individual person or animal can be substituted for the cell lines described, when testing for an individual's sensitivity to nongenotoxic carcinogens. Only reagents and antibodies would therefore be packaged in kits to test individual susceptibility.

Serum Assays

Summary

Normal proliferation of cells is accompanied by a several-fold increase in the expression of the cell cycle control enzymes (CDKs) as the cell exits G ₀ phase and enters the cell cycle. The assay kit taught by the invention allows the detection and quantification of CDK2 in semm by use of an ELISA assay. The semm assay employs a plate capture

technique. The plate is washed and working anti-CDK2 (biotinylated)- Streptavidin- alkaline phosphatase is added to the wells of the plate. A dilution is used to dilute the sample 1:40. The semm is delivered to the ELISA plates which are washed with wash and blocking buffers and then incubated. This technique obviates the need for a secondary detection antibody and can be used with any fluid. The following example teaches an altemative method to detect levels of expression of CDK in semm using polyclonal or monoclonal antibodies to CDK2 and a secondary detection antibody.

EXAMPLE 14

Detection of enhanced levels of expression of cyclin dependent kinases in the blood, plasma or sera of woodchucks with hepatocellularcarcinoma by immunoblotting or microtiter assay

Summarv

This example demonstrates the utility of the determination of enhanced levels of cyclin dependent kinases in the blood, sera or plasma of woodchucks with hepatocellular carcinoma as a means of identification of the presence of abnormal cellular proliferation related to the hepatic cancer.

Woodchucks with hepatic cancer exhibit higher concentrations of the cyclin dependent kinase CDK2 in sera or plasma than normal woodchucks. These results indicate that the determination of semm or plasma concentrations of CDK2 serves as a diagnostic for the presence of abnormal cellular proliferation related to hepatocellular carcinoma in woodchucks.

Materials and Methods

Selection of woodchucks with hepatocellular carcinoma and controls:

Woodchuck cancer patients were selected from a colony of woodchucks that were infected with hepatitis B vims prior to the development of hepatocellular carcinoma. Animals were diagnosed with hepatocellular carcinoma following necropsy and histological examination of hepatic tissue for neoplastic lesions. All diagnoses of cancer were confirmed by an

oncologist with experience with the individual proliferative disease. Control sera were obtained from apparently healthy woodchucks following euthanasia. Histological examination of liver did not reveal any abnormal cellular growth characteristic of hepatocellular carcinoma.

Preparation of plasma or serum sample: Approximately 5 mL of blood was obtained from an easily accessible vein. For the preparation of plasma, clotting of the blood was inhibited by any of the standard anticoagulants. For the preparation of sera, the blood was allowed to clot at room temperature for 1 to 2 hours. The clotted sample was then put into a refrigerator at 4°C for 24 hours. Clot and sera were spun at 3,000 x g for 5 to 10 minutes and the sera were removed and stored at -80°C until assayed.

Immunoblotting semm samples for cell-cycle related proteins:

Commercially obtained antibodies: Polyclonal or monoclonal antibodies to CDK2, CDK4, CDK5, p53, Rb, PCNA, WAF1/CIP1, nm23, mdm2, cyclin A, cyclin B, cyclin Dl, cyclin D2, cyclin D3, cyclin E, alkaline phosphatase or peroxidase-coπjugated anti- rabbit IgG and anti-mouse IgG antibodies were obtained from commercial sources (e.g. Transduction Laboratories, Lexington, KY; Oncogene Science, Inc. Manhasset, NY; Sigma, St. Louis, MO).

Immunoblotting of cell cycle proteins: Five mL of sera was solubilized in SDS gel sample buffer (36) and denatured at 100°C for 8 minutes; SDS PAGE was carried out on the denatured samples as described (36) using 11 % polyacrylamide gels.

The immunoblotting was carried out as described by Towbin et al. (37); however a Milliblot SDE electroblot apparatus (Millipore, Bedford, MA), was used to transfer proteins from polyacrylamide gels to an Immobilon* membrane filter (Millipore,Bedford, MA). Complete transfers were accomplished in 25-30 minutes at 500 mA. Membrane filters were blocked by incubating in TBS (50 M Tris, 150 mM NaCl, pH 7.5) containing 5% commercial nonfat dry milk for 30 minutes at room temperature and incubated 2 hours with 5 mg/mL of the cell cycle protein antibody in TBST (0.05% Tween 20 in TBS). Molecular weights of immuno-stained proteins were estimated by

adding molecular weight standards to reference lanes and staining the membrane filters with amido back 10 B.

To visualize the antibody reactions, the membranes were incubated for 2 hours at room temperature with alkaline phosphatase-conjugated anti-rabbit IgG for rabbit polyclonals or anti-mouse IgG for mouse monoclonals diluted 1: 1000 in TBST and developed for 15 minutes.

Microtiter Assay for cell-cycle related proteins:

Commercially obtained antibodies: Polyclonal or monoclonal antibodies to CDK2 were obtained from Oncogene Science (Manhasset, NY) and Transduction Laboratories (Lexington, KY), respectively. Alkaline phosphatase or peroxidase-conjugated anti-rabbit IgG and anti-mouse IgG antibodies were obtained from Sigma (St. Louis, MO).

Chemicals and Materials:

Reagents:

A. Sodium Carbonate buffer, 0.1 M, pH 9.6.

1) Mix 71.3 mL of 1 M NaHCO ₃ and 28 mL of 1 M Na ₂ CO ₃

2) Add 800 mL dd H ₂ O

3) Adjust pH to 9.6 and Qs to 1.1 L

B. 10X Phosphate buffered saline, 0.15 M, pH 7.2.

1) NaCl, 80.0 g/L

2) KCI, 2.0 g/L

3) Na ₂ HP0 ₄ , 11.5 g/L

4) NaH ₂ PO ₄ , 2.0 g/L

C. Blocking buffer, PBS with 3% BSA

1) IX PBS with 3 g BSA per 100 mL

D. Washing buffer, PBS with 0.2% Triton X-100

1) 1 X PBS with 0.2 mL of Triton X-100 per mL

E. Prep buffer, 25 mM Tris-HCl, pH 8.0 with 10 mM MgC12, 15 mM EGTA, 0.1 %

Triton X-100, 0.1 mM PMSF, 0.1 mM sodium fluoride, 60 mM b- glycerophosphate, 15 mM p-nitrophenylphosphate, 0.1 M sodium orthovanidate,

1 mg/mL leupeptin, 10 mg/mL soybean trypsin inhibitor, 1 mg/mL aprotinin and 10 mg/mL tosyl-phenylalanine.

F. Assay buffer, 50 mM Tris-HCl, pH 7.4 with 10 mM MgCI ₂ , 1 mM DTT.

G. Citrate buffer,

1) Add 9.6 g citric acid (MW 192.12) to 950 mL ddH ₂ O

2) Adjust pH to 4.0 with 5 M NaOH and store at 4°C

H. ABTS stock solution

1)0.5487 g ABTS to 25 mL with ddH ₂ 0 and store at 4°C

I. ABTS substrate, 0.4 mM ABTS

1) 0.05 mL ABTS

2) 0.02 mL diluted H ₂ O ₂ (0.5 M)

3) 5.0 mL citrate buffer

Sample Preparation:

All preparation procedures are performed on individual plasma or serum samples.

Assay;

1. Five mL of plasma or semm is diluted in Prep buffer and mixed 1:1 with blocking buffer.

2. This is added to the wells of a plate and incubated for 5 hr at 4°C with slow constant shaking.

3. Plates are washed 3x with washing buffer and lx with assay buffer.

4. Two-hundred L of primary antibody at a dilution of 1: 1000 in blocking buffer is added to each well and incubated for 2 hr at 4°C.

5. Plates are washed 3x with washing buffer.

6. Two-hundred mL of a peroxidase-conjugated secondary antibody at a dilution of 1:3000 in blocking buffer is added to each well and incubated for 1 hr at 4°C.

7. Wash plates 3x with washing buffer.

8. Add 200 mL of ABTS solution and read the color change on a microtiter plate reader (Botek EL312) at 415 nm.

Results

Figure 41 represents an anti-CDK2 immunoblot of semm from normal woodchucks (lanes 1, 3, 5, 8 and 11) and woodchucks with hepatocellular carcinoma (lanes 2, 4, 6, 7, 9, 10 and 12). Sera from animals with hepatocellular carcinoma exhibited darker staining bands at 33 kDa relative to healthy animals. The bar graph in Figure 42 depicts the results of the microtiter immunoassay of woodchuck plasma or sera concentrations of CDK2 from normal woodchucks and woodchucks with hepatocellular carcinoma. Values presented represent the means of six woodchucks per group; the error bar on the control group represents one standard deviation. Semm CDK2 content was increased an average of 4.3- fold in woodchucks with hepatocellular carcinoma relative to controls. Although other cell cycle related proteins were sometimes seen in the sera of woodchucks with hepatocellular cancers, no consistent association of the presence of an hepatocellular carcinoma and the cell cycle protein was obvious.

EXAMPLE 15

Detection of enhanced levels of expression of cyclin dependent kinases in the serum of dogs with various tumors

Summary

This example demonstrates the utility of the determination of enhanced levels of cyclin dependent kinases in the sera or plasma of dogs with a variety of cancers as a means of identification of the presence of abnormal cellular proliferation related to the presence of the cancer.

It is demonstrated that dogs with cancers exhibit several fold higher concentrations of cyclin dependent kinase CDK2 in sera or plasma than normal dogs. These results indicate that the determination of semm or plasma concentrations of CDK2 can serve as an easily accessible diagnostic biomarker for the presence of abnormal cellular proliferation related to cancer in dogs.

Materials and Methods

Selection of canine cancer patients and controls: Canine cancer patients were selected from patients admitted for physical signs of a proliferative disease. All diagnoses of cancer in the patients were confirmed by an oncologist with experience with the individual proliferative disease and the patient. Control sera were obtained from apparently healthy dogs undergoing normal procedures such as spaying or routine physical exams.

Preparation of plasma or serum sample: This section is as previously described in Example 14.

Immunoblotting serum samples for CDK2 protein: This section is as previously described in Example 14.

Microtiter assay for CDK2 protein: This section is as previously described in

Example 14.

Results

Figure 43 represents an anti-CDK2 immunoblot of semm from normal dogs (lanes 1, 2, 3, 4, 5 and 6) and dogs with a variety of cancers (lanes 7, 8, 9, 10, 11, 12, 13, 14 and 15). Sera from dogs with cancers exhibited dark staining bands at 33 kDa, while the 33 kDa staining bands were not visible for any of the six healthy dogs. The bar graph in Figure 44 depicts the results of the microtiter immunoassay of canine plasma or sera concentrations of CDK2 from normal dogs and dogs diagnosed with a variety of cancers. Values presented represent the means ± SD of 32 normal dogs and five dogs diagnosed with cancer. Serum CDK2 content was increased an average of 20-fold in dogs diagnosed with cancer relative to normal dogs.

EXAMPLE 16

Detection of enhanced levels of expression of cyclin dependent kinases in the plasma or sera of cats with various tumors

Summary

This example demonstrates the utility of the determination of enhanced levels of cyclin dependent kinases in the sera or plasma of cats with a variety of cancers as a means of identification of the presence of abnormal cellular proliferation related to the presence of the cancer.

It is demonstrated that cats with cancers exhibit several fold higher concentrations of cyclin dependent kinase CDK2 in sera or plasma than normal cats. These results indicate that the determination of semm or plasma concentrations of CDK2 can serve as an easily accessible diagnostic biomarker for the presence of abnormal cellular proliferation related to cancer in cats.

Materials and Methods

Selection of feline cancer patients and controls: Feline cancer patients were selected from patients admitted for physical signs of a proliferative disease. All diagnoses of cancer in the patients were confirmed by an oncologist with experience with the individual proliferative disease and the patient. Control sera were obtained from apparently healthy cats undergoing normal procedures such as spaying or routine physical exams.

Preparation of plasma or serum sample: This section is as previously described in Example 14.

Immunoblotting serum samples for CDK2 protein: This section is as previously described in Example 14.

Microtiter assay for CDK2 proteins: This section is as previously described in

Example 14.

Results

Figure 45 represents an anti-CDK2 immunoblot of semm from normal cats (lanes 1, 2, 3, 4, 5 and 6) and dogs with a variety of cancers (lanes 7, 8, 9, 10, 11, 12, 13, 14 and 15). Sera from dogs with cancers exhibited dark staining bands at 33 kDa, while the 33 kDa staining bands were not visible for any of the six healthy dogs.

The bar graph in Figure 46 depicts the results of the microtiter immunoassay of feline plasma or sera concentrations of CDK2 from normal cats and cats diagnosed with a variety of cancers. Values presented represent the means of 32 normal cats and two cats diagnosed with cancer; error bar on the control group represents one standard deviation. Semm CDK2 content was increased an average of 9.3-fold in cats diagnosed with cancer relative to normal cats.

EXAMPLE 17

Detection of enhanced levels of expression of cyclin dependent kinases in the sera or plasma of human males with prostatic cancer

Summary

This example demonstrates the utility of the determination of enhanced levels of cyclin dependent kinases in the sera or plasma of humans with prostatic cancer as a means of identification of the presence of abnormal cellular proliferation related to prostatic cancer.

It is demonstrated that patients with prostatic cancer exhibit higher concentrations of cyclin dependent kinases CDK2 in sera or plasma than normal, age-matched individuals. These results indicate that the determination of semm or plasma concentrations of CDK2 serves as a diagnostic for the presence of abnormal cellular proliferation related to prostatic cancer.

Materials and Methods

Selection of cancer patients and controls: Fifteen cancer patients are randomly selected from files of oncology patients. All diagnoses of cancer in the patients were confirmed by an oncologist with experience with the individual proliferative disease and the patient. Group age-matched controls were selected from the same hospital population. Controls were considered age-matched when their ages were within seven years and had no previous history of a proliferative disease.

Immunoblotting serum samples for CDK2 protein: This section is as previously described in Example 14.

Microtiter assay for CDK2 proteins: This section is as previously described in Example 14.

Results

Figure 47 represents an anti-CDK2 immunoblot of semm from normal human males without cancer (lanes 1 through 9) and prostate cancer patients (lanes 10 through 18). Sera from patients with prostate cancer exhibited dark staining bands at 33 kDa (CDK2), while the 33 kDa staining bands were barely visible for any of the nine healthy human males. The bar graph in Figure 48 depicts the results of the microtiter assay of semm CDK2 content of normal males (controls) and males diagnosed with prostate cancer. Values presented represent means and standard deviations of four healthy males and four males diagnosed with prostate cancer. Mean semm CDK2 concentration of the prostate cancer group was increased 3-fold relative to the noncancer control group.

EXAMPLE 18

Detection of enhanced levels of expression of cyclin dependent kinases in the sera or plasma of human females with breast cancer

Summarv

This example demonstrates the utility of the determination of enhanced levels of cyclin dependent kinases in the sera or plasma of humans with breast cancer as a means of identification of the presence of abnormal cellular proliferation related to breast cancer.

It is demonstrated that patients with breast cancer exhibit higher concentrations of the cyclin dependent kinase CDK2 in sera or plasma than normal, group age-matched individuals. These results indicate that the determination of semm or plasma

concentrations of CDK2 serves as a diagnostic for the presence of abnormal cellular proliferation related to breast cancer.

Materials and Methods

Selection of cancer patients and controls: This section is as previously described in Example 17.

Preparation of plasma or serum sample: This section is as previously described in Example 14.

Immunoblotting serum samples for CDK2 protein: This section is as previously described in Example 14.

Results

Figure 49 represents an anti-CDK2 immunoblot of semm from normal human females without cancer (lanes 1 through 9) and breast cancer patients (lanes 10 through 18). Sera from patients with breast cancer exhibited dark staining bands at 33 kDa (CDK2), while the 33 kDa staining bands were barely visible for any of the nine healthy human females.

Although the present invention has been described in relation to particular embodiments thereof, many other variations and modifications and other uses will become apparent to those skilled in the art. It is preferred, therefore, that the present invention be limited not by the specific disclosure herein, but only by the appended claims.

Literature Cited

1. Farber, E.(1991) Hepatocyte proliferation in stepwise development of experimental liver cell cancer. Dig. Dis. Sci. 36, 973-978 2. Farber, E.(1984) The multistep nature of cancer development. Cancer Res 44, 4217- 4223

3. Kanduc, D.; Aresta, A.; Quagliariello, E. and Farber, E. (1992) Effect of MNU on the methylation pattem of hepatic DNA during compensatory cell proliferation. Biochem. Biophys. Res Commun. 184, 107-111

4. Ames, B. N. (1984) The detection of environmental mutagens and potential 5 carcinogens. Cancer 53, 2034-2040

5. McCann, J. and Ames, B. N. (1976) Detection of carcinogens as mutagens in the

Salmonella/microsome test:assay of 300 chemicals: discussion. Proc Natl. Acad. Sci.U. S. A. 73, 950-954

6. Ames, B. N.; Durston, W. E.; Yamasaki, E. and Lee, F. D. (1973) Carcinogens are 10 mutagens: a simple test system combining liver homogenates for activation and bacteria for detection. Proc. Natl. Acad. Sci. U. S. A. 70, 2281-2285

7. Ames, B. N.; Lee, F. D. and Durston, W. E. (1973) An improved bacterial test system for the detection and classification of mutagens and carcinogens. Proc. Natl. Acad. Sci. U. S. A. 70, 782-786

15 8. Ames, B. N.(1979) Identifying environmental chemicals causing mutations and cancer. Science 204, 587-593

9. Gold, L. S.; Slone, T. H.; Manley, N. B.; Garfinkel, G. B.; Hudes, E. S.; Rohrbach,

L. and Ames, B. N. (1991) The Carcinogenic Potency Database: analyses of 4000 chronic animal cancer experiments published in the general literature and by the 20 U.S. National Cancer institute/National Toxicology Program. Environ. Health

Perspect. 96, 11-15

10. Farber, E. (1988) Initiators, promoters and the uncertainty principle. Tumour. Biol. 9,

165-169 11. Farber, E. and Sarma, D. S. (1986) Chemical carcinogenesis: the liver as a model. Pathol. Immunopathol. Res 5, 1-28

25 12. Moslen, M. T.; Ahluwalia, M. B. and Farber, E. (1985) 1,2-Dibromoethane initiation of hepatic nodules in Sprague-Dawley rats selected with Solt-Farber system. Arch. Toxicol. 58, 118-119

13. Gold, L. S. ; Bernstein, L. and Ames, B. N. (1990) The importance of ranking possible carcinogenic hazards using HERP. Risk. Anal. 10, 625-8; discussion.

30 14. Busser, M. T. and Lutz, W. K. (1987) Stimulation of DNA synthesis in rat and mouse liver by various tumor promoters. Carcinogenesis 8, 1433-1437

15. Wolfle, D.; Munzel, P.; Fischer, G. and Dock, K. W. (1988) Altered growth control of rat hepatocytes after treatment with 3, 4, 3', 4' - tetrachlorobiphenyl in vivo and in vitro. Carcinogenesis 9, 919-924

35 16. Chida, K. ; Hashiba, H.; Sasaki, K. and Kuroki, T. (1986) Activation of protein kinase C and specific phosphorylation of a Mr 90,000 membrane protein of

promotable BALB/3T3 and C3H/IOT1/2 cells by tumor promoters. Cancer Res 46, 1055-1062

17. Smith, B. M. and Colbum, N. H. (1988) Protein kinase C and its substrates in tumor promoter-sensitive and - resistant cells. J. Biol. Chem. 263, 6424-6431

5 18. Novak-Hofer, I.; Kung, W.; Fabbro, D. and Eppenberger, U.(1987) Estrogen stimulates growth of mammary tumor cells ZR-75 without activation of S6 kinase and S6 phosphorylation. Difference from epidermal growth factor and alpha- transforming growth-factor-induced proliferation. Eur. J. Biochem. 164, 445-451

19. Hunter, T.(1987) A thousand and one protein kinases. Cell 50, 823-829

10 20. Hunter, T. and Cooper, J. A. (1985) Protein-tyrosine kinases. Annu. Rev. Biochem. 54, 897-930

21. Hunter, T. ; Alexander, C. B. and Cooper, J. A. (1985) Protein phosphorylation and growth control. Ciba. Found. Symp. 116, 188-204

22. Yarden, Y. and Ullrich, A. (1988) Growth factor receptor tyrosine kinases. Annu. 15 Rev. Biochem. 57, 443-478

23. Yarden, Y. and Ullrich, A. (1988) Molecular analysis of signal transduction by growth factors. Biochemistry 27, 3113-3119

24. Ullrich, A. ; Riedel, H.; Yarden, Y.; Coussens, L.; Gray, A.; Dull, T.; Schlessinger,

J.; Waterfield, M. D. and Parker, P. J. (1986) Protein kinases in cellular signal 20 transduction: tyrosine kinase growth factor receptors and protein kinase C. Cold.

Spring. Harb. Symp. Quant. Biol. 51 Pt 2, 713-724

25. Yarden, Y.; Kuang, W. J.; Yang-Feng, T.; Coussens, L.; Munemitsu, S.; Dull, T.

J.; Chen, E.; Schlessinger, J.; Francke, U. and Ullrich, A. (1987) Human proto- oncogene c-kit: a new cell surface receptor tyrosine kinase for an unidentified 25 ligand. EMBO J. 6, 3341-3351

26. Hunter, T. (1986) Cancer. Cell growth control mechanisms [news]. Nature 322, 14-16

27. Funasaka, Y.; Boulton, T.; Cobb, M.; Yarden, Y.; Fan, B.; Ly an, S. D.;

Williams, D. E.; Anderson, D. M.; Zakut, R.; Mishima, Y. and et al, (1992) c- Kit-kinase induces a cascade of protein tyrosine phosphorylation in normal human 30 melanocytes in response to mast cell growth factor and stimulates mitogen-activated protein kinase but is down-regulated in melanomas. Mol. Biol. Cell 3, 197-209

28. Bouton, A. H.; Kanner, S. B.; Vines, R. R. and Parsons, J. T. (1991) Tyrosine phosphorylation of three cellular proteins correlates with transformation of rat 1 cells by ppδosrc Mol. Carcinog. 4, 145-152

35 29. Keyomarsi, K. and Pardee, A.B. (1993). Redundant cyclin overexpression and gene amplification in breast cancer cells. Proc Natl. Acad. Sci. U. S. A. 90, 1112-1116.

30. Kato, J.Y., Matsuoka, M., Strom, D.K., and Sherr, CJ. (1994). Regulation of cyclin D- dependent kinase 4 (cdk4) by cdk4-activating kinase. Mol. Cell Biol. 14, 2713-2721.

32. Sherr, CJ. (1993). Mammalian Gl cyclins. CeU 73, 1059-1065.

33. Arion, D. ; Meij-e-r-,L. ; Brizuela, L. and Beach, D. (1988) cdc2 is a component of 5 the M phase-specific histone Hi kinase: evidence for identity with MPF. Cell 55,

371-378

34. Arion, D. and Meijer, L. (1989) M-phase-specific protein kinase from mitotic sea urchin eggs: cyclic activation depends on protein synthesis and phosphorylation but does not require DNA or RNA synthesis. Exp. Cell Res 183, 361-375

10 35. Mailer Jl;, ; Gautier, J.; Langan, T. A.; Lohka, M. J.; Shenoy, S.; Shalloway, D. and Nurse, P., (1989) Maturation-promoting factor and the regulation of the cell cycle. J. Cell Sci. Suppl. 12, 53-63

36. Ma, X. F. ; Gibbons, J. A. and Babish, J. G. (1991) Benzo[e]pyrene pretreatment of immature, female C57BL/6J mice results in increased bioactivation of aflatoxin Bl 15 in vitro. Toxicol. Lett. 59, 51-58

37. Laemmli, U. K. and Favre, M. (1973) Maturation of the head of bacteriophage T4. I.

DNA packaging events. J. Mol. Biol. 80, 575-599

38. Towbin, H.; Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. 20 Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4354

39. Smith, P. K.; Krohn, R. I. ; Hermanson, G. T.; Mallia, A. K.; Gartner, F. H.;

Provenzano, M. D.; Fujimoto, E. K.; Goeke, N. M.; Olson, B. J. and Klenk, D. C. (1985) Measurement of protein using bicinchoninic acid [published erratum appears in Anal Biochem 19 87 May 15; 163 (1) :2791Anal. Biochem. 150, 76-85

25 40. Elledge, S. J. and Spottswood, M. R. (1991) A newhuman p34 protein kinase, CDK2, identifiedbycomplementation of a cdc28 mutation in Saccharomycescerevisiae, is a homolog of Xenopus Egl. EMBO J. 10,2653-2659

41. Brizuela, L. ; Draetta, G. and Beach, D. (1989) Activation of human CDC2 protein as a histone HI kinase isassociated with complex formation with the p62 subunitProc 30 Natl. Acad. Sci. U. S. A. 86, 4362-4366

42. Draetta, G.; Luca, F.; Westendorf, J.; Brizuela.L.; Ruderman, J. and Beach, D.

(1989) Cdc2 protein kinaseis complexed with both cyclin A and B: evidence foφroteolytic inactivation of MPF. Cell 56, 829-838

43. Sjolander, S. and Urbaniczky, C. (1991)Integrated fluid handling system for 5 biomolecularinteraction analysis. Anal. Chem. 63, 2338-2345

44. Altschuh, D.; Dubs, M. C; Weiss, E.; Zeder-Lutz,G. and Van Regenmortel, M. H.

(1992) Determination ofkinetic constants for the interaction between a

monoclonalantibody and peptides using surface plasmon resonance. Biochemistry 31, 6298-6304

45. Dubs, M. C; Altschuh, D. andVanRegenmortel, M.H. (1992) Mapping of viral epitopes with conformationallyspecific monoclonal antibodies using biosensor 5 technology. J. Chromatogr. 597, 391-396

46. Dubs, M. C; Altschuh, D. and Van Regenmortel, M. H. (1992) Interaction between vimses and monoclonal antibodies studied by surface plasmon resonance. Immunol. Lett. 31, 59-64

47. Draetta, G. and Beach, D. (1988) Activation of cdc2 protein kinase during mitosis in 10 human cells: cell cycle-dependent phosphorylation and subunit rearrangement. Cell

54, 17-26

48. Ducommun, B.; Brambilla, P.; Felix, M. A.; Franza, B. R.,Jr.; Karsenti, E. and

Draetta, G. (1991) cdc2 phosphorylation is required for its interaction with cyclin. EMBO J. 10, 3311-3319

15 49. Draetta, G. and Beach, D. (1989) The mammalian cdc2 protein kinase: mechanisms of regulation during the cell cycle. J. Cell Sci. Suppl. 12, 21-27

50. Draetta, G. (1990) Cell cycle control in eukaryotes: molecular mechanisms of cdc2 activation. Trends. Biochem. Sci. 15, 378-383

51. Morla, A. O.; Draetta, G.; Beach, D. and Wang, J. Y. (1989) Reversible tyrosine 20 phosphorylation of cdc2: dephosphorylation accompanies activation during entry into mitosis. Cell 58, 193-203

52. Draetta, G. ; Piwnica- Worms, H.; Morrison, D.; Druker, B. ; Roberts, T. and Beach,

D. (1988) Human cdc2 protein kinase is a major cell-cycle regulated tyrosine kinase substrate. Nature 336, 738-744.

25 53. Ducommun, B. and Beach, D. (1993) A Versatile Microtiter Assay for the Universal cdc2 Cell Cycle Regulator. Analytical Biochem. 187, 94-97.