WO1997004316A1 - Determination de la presence d'une proliferation cellulaire anormale par la detection d'une ou de plusieurs kinases dependantes des cyclines - Google Patents

Determination de la presence d'une proliferation cellulaire anormale par la detection d'une ou de plusieurs kinases dependantes des cyclines Download PDF

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Publication number
WO1997004316A1
WO1997004316A1 PCT/US1996/012070 US9612070W WO9704316A1 WO 1997004316 A1 WO1997004316 A1 WO 1997004316A1 US 9612070 W US9612070 W US 9612070W WO 9704316 A1 WO9704316 A1 WO 9704316A1
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cyclin dependent
dependent kinase
cell
cells
tyrosylphosphorylation
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PCT/US1996/012070
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English (en)
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Xingfang Ma
John G. Babish
Joseph Rininger
Brian E. Johnson
Debra S. Whiting
Judith A. St. Leger
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Paracelsian, Inc.
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Priority to AU65931/96A priority Critical patent/AU6593196A/en
Publication of WO1997004316A1 publication Critical patent/WO1997004316A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
    • G01N33/5017—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity for testing neoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • 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.
  • 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.
  • 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).
  • initiation- promotion assay is employed routinely.
  • the test compounds are examined for their ability to promote hepatic tumors or foci formation after initiation with a known genotoxic agent (11,12).
  • genotoxic agent 11,12
  • 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).
  • 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).
  • PTK Protein-tyrosine kinases
  • the cascade of protein tyrosine phosphorylation following the activation of protein tyrosine kinases appears to regulate the proliferative response (27, 28).
  • 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.
  • CDK Cyclin proteins and Cyclin-dependent kinases
  • 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.
  • 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).
  • the assay taught by Ducommen & Beach 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.
  • 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.
  • 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.
  • CDKs cyclin dependent kinases
  • 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).
  • 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.
  • cell cycle inhibitors might be more like the negatively acting tumor suppressors whose loss or inactivation leads to cancer.
  • 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.
  • 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:
  • PSA Prostate specific antigen
  • PAP Prostatic acid phosphatase
  • CEA Carcinoembryonic antigen
  • lung breast, liver, pancreas, stomach and colon cancers.
  • CEA levels are monitored periodically to detect cancer recurrence and are often monitored in surgery patients.
  • 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.
  • AFP Alpha fetoprotein
  • AFP levels are also sometimes found in cancers of the lungs and digestive system.
  • Estrogen and progesterone receptors which have their presence monitored to indicate prognosis of hormone therapy.
  • cancer markers in development examples include:
  • CRP C-reactive peptide
  • Interleukin- 10 Non-Hodgkin's lymphoma
  • CA125 in combination with immunosuppressive acetic Ovarian cancer protein, tissue polypeptide antigen, amylase and alkaline phosphatase
  • IGF-l Insulin-like growth factor
  • 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.
  • 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.
  • 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:
  • 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;
  • 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.
  • 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.
  • FIG. 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.
  • FIG. 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.
  • FIG. 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.
  • FIG. 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.
  • FIG. 11 BIAcore* sensorgram displaying binding of pirnixic acid-treated S-9 protein and control S-9 protein over immobilized anti-cdc2 PSTAIR monoclonal antibody.
  • CDK tyrosylphosphorylated cyclin dependent kinases
  • PSTAIR and C-terminus anti-CDK monoclonal antibodies
  • FIG. 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.
  • FIG. 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.
  • FIG. 1 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.
  • FIG. 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.
  • FIG. 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.
  • CDK tyrosylphosphorylated cyclin dependent kinases
  • PSTAIR and C-terminus anti-CDK polyclonal antibodies
  • 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.
  • FIG. 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.
  • FIG. 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.
  • FIG. 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.
  • TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin
  • Control DMSO vehicle
  • FIG. 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.
  • FIG. 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.
  • TPA 12-0-tetra- decanoylphorbol- 13-acetate
  • Control Control
  • lane IB DMSO vehicle
  • FIG. 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.
  • TPA 12-0-tetra-decanoylphorbol- 13-acetate
  • DMSO DMSO vehicle
  • FIG. 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.
  • TPA 12-0-tetra-decanoylphorbol- 13-acetate
  • FIG. 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.
  • TCDD nM2,3,7,8-tetrachlorodibenzo-p-dioxin
  • Lane 2 is the 20% serum-supplemented control.
  • FIG 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.
  • TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin
  • DMSO vehicle Control
  • 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.
  • FIG. 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.
  • FIG. 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 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.
  • 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 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.
  • 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 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.
  • FIG 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.
  • TCDD 2,3,7,8-tetrachIorodibenzo-p-dioin
  • FIG 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.
  • FIG 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.
  • FIG 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.
  • FIG 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.
  • CDK2 dark staining bands at 33 kDa
  • 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.
  • FIG. 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.
  • CDK2 dark staining bands at 33 kDa
  • 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).
  • 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.
  • 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.
  • cyclin dependent kinase may either be measured directly or, alternatively, by measuring other parameters which are indicative of cyclin dependent kinase concentration.
  • parameters may vary in direct or inverse proportion with cyclin dependent kinase concentration.
  • parameters are measured which are related to either the formation or later metabolic fate of cyclin dependent kinase.
  • mRNA for cyclin dependent kinase could be measured, as could proteases involved in the degradation of cyclin dependent kinase.
  • 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).
  • a control which may be either a historical or concurrent control, standard curve, archival materials, or the like.
  • a control which may be either a historical or concurrent control, standard curve, archival materials, or the like.
  • "before" and “after” measurements are taken to determine the effect of an intervening regimen or of exposure to stimulus.
  • abnormal measurement levels based on historical or archival data or standard curves are determined.
  • a positive indication could be set at one, two or three standard deviations above the mean of a normal control.
  • samples suspected of having undergone transformation to cancerous phenotype e.g., breast, prostate, colon, lung, stomach or pancreas tissues, or lymphocytes, etc.
  • samples suspected of having undergone transformation to cancerous phenotype 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.
  • 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.
  • 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.
  • 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 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 ) ⁇ g per ml of dilution buffer.
  • 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).
  • 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 ⁇ g 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.
  • 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.
  • 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 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).
  • Example 4 show how a genotoxic carcinogen does not enhance tyrosylphosphoylation.
  • p34 cdc2 is the serine/threonine kinase subunit of M-phase promoting factor (MPF)
  • TCDD 2,3,7,8-tetrachlorodibenzo-p- dioxin
  • Anti-phosphotyrosine monoclonal, anti-PSTAIR (CDK), and anti-p34 cdc2 kinase C- terminus polyclonal antibodies are obtained from UBI (Lake Placid, NY).
  • 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.
  • 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.
  • mice 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 ⁇ g/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.
  • 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 vani
  • solubilized hepatic S-9 proteins are then incubated with anti-phosphotyrosine monoclonal antibody (5 ⁇ g/mL) at 4°C for 4 h or ovemight. After the incubation period, add 25 ⁇ L of protein A-Sepharose for each 5 ⁇ g of antibody.
  • 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.
  • 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.
  • TBS Tris buffered saline
  • 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 ⁇ g/mL) in 5 TBST or anti-mouse cdc2 kinase (C-terminus) polyclonal antibody in TBST.
  • PSTAIR anti-human CDK
  • 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.
  • Protein determination Bicinchoninic acid is used for the spectrophotometric determination of protein concentration (38). Mix 100 ⁇ L 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
  • Reagent B consists of 4.9 9 CuSO 4 * 5H 2 0 to 100 mL in double distilled H 2 O.
  • 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).
  • mice with TCDD did not exist in measurable quantities in the hepatic S-9 of com oil treated control mice.
  • dosing of mice with TCDD enhanced the tyrosylphosphorylation of a p34 and p32 to a maximum at 0.5 ⁇ g TCDD/kg.
  • the tyrosylphosphorylation of the kinase(s) becomes attenuated, perhaps due to overt toxicity of TCDD to the mice at these higher doses.
  • CDK p34 c kinase
  • p34 cdc2 is the serine/threonine kinase subunit of M-phase promoting factor (MPF) (29-31).
  • M-phase promoting factor 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.
  • 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.
  • 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.
  • 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.
  • SPR Surface plasmon resonance
  • 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).
  • 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.
  • 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.
  • a typical sensorgram produced on immobilization of anti-cdc2 C-terminus is depicted in Figure 7. Time required for immobilization is approximately 30 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 ⁇ L) 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.
  • 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.
  • 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.
  • FIG. 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.
  • 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.
  • CDK p3 c kinase
  • p34 cdc2 is the serine/threonine kinase subunit of M-phase promoting factor (MPF)
  • 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.
  • Rats are purchased and handled as described in Example 2.
  • 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.
  • CDK tyrosylphosphorylated cyclin dependent kinases
  • 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.
  • p34 cdc2 is the serine/threonine kinase subunit of M-phase promoting factor (MPF) (32-34).
  • M-phase promoting factor 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.
  • 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.
  • PCBs polychlorinated biphenyls
  • 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.
  • FIG. 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.
  • 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.
  • TCDD 2,3,7,8-Tetrachlorodibenzo-p-dioxin
  • 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).
  • DMEN Dulbecco's Modified Eagle's Medium
  • FBS-HI Fetal bovine serum-heat inactivated
  • 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.
  • DMSO dimethyl sulfoxide
  • Enhanced tyrosylphosphorylation of p34/p33 in 3T3 cell lysates 24 hours following exposure to the tumor promoter 12-0-tetra-decanoylphorbol-13- acetate.
  • TPA Tetra-decanoylphorbol-13- acetate
  • 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.
  • Results of semm supplementation 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.
  • 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.
  • BNL CL.2 cells 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.
  • 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.
  • DMSO dimethyl sulfoxide
  • 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.
  • DMSO dimethyl sulfoxide
  • 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.
  • 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.
  • CDK cytosolic cyclin dependent kinases
  • 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.
  • 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.
  • 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.
  • mice 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.
  • CDK or p34 cdc2 kinase respectively.
  • 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.
  • 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.
  • sample tissue protein is diluted in Prep buffer and mixed 1: 1 with blocking buffer.
  • Plates are washed 3x with washing buffer and lx with assay buffer.
  • 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.
  • 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.
  • 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.
  • CDK total cytosolic cyclin-dependent kinases
  • Triton X- 1 00 0. I mM PMSF, 0. 1 mM Na fluoride , 60mM ⁇ -glycerophosphate, 15mM paranitrophenylphosphate, 0. ImM Na orthovanidate, l ⁇ g/ml leupeptin, lO ⁇ g/ml soy bean trypsin inhibitor, I ⁇ g/ml aprotinin, and lO ⁇ g/ml tosyl phenylalanine.
  • 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.
  • tissue S9 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.
  • Plates are washed 3x with washing, buffer and lx with assay buffer.
  • 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 Mon 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • DMSO dimethyl sulfoxide
  • 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.
  • 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 pu ⁇ ose 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.
  • 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.
  • 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.
  • 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.
  • 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-co ⁇ jugated 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.
  • TBS 50 M Tris, 150 mM NaCl, pH 7.5
  • 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.
  • CDK2 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).
  • 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,
  • Plates are washed 3x with washing buffer and lx with assay buffer.
  • 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.
  • 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.
  • 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.
  • 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.
  • Immunoblotting serum samples for CDK2 protein This section is as previously described in Example 14.
  • 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.
  • 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.
  • Microtiter assay for CDK2 proteins This section is as previously described in Example 14.
  • 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.
  • 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.
  • Immunoblotting serum samples for CDK2 protein This section is as previously described in Example 14.
  • 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.
  • CDK2 dark staining bands at 33 kDa
  • 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
  • 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
  • cdc2 is a component of 5 the M phase-specific histone Hi kinase: evidence for identity with MPF. Cell 55,

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Abstract

L'invention concerne des techniques d'analyse in vivo et in vitro permettant de mesurer la concentration intracellulaire ou intratissulaire des kinases dépendantes des cyclines. Elle concerne la façon d'utiliser ces techniques, en particulier pour évaluer la carcinogénicité d'un composé test, des agents antinéoplasiques potentiels et l'efficacité de schémas posologiques pouvant favoriser ou empêcher la croissance cellulaire.
PCT/US1996/012070 1995-07-20 1996-07-19 Determination de la presence d'une proliferation cellulaire anormale par la detection d'une ou de plusieurs kinases dependantes des cyclines WO1997004316A1 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0680609A1 (fr) * 1993-01-21 1995-11-08 Paracelsian, Inc. Produits permettant de mesurer la propension de la croissance cellulaire, et procedes d'utilisation de tels produits
GB2334578A (en) * 1998-02-18 1999-08-25 Univ Liverpool Diagnosis of cancer involving assay of levels of cyclin-dependent kinase (CDK) isoenzymes
GB2334579A (en) * 1998-02-18 1999-08-25 Univ Liverpool Sensitivity of cancer cells to anti-cancer agents involving measurement of properties of signal transduction factors
WO1999042839A2 (fr) * 1998-02-18 1999-08-26 Theryte Limited Traitement du cancer
GB2335739A (en) * 1998-02-18 1999-09-29 Univ Liverpool Screening anti-cancer agents
EP1113780A2 (fr) * 1998-09-17 2001-07-11 Bionexus Techniques permettant de determiner l'activite de melanges complexes
WO2004076686A1 (fr) * 2003-02-26 2004-09-10 Sysmex Corporation Procede pour analyser une cellule

Citations (1)

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Publication number Priority date Publication date Assignee Title
WO1994017413A1 (fr) * 1993-01-21 1994-08-04 Paracelsian, Inc. Produits permettant de mesurer la propension de la croissance cellulaire, et procedes d'utilisation de tels produits

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WO1994017413A1 (fr) * 1993-01-21 1994-08-04 Paracelsian, Inc. Produits permettant de mesurer la propension de la croissance cellulaire, et procedes d'utilisation de tels produits

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S. J. VALLANCE ET AL.: "Monoclonal antibodies to mammalian d-type G1 cyclins.", HYBRIDOMA, vol. 13, no. 1, 1994, pages 37 - 44, XP002018813 *

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0680609A1 (fr) * 1993-01-21 1995-11-08 Paracelsian, Inc. Produits permettant de mesurer la propension de la croissance cellulaire, et procedes d'utilisation de tels produits
EP0680609A4 (fr) * 1993-01-21 1998-07-08 Paracelsian Inc Produits permettant de mesurer la propension de la croissance cellulaire, et procedes d'utilisation de tels produits.
WO1999042839A3 (fr) * 1998-02-18 1999-10-28 Theryte Ltd Traitement du cancer
WO1999042821A3 (fr) * 1998-02-18 1999-11-11 Theryte Ltd Traitement du cancer
WO1999042839A2 (fr) * 1998-02-18 1999-08-26 Theryte Limited Traitement du cancer
WO1999042834A2 (fr) * 1998-02-18 1999-08-26 Theryte Limited Traitement du cancer
WO1999042821A2 (fr) * 1998-02-18 1999-08-26 Theryte Limited Traitement du cancer
GB2335739A (en) * 1998-02-18 1999-09-29 Univ Liverpool Screening anti-cancer agents
GB2334578A (en) * 1998-02-18 1999-08-25 Univ Liverpool Diagnosis of cancer involving assay of levels of cyclin-dependent kinase (CDK) isoenzymes
GB2334579A (en) * 1998-02-18 1999-08-25 Univ Liverpool Sensitivity of cancer cells to anti-cancer agents involving measurement of properties of signal transduction factors
WO1999042834A3 (fr) * 1998-02-18 1999-11-25 Theryte Ltd Traitement du cancer
GB2334579B (en) * 1998-02-18 2003-06-04 Univ Liverpool Treating cancer
US6521407B1 (en) * 1998-02-18 2003-02-18 Theryte Limited Methods for determining chemosensitivity of cancer cells based upon expression of negative and positive signal transduction factors
AU753588B2 (en) * 1998-02-18 2002-10-24 Theryte Limited Treating cancer
EP1113780A4 (fr) * 1998-09-17 2001-11-07 Bionexus Techniques permettant de determiner l'activite de melanges complexes
EP1113780A2 (fr) * 1998-09-17 2001-07-11 Bionexus Techniques permettant de determiner l'activite de melanges complexes
WO2004076686A1 (fr) * 2003-02-26 2004-09-10 Sysmex Corporation Procede pour analyser une cellule
US7501257B2 (en) 2003-02-26 2009-03-10 Sysmex Corporation Molecular diagnostic method of a cancer tissue or a cancer cell

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