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MOLECULAR PHARMACOLOGY 51:69-78 (1997).

Discordant Hepatic Expression of the Cell Division Control Enzyme p34^cdc2 Kinase, Proliferating Cell Nuclear Antigen, p53 Tumor Suppressor Protein, and p21^Waf1 Cyclin-Dependent Kinase Inhibitory Protein after WY14,643 ([4-Chloro-6-(2,3-xylidino)-2-pyrimidinylthio]Acetic Acid) Dosing to Rats

Xinfang Ma, Dana A. Stoffregen, Geoffrey D. Wheelock, Joseph A. Rininger, and John G. Babish

Section of Cellular Physiology, Paracelsian, Inc., 222 Langmuir Laboratories, Cornell Technology Park, Ithaca, New York 14850 (X.M., G.D.W., J.R., J.G.B.), Diagnostic Laboratory, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853 (D.S.), and Institute of Comparative and Environmental Toxicology, Cornell University, Ithaca, New York 14853 (J.G.B.)

The hepatocarcinogen and peroxisome proliferator WY14,643 {[4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid} was examined for its ability to induce changes in the intracellular protein expression of hepatic p34^cdc2 kinase (CDK1), proliferating cell nuclear antigen (PCNA), p53 tumor suppressor protein, and p21^Waf1 CDK inhibiting protein. Young, adult male rats were administered 45 mg-kg/day WY14,643 intraperitoneally for 1, 2, 3, 4, or 5 days or fed diets containing 0% or 0.08% WY14,643 for 1, 2, 3, or 4 weeks. WY14,643 dosing increased concentrations of hepatic proteins of 34- and 37-kDa molecular mass, which were identified through immunoprecipitation as CDK1 and PCNA, respectively. Gel filtration of the hepatic S9 fractions determined by enzyme-linked immunosorbent assay confirmed the increased expression of CDK1 and PCNA immunoreactivity in livers from WY14,643-treated rats. Also, gel filtration revealed that the native CDK1 and PCNA in hepatic S9 from WY14,643-treated rats chromatographed as a major peak with an apparent molecular mass of 70 and 76 kDa, respectively. Immunoblotting of the 70-kDa fraction with anti-CDK1 revealed a single band of molecular mass of 34 kDa. Thus, the CDK1 in the major immunoreactive peak of WY14,643-treated rat liver S9 seems to exist as a heterodimer or homodimer. Immunohistochemistry of formalin-fixed liver demonstrated a cytosolic localization of immunoreactive CDK1 and nuclear localization of immunoreactive PCNA in proliferating cells of WY14,643-treated rat livers. WY14,643 increased hepatic CDK1 content by 1.9-6.3-fold through postdosing days 1-5. Hepatic PCNA content was increased 1.9-5-fold over the same period. In the 4-week feeding study, CDK1 and PCNA expression were increased at all weekly time points by an average of 15-50-fold, respectively. Furthermore, the dietary administration of 0.08% WY14,643 resulted in sustained, overexpression of hepatic p53 tumor suppressor protein from week 1 through week 4 and of p21^Waf1 CDK inhibitory protein from week 3 to week 4.