Reduced expression of the CDK inhibitor p27KIP1 in rat two-stage bladder carcinogenesis and its association with expression profiles of p21WAF1/Cip1 and p53

The cyclin-dependent kinase ( CDK) inhibitorp27 KIP1 exerts its growth suppressive effects by targeting the cyclin-CDK complexes. Reduced protein levels of p27 KIP1 have been reported in numerous human cancers and this has been attributed to increased degradation. However, few reports have addressed the significance of p27 KIP1 expression in chemical carcinogenesisof rodents. In a rat two-stage urinary bladder carcinogenesismodel, with N-butyl-N-(4-hydroxybutyl) nitrosamine(BBN) initiation followed by promotion with sodium L-ascorbate (Na-AsA), we evaluated the expression of p27 KIP1 protein using immunohistochemistry during various stages of urinary bladder carcinogenesis. In addition, we evaluated the mRNA expression profiles for p27 KIP1 , p21 WAF1/Cip1 and p53 in tumors. Fisher 344 rats were initiated with 0.05% BBN in the drinking water for 4 weeks and then administered 5% Na-AsA in the diet. Immunohistochemical examination revealed p27 KIP1 protein to be constitutively expressed in normal urothelium, simple hyperplasia and in most papillary and nodular (PN) hyperplasias and small papillomas, but diminished or absent in large papillomasand in transitional cell carcinomas. An inverse correlation between expression of p27 KIP1 and cell proliferation was generally observed. Quantitation of mRNA by multiplex reverse transcription-PCR showed a significant downregulaton of p27 KIP1 , p21 WAF1/Cip1 and p53 mRNA in tumors. More than 50% reduction in p27 KIP1 mRNA expression was observed in 42 and 47% of tumors at weeks 18 and 24, respectively; similar reduction in p21 WAF1/Cip1 mRNA expression was observed in 58 and 73% of tumors at weeks 18 and 24, and in p53 mRNA expression in 50 and 73% of tumors at weeks 18 and 24, respectively. None of the 25 tumors we examined by PCR- single-strand conformational polymorphismanalysis had p53 mutations. These data imply that abnormal down-regulation of p27 KIP1 , p21 WAF1/Cip1 and/or p53 in tumor cells may contribute to the malignant progression of tumors during rat two-stage bladder carcinogenesis.