Expression and sequence analysis of the p21(WAF1/CIP1) gene in renal cancers

Abstract

Objectives The p21 (WAF1/CIP1) cyclin-dependent kinase inhibitor is an M r 21,000 protein that can arrest cell growth by associating with and inhibiting cyclin-dependent kinase complexes necessary for cells to exit G 1. It is a downstream effector in the p53 growth control pathway and can be transcriptionally activated by increasing levels of p53 protein. The objective of this study was to determine if there are mutations or alterations in the expression of p21 in renal cancers that could contribute to renal cancer cell growth. Methods Twelve renal cancer cell lines were examined for mutations in the coding region of the p21 gene using single-stranded conformation polymorphism analysis and direct deoxyribonucleic acid (DNA) sequencing. Expression of p21 was determined in all 12 cell lines by Northern analysis using a cDNA probe for p21 and Western analyses using a p21-specific antibody. Results Nucleotide base substitutions were detected in the p21 gene in two cell lines, which did not result in amino acid substitutions. P21-specific mRNA was present in 8 of 12 renal cancer cell lines, as determined by Northern analysis, although p21 transcripts could be detected by polymerase chain reaction in all 12 renal cancers. Varying levels of p21-specific protein were detected in 9 of 12 renal cancers. Conclusions These data indicate that mutation of the p21 gene is rare in renal cancer cell lines and that the uncontrolled growth of renal cancer cells is not due to mutation of the p21 gene. However, expression studies found a wide variation in the level of p21 protein in renal cancer cells, suggesting that aberrant regulation of p21 expression may play a role in renal cancer development.