INACTIVATION OF TUMOR-SUPPRESSOR GENES AND DEREGULATION OF THE C-MYC GENE IN UROTHELIAL CANCER CELL-LINES

Recent investigations have demonstrated p53 and Rb alterations in a su bset of transitional cell carcinoma (TCC). Further genetic changes dur ing tumor progression include overexpression of the c-myc gene in a si gnificant number of mainly invasive bladder tumors. To study the possi ble interactions between these genes in TCC, urothelial cancer cell li nes were chosen as an in vitro model. Expression and mutation of p53 w as studied in 15 bladder cancer cell lines by immunocytochemistry, Wes tern blot, polymerase chain reaction single-strand conformation polymo rphism (PCR-SSCP) analysis and direct sequencing of double stranded PC R products of exons 4, 5, 7 and 8 of genomic DNA. C-myc expression and gene structure were studied using Northern and Southern blot techniqu es. Rb protein expression was analyzed by Western blot. Twelve of 15 c ell lines showed either p53 mutations or abnormal protein expression. Consistent with previous studies, five cell lines did not express Rb p rotein. None of the cell lines studied retained both tumor suppressor genes in a functional form. The c-myc gene appeared to be intact in al l cell lines and copy numbers were close to normal. Northern analysis demonstrated that all cell lines expressed c-myc mRNA but evidence for altered regulation was found in at least two cell lines. Our data sug gest that amplification or translocation are not the underlying mechan ism for c-myc overexpression in urothelial tumors. No correlation betw een loss of Rb protein and c-myc expression was observed. The results presented here for the cell lines match well those obtained in vivo. T hus, these cell lines may provide a suitable model for further analysi s of molecular alterations in urothelial cancer.