Inactivation of tumor suppressor genes and deregulation of the c-myc gene in urothelial cancer cell lines

Abstract

Recent investigations have demonstrated p53 and Rb alterations in a subset of transitional cell carcinoma (TCC). Further genetic changes during tumor progression include overexpression of the c-myc gene in a significant number of mainly invasive bladder tumors. To study the possible interactions between these genes in TCC, urothelial cancer cell lines were chosen as an in vitro model. Expression and mutation of p53 was studied in 15 bladder cancer cell lines by immunocytochemistry, Western blot, polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis and direct sequencing of double stranded PCR products of exons 4, 5, 7 and 8 of genomic DNA. C-myc expression and gene structure were studied using Northern and Southern blot techniques Rb protein expression was analyzed by Western blot. Twelve of 15 cell lines showed either p53 mutations or abnormal protein expression. Consistent with previous studies, five cell lines did not express Rb protein. None of the cell lines studied retained both tumor suppressor genes in a functional form. The c-myc gene appeared to be intact in all 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 suggest that amplification or translocation are not the underlying mechanism for c-myc overexpression in urothelial tumors. No correlation between loss of Rb protein and c-myc expression was observed. The results presented here for the cell lines match well those obtained in vivo. Thus, these cell lines may provide a suitable model for further analysis of molecular alterations in urothelial cancer.

Frequent and heterogenous expression of cyclin-dependent kinase inhibitor WAF1/p21 protein and mRNA in urothelial carcinoma

“…Accordingly, when averaged across all tumour stages, increased p21 expression did not correlate with either the presence or the absence of p53 accumulation (Table 3). In urothelial carcinoma accumulation of p53 protein, particularly in advanced tumours, is usually due to mutations in the gene (Cordon-Cardo et al, 1994;Williamson et al, 1994;Grimm et al, 1995;Uchida et al, 1995;Vet et al, 1995). This suggests that in the majority of tumours staining positive for both proteins, p21 was not induced by p53.…”

DNA methylation and expression of LINE-1 and HERV-K provirus sequences in urothelial and renal cell carcinomas

“…Human bladder carcinoma cell lines (Grimm et al, 1995), male teratocarcinoma Tera-1, NCCIT, and GH, female teratocarcinoma Pa 1, amnion AV3, choriocarcinoma JAR, Molt 4 T-cell lymphoma and rhabdomyosarcoma A204 cell lines were cultured as described (Löwer et al, 1993). Figure 2 Southern blot analysis of LINE-1 methylation in renal carcinoma.…”

Decreased expression of p57KIP2 mRNA in human bladder cancer

“…The human bladder cancer cell lines, J82, VMCubI, VMCubII, VMCubIII, T24, 647V, 5637, HT1376, RT-4, 639V, TCCsup, SW1710, 253J and BFTC909 were cultured as described previously (Grimm et al, 1995).…”