Growth arrest and suppression of tumorigenicity of bladder carcinoma cell lines induced by theP16/CDKN2 (p16INK4A,MTS1) gene and other loci on human chromosome 9

Abstract

Wild-type P16ICDKNZ MTSI) cDNA, directed by the cytomegalovirus (CMV) immediate early promoter, was transfected into RT4 and RTI I 2 bladder-carcinoma cell lines bearing a mutated endogenous P16ICDKNZ gene and lacking endogenous P 16lCDKN2 respectively. In both cases, only trans- Bladder carcinoma is one of the most common malignancies, occurring as the fourth most frequent neoplasm in men and the ninth most frequent in women in the United States. Cytogenetic and molecular genetic analyses have documented the involvement of oncogenes, such as c-Ha-ras, c-myc and c-erb-2-neu, of epidermal growth factor and of tumor-suppressor genes in bladder carcinoma (Cordon-Cardo et at., 1994). A large proportion (60%) of bladder cancers shows deletion or mutational inactivation of the p53 gene on chromosome 1 7~1 3 .Likewise, alterations in the RB gene on chromosome 13q14 are observed in about 30% of bladder cancers. Alterations in both p53 and RB genes correlate with advanced stages in the progression of the disease. Although no other known tumorsuppressor genes have yet been involved in bladder carcinoma, several studies have reported cytogenetic deletions or nonrandom LOH for alleles located on several human chromosomes. Monosomy of chromosome 9 is a frequent change, reaching 94% of cases in some investigations (Cairns et al., 1993). LOH at various loci on chromosome 9 was observed not only in tumors of high grade or stage but also in more than 50% of superficial tumors of Ta and Tis initial stages, indicating that allelic loss on chromosome 9 is an early genetic event in the development of bladder cancer. Deletion of loci at 91321-22 have been described in malignant mesothelioma, head-andneck cancer, non-small-cell and small-cell lung cancer, melanoma, glioma and acute lymphoblastic leukemia, glioblastoma and bladder carcinoma. The P161CDxN2 (plQNK4"", MTSI) gene has recently been cloned and mapped to chromosome 9p21 (Serrano et al., 1993; Kamb et al., 1994). The gene encodes p16, a cell-cycle regulatory protein that binds to CDK4 and inhibits phosphorylation of the RB protein by the CDK41cyclin D complex. Hornozygous or hemizygous deletions of the P161CDKN2 gene were frequently detected in primary glioblastoma and pancreatic adenocarcinoma, as well as in tumor cell lines derived from several tumor histotypes, including bladder carcinoma (Kamb et al., 1994;Nobori et al., 1994;Okamoto et al., 1994), but intragenic mutations of this gene were rare both in primary tumors and in tumor cell lines (Okamoto et al., 1994;Cairns et al., 1994;Spruck et al., 1994). The only exceptions were pancreatic adenocarcinoma (Caldas et al., 1994) and esophageal squamous-cell carcinoma (Mori et al., 1994) where sequence changes were detected in 38% and 52% of primary cases respectively. P16/CDKN2-gene alterations, however, were generally found to be 2 to 3 times more common in tumor cell lines than in primary tumors. This observation suggests that, although PI6ICDKN2 may play a role in tumorigenesis, its inactivation is selected by in vitro ...

The p16 (CDKN2A) gene is involved in the growth of neuroblastoma cells and its expression is associated with prognosis of neuroblastoma patients

“…There have been several reports showing that induction of p16 gene expression resulted in inhibition of cell cycle progression and decrease in the growth rate of cancer cells (Fueyo et al, 1996;Wu et al, 1996;Adachi et al, 1997). In this study, a signi®cant degree of G1 arrest was induced in neuroblastoma cell lines lacking p16 expression with and without 5'CpG island methylation by transfection of a wild-type p16 vector.…”

Measurement of Relative Copy Number of CDKN2A/ARF and CDKN2B in Bladder Cancer by Real-Time Quantitative PCR and Multiplex Ligation-Dependent Probe Amplification

“…For example, gene replacement studies have shown a marked effect on TCC tumor cell phenotype. 32 Studies of papillary superficial TCC in vitro have shown that many of these tumors retain at least one copy of p16 and that the cells derived from them have limited lifespan in culture with typical up-regulation of p16 protein at senescence. Loss of p16 or Rb expression is required for establishment of such tumor cells as immortal lines in vitro.…”

“…Introduction of wild-type p16 into bladder tumor cell lines lacking functional p16, induced cell cycle arrest and suppression of tumorigenicity. 32 Further evidence for a suppressor function of p16 in the urothelium has come from studies of cultured primary bladder tumor biopsies where the majority of superficial papillary tumors showed limited in vitro lifespan, which was associated with elevated levels of p16 expression at senescence. In contrast, cultures derived from muscle invasive tumors commonly bypassed Supported by Cancer Research UK.…”

Molecular Mechanisms and Pathways in Bladder Cancer Development and Progression

“…It has been shown that transfecting wildtype p16 into BC cell lines that are deficient in p16 produces growth arrest, thus supporting its role as a tumor suppressor gene. 48 One potential mechanism by which p16 or CDKN2 function is lost is by gene silencing via methylation of the promoter region of p16. 49 Interestingly, another cycle regulatory gene, p14, is also located on chromosome 9p21 and shares part of the same coding region as p16, in the INK4a locus.…”