DNA methylation and the mechanisms of CDKN2A inactivation in transitional cell carcinoma of the urinary bladder

Alterations of the CDKN2A locus on chromosome 9p21 encoding the p16(INK4A) cell cycle regulator and the p14(ARF1) p53 activator proteins are frequentl y found in bladder cancer. Here, we present an analysis of 86 transitional cell carcinomas (TCC) to elucidate the mechanisms responsible for inactivat ion of this locus. Multiplex quantitative PCR analysis for five microsatell ites around the locus showed that 34 tumors (39%) had loss of heterozygosit y (LOH) generally encompassing the entire region. Of these, 17 tumors (20%) carried homozygous deletions of at least one CDKN2A exon and of flanking m icrosatellites, as detected by quantitative PCR. Analysis by restriction en zyme PCR and methylation-specific PCR showed that only three specimens, eac h with LOH across 9p21, had bona fide hypermethylation of the CDKN2A exon 1 alpha CpG-island in the remaining allele. Like most other specimens, these three specimens displayed substantial genome-wide hypomethylation of DNA a s reflected in the methylation status of LINE L1 sequences. The extent of D NA hypomethylation was significantly more pronounced in TCC with LOH and/or homozygous deletions at 9p21 than in those without (26% and 28%, respectiv ely, on average, versus 11%, p < 0.0015). No association of LOH or homozygo us deletions at 9p21 with tumor stage or grade was found. The data indicate that DNA hypermethylation may be rare in TCC and that deletions are the mo st important mechanism for inactivation of the CDKN2A locus. The predominan ce of allelic loss may be explained by its correlation with genome-wide DNA hypomethylation, which is thought to favor chromosomal instability and ill egitimate recombination.