Purpose: The growth suppressive genes p16INK4a and p14ARF located on the 9p21 gene cluster have active roles in the Rb and p53 growth control pathways, respectively. p16INK4a is a cyclin dependent kinase inhibitor functioning upstream of Rb. p14ARF restrains cell growth by abrogating Mdm2 inhibition of p53 activity, thereby, facilitating p53 mediated cell cycle arrest and apoptosis. To elucidate specific targets and aberrations affecting the 9p21 chromosomal region in bladder cancer tumorigenesis alterations in the p16INK4a and p14ARF genes were analyzed.
Materials and methods: A total of 53 transitional cell carcinomas from 44 patients with bladder cancer were collected. Genetic alterations of p16INK4a and p14ARF genes were analyzed by Southern hybridization, polymerase chain reaction (PCR)-single strand conformational polymorphism analysis and methylation specific PCR. In addition, mRNA expression status was detected by reverse transcriptase-PCR.
Results: Homozygous deletion of p16INK4a and p14ARF genes was observed in 23% (12 of 53 samples) and 43% (23 of 53), respectively. Most deletions occurred exclusively on the E1 beta-p14ARF region. Concomitant deletion of p16INK4a and p14ARF genes was found in only 2 samples. One mutation was detected in exon 2 of p14ARF plus p16INK4a genes. Aberrant methylation of p16INK4a gene was found in 60% (24 of 40 tumors). However, no p14ARF gene methylation was detected in any case. The result of comparative reverse transcriptase-PCR showed that suppressed mRNA expression correlated with genetic alterations of p14ARF and p16INK4a genes in most tumor samples examined.
Conclusions: Results indicate that p14ARF is a primary target of homozygous deletion, whereas p16INK4a is the hot spot of hypermethylation on the 9p21 region in bladder cancer. The frequent inactivation of the p14ARF and p16INK4a genes may be an important mechanism for the dysfunction of p53 and Rb growth regulatory pathways during bladder cancer development.