Promoter methylations of p16INK4a and p14ARF genes in early and advanced gastric cancer. Correlations of the modes of their occurrence with histologic type.

The INK4a/ARF locus encodes two cell cycle-regulatory proteins, p16INK4a and p14ARF. These share an exon using different reading frames, and act through Rb and p53 pathways. Recently, it has been found that silencing of p16INK4a and p14ARF expressions by aberrant methylation of the CpG islands in the promoter regions is an alternative mechanism that inactivates possible tumor suppressor functions in various tumors. To clarify the features of gastric cancers with promoter methylation of p16INK4a and p14ARF, we investigated the methylation status in gastric cancer cell lines and primary gastric cancers using methylation-specific PCR (MSP), and correlated the methylation status with microsatellite instability (MSI), DNA ploidy pattern, p53 immunohistochemistry, and various clinicopathologic factors, paying attention to the correlations with the histologic types. Of 10 cell lines studied, silencing of the expression of p16INK4a and p14ARF due to promoter methylation was detected by MSP and RT-PCR in six (60%) and two (20%) cell lines, respectively. p14ARF silencing was detected only in cell lines derived from gastric cancer of the diffuse type, while p16INK4a silencing was found in cell lines derived from both diffuse and intestinal types. In 59 primary gastric cancers, promoter methylation of p16INK4a and p14ARF was found in 10 (17%) and 14 (24%) of the tumors independently, there being an association with DNA diploidy, but not with p53 immunohistochemistry. p16INK4a methylation was found irrespective of tumor stages and histology. Whereas p14ARF methylation was found more frequently in intestinal type cancers in an early stage and in diffuse type cancers in an advanced stage, MSI tended to be related especially to p14ARF methylation in cancers of the intestinal type. Thus, the significance of p14ARF methylation differed between intestinal and diffuse types, while such a difference was not observed in p16INK4a methylation.