RAF may induce cell proliferation through hypermethylation of tumor suppressor gene promoter in gastric epithelial cells

The extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK-MAPK) is critical in human malignancies. It remained to be established whether DNA methyltransferases (Dnmt) and proliferating cell nuclear antigen (PCNA) involved in DNA methylation during RAF-transformed cell proliferation. The plasmid of constitutively active RAF was used to transfect gastric cell GES-1 and cancer cell AGS. RAF promoted cell proliferation, growth in soft agar and induced cell cycle progress faster than empty plasmid by accelerating G1/S transition in both cell lines, a massive induction of cyclin D1 and PCNA expression was observed, along with reduced expression of p16(INK4A), p21(WAF1) and p27(KIP1). Methylation-specific polymerase chain reaction and bisulfite sequencing showed that the promoter of p16(INK4A) was methylated in RAF-transformed cells, treatment with 5-aza-dC or PD98059 restored the expression of p16(INK4A), increased p21(WAF1) and p27(KIP1) partially, associated with upregulation of the activity of Dnmt in RAF-transformed cell GES-1, and also decreased the hypermethylation status of p16(INK4A), but not all CpG islands of p21(WAF1) and p27(KIP1). These data suggest that RAF may induce cell proliferation through hypermethylation of tumor suppressor gene p16(INK4A), while the epigenetic inactivation of p21(WAF1) and p27(KIP1) may be not a key factor in RAF-transformed cells. (Cancer Sci 2009; 100: 117-125).