Distinct susceptibility of induction of methylation of p16ink4a and p19arf CpG islands by X-radiation and chemical carcinogen in mice.

Inactivation of the tumor suppressor genes p16(ink4a) and p19(arf)/p14(arf) by hypermethylation of promoter CpG islands occurs frequently in various tumors. The aim of this study is to investigate the difference of susceptibility of methylation induced by carcinogens between p16(ink4a) and p19(arf). The methylation status of both genes was analyzed by denaturing high performance liquid chromatography (DHPLC) and bisulfite-sequencing, respectively. The expression level of P16 protein was analyzed by immunohistochemistry. Results showed that p16(ink4a) methylation was detected in the glandular stomach, small intestine and other organs of mice following X-radiation and subsequent bone marrow transplantation (BMT), but not in mock control mice. We found that the intestinal tract was the most sensitive organ for X-ray induced p16(ink4a) methylation. Loss of P16 protein expression was observed in the intestinal tissues of X-irradiated mice, but not in the mock control mice. Interestingly, p19(arf) methylation was not observed in the gastrointestinal tissues of the negative control mice following X-radiation/BMT. However, administration of N-nitrosomethylurea and/or Helicobacter felis infection promoted methylation of p19(arf) CpG islands in the gastrointestinal tracts, but did not promote p16(ink4a) methylation. In addition, p16(ink4a) methylation was detected not only in the X-irradiated GFP-negative tissue cells, but also in the GFP-positive bone marrow-derived cells that were transplanted into the BMT mice after X-radiation. In conclusion, the methylation susceptibility of p16(ink4a) and p19(arf) to carcinogen treatments was remarkably different: X-radiation indirectly induces systemic p16(ink4a) methylation, especially in the intestine; whereas N-nitrosomethylurea and/or H. felis infection induce p19(arf) methylation in their target organs.