ABSTRACT
Objective To investigate methylation of the P15INK4B and p27kipl genes in human mantle cell lymphoma cell line Mino,to evaluate the effects of decitabine on demethylation of p15INK4B and p27kip1 genes and on apoptosis of Mino cells and its relative mechanism. Methods Mino cells were after treated with various concentration of decitabine, the cell viability, cell cycle distribution or the apoptosis of Mino cells was respectively analyzed by trypan dye-exclusion assay or flow cytometry.The mRNA and protein expression of p15INK4B 、p27kip1 and bcl-2 were studied by RT-PCR or Western blot, respectively.Methylation of the p15INK4B and p27kip1 genes in Mino cells were determined by PCR using the methylation specific primer(MSP).Results Decitabine significantly inhibit the cell growth,induced G1 arrest and promoted apoptosis of Mino cells. The expression of p15INK4B and p27kipl mRNA were both significantly increased,wheres bcl-2 mRNA was decreased, After treatment with 6.4,3.2,1.6 mmol/L decitabine for 72 h,the methylationg of p15INK4B gene were decreased to 25.2 ％ 、48.2 ％ and 65.0 ％respectively,in the meantime,the methylationg of p27kip1 gene was decreased to 20.21％ 、50.2 ％ and 70.0 ％. Conclusion The hp15INK4B and p27kip1 genes of Mino cells are methylated and down-regulated.Decitabine can inhibit the proliferation and induce the apoptosis of Mino cell lines,with the reduction of bcl-2 gene and demethylation of p15INK4B and p27kip1 genes.
ABSTRACT
PURPOSE: p16Ink4A and p15lnk4B, encoded by the genes located on chromosome 9p21, are cyclin-dependent kinase 4 inhibitors and are the upstream regulators of pRB (retinoblastoma protein) function and are involved in the regulation of cell cycle in mammalian cells. It has been demonstrated that p16 and p15 genes are frequently deleted, mutated, and hypermethylated in many malignancies and cancer cell lines. This study was performed to investigate the genetic alteration and immunohistochemical profile of p16 and p15 in gastric carcinomas. MATERIALS AND METHODS: We examined 30 primary gastric cancer samples using PCR- SSCP (Polymerase chain reaction-single strand conformation polymorphism), DNA sequencing, PCR-based hypermethylation assay, and immunohistochemistry. RESULTS: No homozygous deletion was detected in either pl6 or p15 gene, and only one gastric carcinoma sample showed mutation of p16 gene and p15 gene. However, hyper-methylation of 5' CpG islands was observed in 53.6% of exon1 of p16 gene and in 46.4% of exon 1 of pl5 gene. By immunohistochemistry of p16, nuclear under-expression was observed in 58.6%, whereas nuclear over-expression was detected in 31% of formalin-fixed, paraffin-embedded gastric cancer tissues. CONCLUSIONS: Our results suggest that the p16 and p15 tumor suppressor genes may play an important role in gastric carcinogenesis and may be inactivated not by deletions or mutations but mainly by hypermethylation of their 5' CpG islands. There was a good correlation between methylation study and immunohistochemical results in p16 genes.