Study on the role of DNA methyltransferases in regulation of the expression of genes associated with the control of cell cycle

为了估计甲基化是否参与了与细胞周期调节相关的APC、p16、FEZ1、KAI1和survivine基因的下调，我们检测了8种细胞系（SAS、HSC-2、HSC-3、HSC-4、Ca9-22、OK-92、HO-1-u-1和HO-1-N-1）中被认为与癌变有关的甲基转移酶DNMT1、DNMT3A和DNMT3B的表达水平，这些细胞系来源于口腔癌和临床组织样本。此外，还检测了5-Aza-C（其中一种去甲基化剂）对基因表达的恢复。50例口腔鳞状细胞癌（OSCC）中有24例（48.0%）检测到CDKN2A/p16基因高甲基化，8株细胞系中有6株（75.0%）检测到5-Aza-C修复。50例患者中有15例（30.0%）出现APCgene下调表达，12例（24.0%）出现CPG岛高甲基化。31例患者中fez1基因表达抑制和高甲基化分别为11例（35.5%）和8例（25.8%）。在31例中，检测到高甲基化，并在所有8个细胞系中发现5-Aza-C基因表达的恢复。101例OSCC中84例检测到KAI1基因沉默或表达抑制。然而，没有发现启动子区域CPG岛的超甲基化，也没有发现5-Aza-C恢复基因表达。在58%的OSCC病例和37%的癌前病变、白斑中检测到存活基因的过表达。另一方面，9个正常口腔上皮组织的RT-PCRin均未检测到存活基因的表达，9个正常标本中的4个经甲基化特异性PCR证实DNA甲基化，8个细胞系中的2个经5- aza - c处理后基因表达恢复。DNA甲基转移酶（DNMTs）的过表达；DNMT1、DNMT3A和DNMT3B被认为与癌变有关，在OSCC中检测频率较高。DNMTI、DNMT3A和DNMT3B在OSCC中的表达率分别为72%、56%和64%。抑癌基因和癌基因的高甲基化情况与甲基转移酶的表达水平无显著相关性。我们的数据表明，该基因的高甲基化可能是口腔癌发生中基因失活的主要机制之一，除了甲基转移酶外，一些参与抑制基因表达的转录因子可能在口腔癌发生中发挥重要作用。少

To estimate the involvement of methylation in the down-regulation of the genes, APC, p16, FEZ1, KAI1, and survivine, wehich were associated with the regulation of cell cycle, we examined the expression revels of them and methyltransferases, DNMT1, DNMT3A, and DNMT3B, which were believed to be associated with carcinogenesis, in 8 cell lines, SAS, HSC-2, HSC-3, HSC-4, Ca9-22, OK-92, HO-1-u-1, and HO-1-N-1, which were derived from oral carcinoma, and clinical tissue samples of oral cancer. Additionally, restoration of the gene expression by 5-Aza-C, one of the demethylation agents, was examined. The hyper methylation of CDKN2A/p16 gene was detected in 24 (48.0%) of 50 oral squamous cell carcinoma cases (OSCC) and the restoration by 5-Aza-C was found in 6 (75.0%) of 8 cell lines. The down expression of APCgene was detected in 15 (30.0%) of 50cases, and hypermethylation of CPG island was found 12 (24.0%) of the all cases. Suppressed expression and hypermethylation of FEZ1gene was detected i … More n 11 (35.5%) and in 8 (25.8%) of 31 case, respectively. Out of the 31 cases, hypermethylation was detected and restoration of the gene expression by 5-Aza-C was found in all of the 8 cell lines. Silence or suppressed expression of KAI1 gene was detected in 84 of 101 OSCC cases. However, neither hypermethylation of CPG island of the promoter region nor restoration of the gene expression by 5-Aza-C was not found. The over-expression of surviving gene was detected in 58% of OSCC cases and in 37% of precancerous lesion, leukoplakia. On the other hand, the expression of surviving gene was not detected by RT-PCRin all of the 9 normal oral epitherium tissues and DNA methylation was confirmed by methylation specific PCR in 4 of the 9 normal specimens and the gene expression was restored by 5-Aza-Ctreatment in 2 of 8 cell lines. The over-expression of DNA methyltransferase, DNMTs ; DNMT1, DNMT3A, and DNMT3B, which were believed to be associated with carcinogenesis, were high frequently detected in OSCC. The rates of the expression of DNMTI, DNMT3A, and DNMT3B in OSCC were 72%, 56%, and 64%, respectively. There was no significant correlation between the expression levels and the situations of hypermethylation of the tumor suppressor and oncogenes examined and the expression levels of methyltransferases. Our data suggest that hypermethylation of the gene may be one of the major mechanisms for inactivation of the genes in oral carcinogenesis and that besides methyltransferases, some transcription factors involved in the suppression of gene expression might play an important roles in ora oncogenesis. Less