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

为了评估甲基化在APC、p16、FEZ1、KAI1和Survivin等与细胞周期调控相关的基因下调中的作用，我们检测了它们和甲基转移酶DNMT1、DNMT3a和Dnmt3b的表达水平，这些甲基转移酶被认为与口腔癌的发生有关，8个细胞系，SAS，HSC-2，HSC-3，HSC-4，Ca9-22，OK-92，HO-1-U-1和HO-1-N-1，以及口腔癌的临床组织样本。此外，还检测了去甲基化试剂5-aza-C对基因表达的恢复作用。50例口腔鳞状细胞癌(OSCC)中24例(48.0%)CDKN2A/p16基因高甲基化，6例(75.0%)经5-aza-C治疗后恢复正常。APC基因低表达15例(30.0%)，CpG岛高甲基化12例(24.0%)。…检测到FEZ1基因表达抑制和高甲基化多发11例(35.5%)，多发8例(25.8%)。31例中，8株细胞均出现甲基化，经5-aza-C处理后，基因表达均恢复正常。101例口腔鳞癌中有84例KAI1基因表达下调或沉默。然而，没有发现启动子区域CpG岛的高甲基化，也没有发现5-aza-C恢复基因表达。Survivin基因在58%的口腔鳞状细胞癌和37%的癌前病变白斑中过度表达。反转录-聚合酶链式反应检测9例正常口腔上皮组织中未检测到存活基因的表达，甲基化特异性聚合酶链式反应证实4例正常口腔黏膜组织中有4例DNA甲基化，经5-aza-C处理后，8例口腔上皮细胞中有2例表达恢复。DNA甲基转移酶Dnmts、DNMT1、DNMT3a和Dnmt3b在口腔鳞癌中的过度表达频率较高。DNMTI、DNMT3A和DNMT3B在口腔鳞癌中的阳性表达率分别为72%、56%和%。抑癌基因和癌基因的高甲基化程度与甲基转移酶的表达水平之间无明显相关性。我们的结果提示，该基因的高甲基化可能是口腔癌发生过程中基因失活的主要机制之一，除甲基转移酶外，参与抑制基因表达的一些转录因子可能在口腔癌的发生中起重要作用。较少

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