Eads C A, Danenberg K D, Kawakami K, Saltz L B, Danenberg P V, Laird P W
Department of Surgery, University of Southern California, School of Medicine, Norris Comprehensive Cancer Center, Los Angeles 90033, USA.
Cancer Res. 1999 May 15;59(10):2302-6.
The molecular basis of aberrant hypermethylation of CpG islands observed in a subset of human colorectal tumors is unknown. One potential mechanism is the up-regulation of DNA (cytosine-5)-methyltransferases. Recently, two new mammalian DNA methyltransferase genes have been identified, which are referred to as DNMT3A and DNMT3B. The encoded proteins differ from the predominant mammalian DNA methyltransferase DNMT1 in that they have a substantially higher ratio of de novo to maintenance methyltransferase activity. We have used a highly quantitative 5' nuclease fluorogenic reverse transcription-PCR method (TaqMan) to analyze the expression of all three DNA methyltransferase genes in 25 individual colorectal adenocarcinoma specimens and matched normal mucosa samples. In addition, we examined the methylation patterns of four CpG islands [APC, ESR1 (estrogen receptor), CDKN2A (p16), and MLH1] to determine whether individual tumors show a positive correlation between the level of DNA methyltransferase expression and the frequency of CpG island hypermethylation. All three methyltransferases appear to be up-regulated in tumors when RNA levels are normalized using either ACTB (beta-actin) or POLR2A (RNA pol II large subunit), but not when RNA levels are normalized with proliferation-associated genes, such as H4F2 (histone H4) or PCNA. The frequency or extent of CpG island hypermethylation in individual tumors did not correlate with the expression of any of the three DNA methyltransferases. Our results suggest that deregulation of DNA methyltransferase gene expression does not play a role in establishing tumor-specific abnormal DNA methylation patterns in human colorectal cancer.
在一部分人类结肠直肠癌中观察到的CpG岛异常高甲基化的分子基础尚不清楚。一种潜在机制是DNA（胞嘧啶-5）-甲基转移酶的上调。最近，已鉴定出两个新的哺乳动物DNA甲基转移酶基因，分别称为DNMT3A和DNMT3B。编码的蛋白质与主要的哺乳动物DNA甲基转移酶DNMT1不同，因为它们的从头甲基转移酶活性与维持甲基转移酶活性的比例要高得多。我们使用了一种高度定量的5'核酸酶荧光逆转录PCR方法（TaqMan）来分析25个个体结肠腺癌标本和匹配的正常黏膜样本中所有三种DNA甲基转移酶基因的表达。此外，我们检查了四个CpG岛[APC、ESR1（雌激素受体）、CDKN2A（p16）和MLH1]的甲基化模式，以确定单个肿瘤是否在DNA甲基转移酶表达水平与CpG岛高甲基化频率之间显示出正相关。当使用ACTB（β-肌动蛋白）或POLR2A（RNA聚合酶II大亚基）对RNA水平进行标准化时，所有三种甲基转移酶在肿瘤中似乎都上调，但当使用与增殖相关的基因（如H4F2（组蛋白H4）或PCNA）对RNA水平进行标准化时则不然。单个肿瘤中CpG岛高甲基化的频率或程度与三种DNA甲基转移酶中的任何一种的表达均无相关性。我们的结果表明，DNA甲基转移酶基因表达失调在人类结肠直肠癌中建立肿瘤特异性异常DNA甲基化模式中不起作用。
