Vertino P M, Issa J P, Pereira-Smith O M, Baylin S B
Oncology Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231.
Cell Growth Differ. 1994 Dec;5(12):1395-402.
De novo methylation of normally unmethylated CpG islands and increased expression of DNA (cytosine-5)-methyltransferase (DNA MTase) are common characteristics of immortalized cell lines and human tumors. To examine the acquisition of these properties with respect to cellular immortalization, we studied CpG island methylation and DNA MTase expression in aging normal human fibroblasts and their SV40-infected preimmortal (precrisis) and immortal (postcrisis) derivatives. The levels of DNA MTase enzyme activity decreased by 50% as normal fibroblasts were cultured to senescence. By contrast, DNA MTase activity did not decrease in SV40-infected pre- or postcrisis cells but remained similar to that of young fibroblasts and 2-4-fold higher than that of senescent fibroblasts. DNA MTase mRNA levels paralleled those of enzyme activity. Several loci were examined to determine the relationship between the dynamics of DNA MTase expression and the appearance of de novo CpG island methylation. Ten CpG island loci examined were unmethylated in normal young fibroblasts. By contrast, four of these loci (the CALC1, MyoD, and IGF-2 genes on chromosome 11p and the estrogen receptor gene on chromosome 6q) were de novo methylated in fully immortalized, postcrisis cells. Two of these four were actually methylated in extended life span precrisis cells, and one, the estrogen receptor locus, exhibited de novo methylation with aging in normal fibroblasts. The data indicate that an ability to maintain DNA MTase levels is acquired with SV40-induced escape from senescence. Furthermore, aberrant CpG island methylation can be established prior to immortalization, either as a function of normal aging or in response to SV40-induced escape from senescence.
正常情况下未甲基化的CpG岛发生从头甲基化以及DNA（胞嘧啶-5）-甲基转移酶（DNA MTase）表达增加是永生化细胞系和人类肿瘤的共同特征。为了研究细胞永生化过程中这些特性的获得情况，我们研究了衰老的正常人成纤维细胞及其感染SV40的永生化前（危机前）和永生化后（危机后）衍生物中的CpG岛甲基化和DNA MTase表达。随着正常成纤维细胞培养至衰老，DNA MTase酶活性水平降低了50%。相比之下，感染SV40的危机前或危机后细胞中的DNA MTase活性并未降低，而是与年轻成纤维细胞的活性相似，且比衰老成纤维细胞的活性高2至4倍。DNA MTase mRNA水平与酶活性水平平行。检测了几个位点以确定DNA MTase表达动态与从头CpG岛甲基化出现之间的关系。在正常年轻成纤维细胞中检测的10个CpG岛位点未甲基化。相比之下，其中4个位点（11号染色体上的CALC1、MyoD和IGF-2基因以及6号染色体上的雌激素受体基因）在完全永生化的危机后细胞中发生了从头甲基化。这4个位点中的2个实际上在寿命延长的危机前细胞中就已甲基化，其中一个，即雌激素受体位点，在正常成纤维细胞衰老过程中出现了从头甲基化。数据表明，随着SV40诱导的细胞从衰老中逃逸，获得了维持DNA MTase水平的能力。此外，异常的CpG岛甲基化可在永生化之前建立，这要么是正常衰老的结果，要么是对SV40诱导的细胞从衰老中逃逸的反应。
