Worton K S, Kerbel R S, Andrulis I L
Mount Sinai Hospital Research Institute, Toronto, Ontario, Canada.
Cancer Res. 1991 Feb 1;51(3):985-9.
Successful chemotherapeutic treatment of drug-responsive cancers can be compromised by the acquisition of drug resistance. Standard remission induction therapy for childhood acute lymphoblastic leukemia includes L-asparaginase, since the leukemic cells lack asparagine synthetase (AS) activity and require exogenous asparagine. We have used the Chinese hamster ovary cell line N3, which lacks AS activity, as a model to examine a novel mechanism involved in the development of drug resistance in acute lymphoblastic leukemia. Expression of AS in Chinese hamster ovary cells is associated with hypomethylation in the 5' region of the gene. Activation of AS in concert with hypomethylation occurs spontaneously at a frequency of about 10(-6); we have found that treatment with the hypomethylating drug 5-azacytidine induces a reversion frequency of 10(-2). To investigate the possibility that chemotherapeutic drugs induce similar changes, the asparagine auxotrophic cell line N3 was treated with the chemotherapeutic agents L-asparaginase, vincristine, and 1-beta-D-arabinofuranosylcytosine and with the mutagen ethyl methanesulfonate. Both L-asparaginase and ethyl methanesulfonate increased the frequency of reversion to asparagine prototrophy to about 10(-5), whereas vincristine and 1-beta-D-arabinofuranosylcytosine had no such effect. Asparagine prototrophy correlated with the demethylation of CpG sites in the 5' region of the AS gene and with the appearance of AS mRNA in revertants. In addition to the specific effect seen with the AS gene, L-asparaginase and ethyl methanesulfonate induced global reductions in methylation of up to 25 and 10%, respectively. The ability of chemotherapeutic drugs to inhibit DNA methylation and thereby activate previously silent genes may enable them to promote the aggressiveness of cancers in vivo, including the expression of drug resistance.
对药物敏感的癌症进行成功的化疗可能会因获得耐药性而受到影响。儿童急性淋巴细胞白血病的标准缓解诱导疗法包括使用L-天冬酰胺酶，因为白血病细胞缺乏天冬酰胺合成酶（AS）活性，需要外源性天冬酰胺。我们使用缺乏AS活性的中国仓鼠卵巢细胞系N3作为模型，来研究急性淋巴细胞白血病耐药性发展中涉及的一种新机制。中国仓鼠卵巢细胞中AS的表达与该基因5'区域的低甲基化有关。AS的激活与低甲基化协同发生的频率约为10^(-6)；我们发现用去甲基化药物5-氮杂胞苷处理可诱导10^(-2)的回复频率。为了研究化疗药物是否会诱导类似变化，将天冬酰胺营养缺陷型细胞系N3用化疗药物L-天冬酰胺酶、长春新碱和1-β-D-阿拉伯呋喃糖基胞嘧啶以及诱变剂甲磺酸乙酯进行处理。L-天冬酰胺酶和甲磺酸乙酯都将回复到天冬酰胺原养型的频率提高到了约10^(-5)，而长春新碱和1-β-D-阿拉伯呋喃糖基胞嘧啶则没有这种效果。天冬酰胺原养型与AS基因5'区域CpG位点的去甲基化以及回复子中AS mRNA的出现相关。除了在AS基因上看到的特异性效应外，L-天冬酰胺酶和甲磺酸乙酯分别诱导整体甲基化降低高达25%和10%。化疗药物抑制DNA甲基化从而激活先前沉默基因的能力可能使它们能够促进体内癌症的侵袭性，包括耐药性的表达。
