Enforced expression of wild-type p53 curtails the transcription of the O(6)-methylguanine-DNA methyltransferase gene in human tumor cells and enhances their sensitivity to alkylating agents.
Enforced expression of wild-type p53 curtails the transcription of the O(6)-methylguanine-DNA methyltransferase gene in human tumor cells and enhances their sensitivity to alkylating agents.
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发表时间：
2001-05
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通讯作者：
K. Srivenugopal;Jiang Shou;S. Mullapudi;Frederick F Lang;Jasti S. Rao;Francis Ali-Osman
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作者：
K. Srivenugopal;Jiang Shou;S. Mullapudi;Frederick F Lang;Jasti S. Rao;Francis Ali-Osman
我们使用等基因人肿瘤细胞系研究野生型（wt） p53对O(6)-甲基鸟嘌呤-DNA甲基转移酶（MGMT）表达的特异性和直接影响，MGMT是一种DNA修复蛋白，赋予肿瘤对许多抗癌烷基化剂的抗性。在四环素调控的系统中，我们设计了一种p53缺失、mgmt精通的肺肿瘤细胞系（H1299）来表达p53。在该细胞系中，四环素停药诱导的高水平p53伴随着G(1)细胞周期阻滞，没有明显的凋亡。p53的积累导致MGMT mRNA、蛋白和酶活性的逐渐和急剧下降，其水平在诱导的第3天无法检测到。然而，MGMT蛋白的丢失并不是由于其降解，因为泛素促进的MGMT体外降解，介导修复蛋白的细胞处置，并没有被p53改变。运行转录分析显示MGMT基因转录率显著降低。在另外两种人类等基因细胞系中证实了wt p53对MGMT表达的负调控，即含有地塞米松诱导的wt p53的GM47.23胶质母细胞瘤细胞系和wt p53被人乳头瘤病毒E6蛋白灭活的H460肺癌细胞系。此外，一组四种人类肿瘤细胞系，包括具有wt p53状态的胶质瘤，显示MGMT基因转录水平明显低于p53突变的细胞。在这些模型中诱导wt p53分别导致对1,3-双（2-氯乙基）-1-亚硝基脲和替莫唑胺的敏感性增加3倍和2倍，这两种物质在DNA中产生可修复的O(6)-烷基加合物。这些结果表明，p53是MGMT基因表达的负调控因子，可以在人类肿瘤中产生MGMT-耗尽状态，类似于O(6)-苄基鸟嘌呤（一种有效的MGMT抑制剂，目前正在临床试验中）。因此，我们的研究揭示了与p53基因治疗相关的额外益处，并为将mgmt导向的烷基化剂与p53基因转移相结合以提高抗肿瘤疗效提供了强有力的生化基础。
We used isogenic human tumor cell lines to investigate the specific and direct effects of wild-type (wt) p53 on the expression of O(6)-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein that confers tumor resistance to many anticancer alkylating agents. A p53-null, MGMT-proficient lung tumor cell line (H1299) was engineered to express wt p53 in a tetracycline-regulated system. High levels of p53 induction achieved by tetracycline withdrawal were accompanied by G(1) cell cycle arrest without significant apoptosis in this cell line. p53 accumulation resulted in a gradual and dramatic loss of MGMT mRNA, protein, and enzyme activity, whose levels were undetectable by day 3 of induction. The loss of MGMT protein was, however, not due to its degradation because the ubiquitin-promoted in vitro degradation of MGMT, which mediates the cellular disposal of the repair protein, was not altered by p53. Run-on transcription assays revealed a significant reduction in the rate of MGMT gene transcription. The negative regulation of MGMT expression by wt p53 was confirmed in two other human isogenic cell lines, namely, the GM47.23 glioblastoma, which contains a dexamethasone-inducible wt p53, and the H460 lung cancer cell line, in which wt p53 had been inactivated by the human papillomavirus E6 protein. Furthermore, a panel of four human tumor cell lines, including gliomas with wt p53 status, displayed markedly lower levels of MGMT gene transcripts than those having p53 mutations. Induction of wt p53 in these models led to a 3- and 2-fold increase in sensitivity to 1,3-bis(2-chloroethyl)-1-nitrosourea and temozolomide, respectively, which generate the MGMT-repairable O(6)-alkyl adducts in DNA. These results demonstrate that p53 is a negative regulator of MGMT gene expression and can create a MGMT-depleted state in human tumors similar to that achieved by O(6)-benzylguanine, a potent inhibitor of MGMT currently undergoing clinical trials. Thus, our study exposes an additional benefit associated with p53 gene therapy and provides a strong biochemical rationale for combining the MGMT-directed alkylators with p53 gene transfer to achieve improved antitumor efficacy.