Altered MGMT in Acquired Drug Resistance

获得性耐药性中 MGMT 的改变

基本信息

批准号：
6383921
负责人：
LILI LIU
金额：
$ 20.66万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-06-01 至 2005-05-31
项目状态：
已结题

项目摘要

One of the mechanisms responsible for the therapeutic failure of alkylating agents is the DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGT), encoded by the MGMT gene. AGT removes the alkyl group from 06-alkylguanine in a fast and single step reaction, thereby preventing the formation of DNA cross-links by chloroethylating agents such as BCNU. Currently, a strategy involving the inactivation of AGT by 06-benzylguanine (BG) followed by BCNU treatment has shown evidence of significantly increased antitumor effect of BCNU. Ongoing clinical trials are evaluating its efficacy in tumor chemotherapy. However, repeated administration of BG and BCNU will raise the possibility that BG resistant cells develop, subsequently, resulting in the failure of chemotherapy. In our recent studies, we selected two MMR deficient colon cancer cells for resistance to BG and BCNU and found two different mutations at amino acid 165 of AGT, to form K165E and K165N mutant AGT in these two cell lines. The cells harboring the K165 mutations have dramatically decreased AGT activity but remarkably increased resistance to BG+BCNU. Thus, we hypothesize that MMR deficiency leads to a high mutation frequency in DNA repair gene such as AGT gene and that two K165 mutant AGTs predominantly confer acquired resistance either to the combination BG+BCNU and BG+TMZ or alkylating agents alone. To test this hypothesis, it is necessary to distinguish acquired resistance caused by mutated AG from other resistance factors. This is of concern because the two BG-resistant AGTs were identified in cell lines with MMR defects. Once cells lose MMR, their sensitivity to various chemotherapeutic agents is decreased directly by impairing the ability to recognize or process DNA damage and indirectly by increasing the mutation rate throughout the genome. Therefore, it is possible that not only does mutation in AGT confer drug resistance, but other mechanisms of drug resistance as well. Thus, these specific objectives are proposed: to define whether K165 mutant AGTs are the major factor of acquired resistance to BG and BCNU, despite low AGT activity; to define whether colon cancer tumors with MMR deficiency are more likely to acquire resistance to BG+BCNU through mutations in MGMT than MMR wt tumors; and to determine whether BG-resistant AGT could be selected in the xenograft setting after mice carrying the tumor received multiple treatments with BG and BCNU. The long-term goal is to define the conditions in which MGMT mutations are observed in human tumors after clinical use of BG and BCNU. Overall, this project promises to provide novel information on the induction of BG-resistant AGT in drug treated MMR defective tumors and the impact of the altered AGT-resistance to BG+BCNU.

烷化剂治疗失败的机制之一是由 MGMT 基因编码的 DNA 修复蛋白 O6-烷基鸟嘌呤-DNA 烷基转移酶 (AGT)。 AGT 以快速且一步的反应从 06-烷基鸟嘌呤中除去烷基，从而防止氯乙基化剂（例如 BCNU）形成 DNA 交联。目前，涉及通过 06-苄基鸟嘌呤 (BG) 灭活 AGT，然后进行 BCNU 治疗的策略已显示出 BCNU 显着增强的抗肿瘤作用的证据。正在进行的临床试验正在评估其在肿瘤化疗中的功效。然而，重复给予BG和BCNU会增加产生BG耐药细胞的可能性，从而导致化疗失败。在我们最近的研究中，我们选择了两种MMR缺陷的结肠癌细胞来抵抗BG和BCNU，并在AGT的165位氨基酸处发现了两种不同的突变，从而在这两种细胞系中形成K165E和K165N突变体AGT。携带 K165 突变的细胞显着降低了 AGT 活性，但显着增加了对 BG+BCNU 的抵抗力。因此，我们假设 MMR 缺陷导致 DNA 修复基因（例如 AGT 基因）的高突变频率，并且两个 K165 突变 AGT 主要赋予对 BG+BCNU 和 BG+TMZ 组合或单独烷化剂的获得性抗性。为了检验这一假设，有必要将突变AG引起的获得性耐药与其他耐药因素区分开来。这是值得关注的，因为在具有 MMR 缺陷的细胞系中发现了两种 BG 抗性 AGT。一旦细胞失去 MMR，它们对各种化疗药物的敏感性就会通过削弱识别或处理 DNA 损伤的能力而直接降低，并通过增加整个基因组的突变率而间接降低。因此，有可能不仅 AGT 突变会产生耐药性，而且其他机制也会产生耐药性。因此，提出了这些具体目标：确定 K165 突变 AGT 是否是 BG 和 BCNU 获得性耐药的主要因素，尽管 AGT 活性较低；确定 MMR 缺陷的结肠癌肿瘤是否比 MMR wt 肿瘤更有可能通过 MGMT 突变获得 BG+BCNU 耐药性；并确定在携带肿瘤的小鼠接受 BG 和 BCNU 多次治疗后，是否可以在异种移植环境中选择 BG 抗性 AGT。长期目标是确定临床使用 BG 和 BCNU 后在人类肿瘤中观察到 MGMT 突变的条件。总体而言，该项目有望提供关于在药物治疗的 MMR 缺陷肿瘤中诱导 BG 抗性 AGT 以及改变的 AGT 抗性对 BG+BCNU 的影响的新信息。