Reciprocal relationship between O6-methylguanine-DNA methyltransferase P140K expression level and chemoprotection of hematopoietic stem cells.
Reciprocal relationship between O6-methylguanine-DNA methyltransferase P140K expression level and chemoprotection of hematopoietic stem cells.
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DOI：
10.1158/0008-5472.can-08-0320
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发表时间：
2008-08-01
期刊：
影响因子：
11.2
通讯作者：
Williams DA
中科院分区：
文献类型：
--
作者：
Milsom MD;Jerabek-Willemsen M;Harris CE;Schambach A;Broun E;Bailey J;Jansen M;Schleimer D;Nattamai K;Wilhelm J;Watson A;Geiger H;Margison GP;Moritz T;Baum C;Thomale J;Williams DA
已经提出将P140 K突变体O 6-甲基鸟嘌呤-DNA甲基转移酶（MGMTP 140 K）逆转录病毒介导的递送到造血干细胞（HSC）中作为一种手段来保护免受由化疗结合O 6-烷化剂（例如，替莫唑胺）与内源性MGMT的假底物抑制剂（如O 6-苄基鸟嘌呤）。由于MGMT对O 6-烷基鸟嘌呤加合物的解毒是化学计量的，因此已经提出，较高水平的MGMT将对基因修饰的HSC提供更好的保护。然而，实现这一目标可能与基因治疗领域目前的努力相冲突，基因治疗领域的目标是掺入较弱的增强子元件以避免插入诱变。使用一组表达一系列MGMTP 140 K活性的自失活γ-逆转录病毒载体，我们表明通过较弱的细胞启动子/增强子表达MGMTP 140 K足以在用O 6-苄基鸟嘌呤/替莫唑胺治疗后进行体内保护/选择。相反，最高水平的MGMTP 140 K活性没有促进有效的体内保护，尽管介导的O 6-烷基鸟嘌呤加合物的解毒。此外，非常高的MGMTP 140 K表达与HSC中的竞争性再增殖缺陷相关。从机制上讲，我们发现细胞增殖的缺陷与MGMTP 140 K的表达升高有关，但与野生型MGMT无关。这种增殖缺陷与MGMTP 140 K向细胞核/染色质的定位增加相关。这些数据表明，非常高的MGMTP 140 K表达对细胞增殖、植入和化学保护具有有害作用。这些研究与正在进行的使用MGMTP 140 K的临床基因治疗研究具有直接的翻译相关性，而新的机制发现与MGMT对DNA修复的基本理解相关。
Retroviral-mediated delivery of the P140K mutant O6-methylguanine-DNA methyltransferase (MGMTP140K) into hematopoietic stem cells (HSC) has been proposed as a means to protect against dose-limiting myelosuppressive toxicity ensuing from chemotherapy combining O6-alkylating agents (e.g., temozolomide) with pseudosubstrate inhibitors (such as O6-benzylguanine) of endogenous MGMT. Because detoxification of O6-alkylguanine adducts by MGMT is stoichiometric, it has been suggested that higher levels of MGMT will afford better protection to gene-modified HSC. However, accomplishing this goal would potentially be in conflict with current efforts in the gene therapy field, which aim to incorporate weaker enhancer elements to avoid insertional mutagenesis. Using a panel of self-inactivating gamma-retroviral vectors that express a range of MGMTP140K activity, we show that MGMTP140K expression by weaker cellular promoter/enhancers is sufficient for in vivo protection/selection following treatment with O6-benzylguanine/temozolomide. Conversely, the highest level of MGMTP140K activity did not promote efficient in vivo protection despite mediating detoxification of O6-alkylguanine adducts. Moreover, very high expression of MGMTP140K was associated with a competitive repopulation defect in HSC. Mechanistically, we show a defect in cellular proliferation associated with elevated expression of MGMTP140K, but not wild-type MGMT. This proliferation defect correlated with increased localization of MGMTP140K to the nucleus/chromatin. These data show that very high expression of MGMTP140K has a deleterious effect on cellular proliferation, engraftment, and chemoprotection. These studies have direct translational relevance to ongoing clinical gene therapy studies using MGMTP140K, whereas the novel mechanistic findings are relevant to the basic understanding of DNA repair by MGMT.