Milsom Michael D, Jerabek-Willemsen Moran, Harris Chad E, Schambach Axel, Broun Emily, Bailey Jeff, Jansen Michael, Schleimer David, Nattamai Kalpana, Wilhelm Jamie, Watson Amanda, Geiger Hartmut, Margison Geoffrey P, Moritz Thomas, Baum Christopher, Thomale Jürgen, Williams David A
Division of Experimental Hematology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA.
Cancer Res. 2008 Aug 1;68(15):6171-80. doi: 10.1158/0008-5472.CAN-08-0320.
Retroviral-mediated delivery of the P140K mutant O(6)-methylguanine-DNA methyltransferase (MGMT(P140K)) into hematopoietic stem cells (HSC) has been proposed as a means to protect against dose-limiting myelosuppressive toxicity ensuing from chemotherapy combining O(6)-alkylating agents (e.g., temozolomide) with pseudosubstrate inhibitors (such as O(6)-benzylguanine) of endogenous MGMT. Because detoxification of O(6)-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 MGMT(P140K) activity, we show that MGMT(P140K) expression by weaker cellular promoter/enhancers is sufficient for in vivo protection/selection following treatment with O(6)-benzylguanine/temozolomide. Conversely, the highest level of MGMT(P140K) activity did not promote efficient in vivo protection despite mediating detoxification of O(6)-alkylguanine adducts. Moreover, very high expression of MGMT(P140K) was associated with a competitive repopulation defect in HSC. Mechanistically, we show a defect in cellular proliferation associated with elevated expression of MGMT(P140K), but not wild-type MGMT. This proliferation defect correlated with increased localization of MGMT(P140K) to the nucleus/chromatin. These data show that very high expression of MGMT(P140K) has a deleterious effect on cellular proliferation, engraftment, and chemoprotection. These studies have direct translational relevance to ongoing clinical gene therapy studies using MGMT(P140K), whereas the novel mechanistic findings are relevant to the basic understanding of DNA repair by MGMT.
有人提出，通过逆转录病毒介导将P140K突变型O(6)-甲基鸟嘌呤-DNA甲基转移酶（MGMT(P140K)）导入造血干细胞（HSC），以此来预防因化疗联合使用O(6)-烷基化剂（如替莫唑胺）和内源性MGMT的假底物抑制剂（如O(6)-苄基鸟嘌呤）而产生的剂量限制性骨髓抑制毒性。由于MGMT对O(6)-烷基鸟嘌呤加合物的解毒作用是化学计量的，因此有人认为更高水平的MGMT将为基因修饰的HSC提供更好的保护。然而，要实现这一目标可能会与基因治疗领域目前的努力相冲突，目前的努力旨在引入较弱的增强子元件以避免插入诱变。我们使用一组表达一系列MGMT(P140K)活性的自失活γ-逆转录病毒载体，结果表明，较弱的细胞启动子/增强子介导的MGMT(P140K)表达足以在接受O(6)-苄基鸟嘌呤/替莫唑胺治疗后实现体内保护/选择。相反，尽管MGMT(P140K)活性最高水平介导了O(6)-烷基鸟嘌呤加合物的解毒作用，但并未促进有效的体内保护。此外，MGMT(P140K)的极高表达与HSC的竞争性再增殖缺陷有关。从机制上讲，我们发现细胞增殖缺陷与MGMT(P140K)而非野生型MGMT的表达升高有关。这种增殖缺陷与MGMT(P140K)在细胞核/染色质中的定位增加相关。这些数据表明，MGMT(P140K)的极高表达对细胞增殖、植入和化学保护具有有害影响。这些研究与正在进行的使用MGMT(P140K)的临床基因治疗研究直接相关，而新的机制发现与对MGMT进行DNA修复的基本理解相关。
