Roth Timothy J, Xu Yi, Luo Meihua, Kelley Mark R
Department of Physiology and Biophysics, Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, 702 Barnhill Dr, Room 2600, Indianapolis, Indiana 46202, USA.
Cancer Gene Ther. 2003 Aug;10(8):603-10. doi: 10.1038/sj.cgt.7700605.
Chemotherapeutic DNA alkylating agents are common weapons employed to fight both pediatric and adult cancers. In addition to cancerous cells, nontarget tissues are subjected to the cytotoxicity of these agents, and dose-limiting toxicity in the form of myelosuppression is a frequent result of treatment. One approach to prevent myelosuppression that results from the use of chemotherapeutic agents is to increase the levels of DNA repair proteins in bone marrow cells. Here we report our second successful attempt to create a fusion protein that possesses both direct reversal and base excision repair pathway DNA repair activities. The chimeric protein is composed of the human O(6)-Methylguanine-DNA Methyltransferase (MGMT) and the yeast Apn1 proteins and retains both MGMT and AP endonuclease activities as determined by biochemical analysis. We have also demonstrated that the chimeric protein is able to protect mammalian cells from the DNA alkylating agents 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) and methyl methanesulfonate (MMS). The protection by the chimeric protein against BCNU is even greater than MGMT alone, which has potential translational significance given that MGMT is currently in clinical trials. Additionally, we show that the chimeric MGMT-Apn1 protein can protect mammalian cells from dual treatments of BCNU and MMS and that this effect is greater than that provided by MGMT alone. The data support our previous finding that a protein with multiple DNA repair activities can be constructed and that this and other constructs may play an important clinical role in guarding against dose-limiting effects of chemotherapy, particularly in situations of multiple drug use.
化疗性DNA烷化剂是用于治疗儿童和成人癌症的常用武器。除癌细胞外，非靶组织也会受到这些药物的细胞毒性影响，而骨髓抑制形式的剂量限制性毒性是治疗的常见结果。一种预防因使用化疗药物导致骨髓抑制的方法是提高骨髓细胞中DNA修复蛋白的水平。在此，我们报告了第二次成功创建一种具有直接修复和碱基切除修复途径DNA修复活性的融合蛋白的尝试。该嵌合蛋白由人O(6)-甲基鸟嘌呤-DNA甲基转移酶（MGMT）和酵母Apn1蛋白组成，生化分析表明其保留了MGMT和AP内切核酸酶活性。我们还证明了该嵌合蛋白能够保护哺乳动物细胞免受DNA烷化剂1,3-双（2-氯乙基）-1-亚硝基脲（BCNU）和甲基磺酸甲酯（MMS）的损伤。该嵌合蛋白对BCNU的保护作用甚至大于单独的MGMT，鉴于MGMT目前正在进行临床试验，这具有潜在的转化意义。此外，我们表明嵌合的MGMT-Apn1蛋白可以保护哺乳动物细胞免受BCNU和MMS的联合处理，且这种效果大于单独的MGMT所提供的保护。这些数据支持了我们之前的发现，即可以构建具有多种DNA修复活性的蛋白，并且这种蛋白及其他构建体可能在预防化疗的剂量限制性效应方面发挥重要的临床作用，特别是在联合使用多种药物的情况下。
