Lentivirus-mediated expression of mutant MGMTP140K protects human CD34+ cells against the combined toxicity of O6-benzylguanine and 1,3-bis(2-chloroethyl)-nitrosourea or temozolomide
Lentivirus-mediated expression of mutant MGMTP140K protects human CD34+ cells against the combined toxicity of O6-benzylguanine and 1,3-bis(2-chloroethyl)-nitrosourea or temozolomide
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DOI：
10.1089/1043034041648417
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发表时间：
2004-08-01
影响因子：
4.2
通讯作者：
Chinnasamy, N
中科院分区：
文献类型：
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作者：
Chinnasamy, D;Fairbairn, LJ;Chinnasamy, N
慢病毒载体能够有效地转导未分裂和缓慢分裂的细胞，包括造血干细胞，从而实现稳定的整合和持续的转基因表达。我们构建了基于人类免疫缺陷病毒1型的自失活慢病毒载体，表达人O-6-烷基鸟嘌呤-脱氧核糖核酸甲基转移酶野生型或O-6-苄基鸟嘌呤(O-6-BegE)抗性突变形式，并转导K562和粒细胞集落刺激因子动员的人外周血DNA-O-6-methylguanine：[protein]-L-cysteineS-methyltransferase，(+)细胞。Western印迹、免疫细胞化学和生化检测表明，转导后的K562细胞表达高水平的MGMT。集落形成存活实验显示对O-6-BEGE加1，3-二(2-氯乙基)-亚硝脲(BCNU)或替莫唑胺(TMZ)毒性有明显的保护作用。同样，单次转导CD34(+)细胞可导致MGMT表达水平增加13-14倍。与未转导的细胞相比，转导突变MGMT(P140K)的CD34(+)细胞对O-6-BegE与TMZ或BCNU的联合毒性表现出显著的抵抗力：O-6-BegE+TMZ处理后的集落形成细胞存活率增加了近9倍，O-6-BegE+BCNU处理后的IC50值显示出类似于5倍的增加。这些结果表明，慢病毒介导的MGMT(P140K)的表达能有效地保护造血室免受O-6-BEG与TMZ或BCNU的联合毒性。
Lentiviral vectors are capable of efficiently transducing nondividing and slowly dividing cells, including hematopoietic stem cells, resulting in stable integration and sustained transgene expression. We constructed human immunodeficiency virus type 1-based self-inactivating lentiviral vectors to express either wild-type or an O-6-benzylguanine (O-6-beG)-resistant mutant form of the human O-6-alkylguanine-DNA methyltransferase (MGMT; DNA-O-6-methylguanine:[protein]-L-cysteineS-methyltransferase, EC 2.1.1.63) and transduced K562 and granulocyte colony-stimulating factor-mobilized human peripheral blood CD34(+) cells. After transduction, K562 cells expressed high levels of MGMT as determined by Western blot, immunocytochemistry, and biochemical assay. A colony-forming survival assay showed significant protection against O-6-beG plus 1,3-bis(2-chloroethyl)-nitrosourea(BCNU) or temozolomide (TMZ) toxicity. Similarly, a single transduction of CD34(+) cells resulted in a 13- to 14-fold increase in the level of MGMT expression. In comparison with non-transduced cells, mutant MGMT(P140K)-transduced CD34(+) cells showed significant resistance against the combined toxicity of O-6-beG with either TMZ or BCNU: there was an similar to9-fold increase in the survival of colony-forming cells as indicated by the IC50 values after O-6-beG plus TMZ treatment and an similar to5-fold increase in the case of O-6-beG plus BCNU treatment. These results show that lentivirus-mediated expression of MGMT(P140K) can efficiently protect the hematopoietic compartment against the combined toxicity of O-6-beG plus TMZ or BCNU.