Allay J A, Koç O N, Davis B M, Gerson S L
Departments of Medicine and Biology, Molecular Virology Training Program, Case Western Reserve School of Medicine, Cleveland, Ohio 44106, USA.
Clin Cancer Res. 1996 Aug;2(8):1353-9.
Myelosuppression is the dose-limiting toxicity for nitrosourea chemotherapy due to low levels of the DNA repair protein O6-alkylguanine-DNA alkyltransferase in myeloid precursors. We have shown that high-efficiency myeloproliferative sarcoma virus (vM5MGMT)-mediated transduction of the human MGMT cDNA into murine bone marrow (BM) cells leads to high MGMT expression and increased progenitor resistance to 1,3-bis-(2-chloroethyl) nitrosourea (BCNU) in vitro immediately after infection and after BM transplantation. These experiments were designed to increase MGMT expression in human hematopoietic progenitors. CD34(+) BM cells were isolated over an immunoaffinity column (CEPRATE, CellPro, Inc.), resulting in a mean 66-fold enrichment in clonogenic progenitors (colony-forming unit granulocyte-macrophage + burst-forming unit erythroid + colony-forming unit granulocyte erythroid macrophage = megakaryocyte), with an average progenitor yield of 58 +/- 11.5% and a final population that was 54% CD34(+). Seventy % of progenitors derived from CD34(+) cells were transduced after coculture with AM12-vM5MGMT retroviral producers. vM5MGMT-transduced progenitors were over 2-fold more resistant to concentrations of BCNU between 30 and 50 micrometer than were concurrently LacZ-transduced progenitors (P < 0.003). In vitro selection of transduced, cytokine-stimulated CD34(+) cells with 20 micrometer BCNU resulted in survival of 4.7% of MGMT+ clonogenic progenitors compared to 0.05% of LacZ+ progenitors. These studies indicate that MGMT-transduced human hematopoietic progenitors have increased resistance to nitrosoureas, and in a clinical transplant setting, this strategy may reduce alkylating agent myelosuppression.
骨髓抑制是亚硝基脲化疗的剂量限制性毒性，这是由于髓系前体细胞中DNA修复蛋白O6-烷基鸟嘌呤-DNA烷基转移酶水平较低所致。我们已经表明，高效髓性增殖性肉瘤病毒（vM5MGMT）介导的人MGMT cDNA转导至小鼠骨髓（BM）细胞中，会导致MGMT高表达，并在感染后及骨髓移植后立即在体外使祖细胞对1,3-双（2-氯乙基）亚硝基脲（BCNU）的抗性增加。这些实验旨在提高人造血祖细胞中的MGMT表达。通过免疫亲和柱（CEPRATE，CellPro公司）分离CD34(+) BM细胞，克隆形成祖细胞（集落形成单位粒细胞-巨噬细胞+爆式红系集落形成单位+集落形成单位粒细胞红系巨噬细胞=巨核细胞）平均富集66倍，祖细胞平均产率为58±11.5%，最终群体中54%为CD34(+)。与AM12-vM5MGMT逆转录病毒生产者共培养后，70%源自CD34(+)细胞的祖细胞被转导。vM5MGMT转导的祖细胞对30至50微米浓度的BCNU的抗性比同时转导LacZ的祖细胞高2倍以上（P<0.003）。用20微米BCNU对转导的、细胞因子刺激的CD34(+)细胞进行体外选择，结果MGMT+克隆形成祖细胞的存活率为4.7%，而LacZ+祖细胞为0.05%。这些研究表明，MGMT转导的人造血祖细胞对亚硝基脲的抗性增加，在临床移植环境中，这种策略可能会降低烷化剂引起的骨髓抑制。
