Liu Shuqian, Song Lifu, Bevins Robert, Birhiray Ogheneruemu, Moscow Jeffrey A
Department of Pediatrics, University of Kentucky, Lexington, KY 40536, USA.
Hum Gene Ther. 2002 Sep 20;13(14):1777-82. doi: 10.1089/104303402760293600.
Increased expression of the reduced folate carrier confers sensitivity to the antifolate drug methotrexate because it results in increased cellular uptake of this drug, and increased resistance to trimetrexate, a lipid-soluble antifolate drug, because it enables cells to take up exogenous folates that rescue cells from antifolate cytotoxicity. We therefore hypothesized that the reduced folate carrier could act as a suicide gene after treatment with methotrexate and as a selectable marker after exposure to trimetrexate. To test this hypothesis, we constructed replication-defective retroviruses containing the murine-reduced folate carrier (mRFC). Murine bone marrow cells transduced with the mRFC-containing retrovirus showed increased sensitivity to methotrexate and increased resistance to trimetrexate compared to empty vector-transduced controls in colony forming assays. Furthermore, colonies surviving trimetrexate and methotrexate treatment showed an enrichment of the mRFC gene after exposure to trimetrexate and a decrease after exposure to methotrexate. Lethally irradiated mice transplanted with bone marrow cells transduced with the mRFC-retrovirus and treated with the antifolate drugs after hematopoietic recovery demonstrated a relative increase in the number of cells containing the mRFC transgene after trimetrexate treatment and a decrease after methotrexate treatment. Therefore, these studies demonstrate the potential of the reduced folate carrier gene to play a dual role in gene therapy applications.
还原型叶酸载体的表达增加赋予了对抗叶酸药物甲氨蝶呤的敏感性，因为这导致该药物的细胞摄取增加；同时赋予了对三甲曲沙（一种脂溶性抗叶酸药物）的抗性增加，因为它使细胞能够摄取外源性叶酸，从而使细胞免受抗叶酸细胞毒性的影响。因此，我们推测还原型叶酸载体在用甲氨蝶呤治疗后可作为自杀基因，在接触三甲曲沙后可作为选择标记。为了验证这一假设，我们构建了含有小鼠还原型叶酸载体（mRFC）的复制缺陷型逆转录病毒。在集落形成试验中，与转导空载体的对照相比，用含mRFC的逆转录病毒转导的小鼠骨髓细胞对甲氨蝶呤的敏感性增加，对三甲曲沙的抗性增加。此外，在三甲曲沙和甲氨蝶呤处理后存活的集落在接触三甲曲沙后显示mRFC基因富集，在接触甲氨蝶呤后减少。用mRFC逆转录病毒转导的骨髓细胞移植并在造血恢复后用抗叶酸药物治疗的致死性照射小鼠，在三甲曲沙治疗后含有mRFC转基因的细胞数量相对增加，在甲氨蝶呤治疗后减少。因此，这些研究证明了还原型叶酸载体基因在基因治疗应用中发挥双重作用的潜力。
