Capiaux Gina M, Budak-Alpdogan Tulin, Takebe Naoko, Mayer-Kuckuk Philipp, Banerjee Debabrata, Maley Frank, Bertino Joseph R
Graduate School of Medical Sciences, Cornell University, New York, NY 10021, USA.
Hum Gene Ther. 2003 Mar 20;14(5):435-46. doi: 10.1089/104303403321467207.
Gene transfer-based myeloprotection strategies against chemotherapy require the development of effective drug resistance genes or gene combinations. Our laboratory has previously generated drug-resistant mutants of dihydrofolate reductase (DHFR F/S) and thymidylate synthase (TS G52S) for myeloprotection against methotrexate (MTX) and 5-fluorouracil (5-FU), respectively. For the purpose of conferring dual myeloprotection against both MTX and 5-FU, we have generated two retroviral constructs encoding both DHFR F/S and TS G52S as a fusion protein (DHFR F/S-TS G52S) or as individual proteins from a bicistronic gene. The DHFR F/S-TS G52S fusion protein is functional and exhibits kinetic properties similar to that of the individual mutant enzymes. NIH 3T3 cells and mouse bone marrow progenitors retrovirally transduced with the fusion DHFR F/S-TS G52S cDNA provided similar levels of resistance to MTX and 5-FU as cells expressing the individual mutant enzymes and higher levels of resistance to MTX than cells expressing DHFR F/S from the 3' end of a bicistronic gene. As MTX and 5-FU are used in combination therapy for diseases such as breast and colon cancer, this fusion gene may be useful in the clinic to reduce myelosuppressive toxicity associated with this drug combination.
基于基因转移的针对化疗的骨髓保护策略需要开发有效的耐药基因或基因组合。我们实验室之前分别生成了二氢叶酸还原酶（DHFR F/S）和胸苷酸合成酶（TS G52S）的耐药突变体，用于分别抵抗甲氨蝶呤（MTX）和5-氟尿嘧啶（5-FU）对骨髓的抑制作用。为了赋予对MTX和5-FU的双重骨髓保护作用，我们构建了两种逆转录病毒载体，一种编码DHFR F/S和TS G52S作为融合蛋白（DHFR F/S-TS G52S），另一种从双顺反子基因编码这两种蛋白作为单独的蛋白质。DHFR F/S-TS G52S融合蛋白具有功能，并且表现出与单个突变酶相似的动力学特性。用融合的DHFR F/S-TS G52S cDNA进行逆转录病毒转导的NIH 3T3细胞和小鼠骨髓祖细胞，对MTX和5-FU的耐药水平与表达单个突变酶的细胞相似，并且对MTX的耐药水平高于从双顺反子基因3'端表达DHFR F/S的细胞。由于MTX和5-FU联合用于治疗乳腺癌和结肠癌等疾病，这种融合基因可能在临床上有助于降低与这种药物组合相关的骨髓抑制毒性。
