Galipeau J, Benaim E, Spencer H T, Blakley R L, Sorrentino B P
Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
Hum Gene Ther. 1997 Oct 10;8(15):1773-83. doi: 10.1089/hum.1997.8.15-1773.
Chemoresistance gene transfer is an experimental method to protect hematopoietic cells from the toxicity of anticancer drugs. Because multiple drugs are usually given together in cancer therapy, this strategy will ultimately require vectors expressing multiple chemoresistance genes. For this reason, we designed a bicistronic retroviral vector (HaMID) containing a modified human multidrug resistance-1 cDNA and a mutant human dihydrofolate reductase cDNA bearing a leucine to tyrosine substitution at codon 22 (L22Y). To determine if this vector would confer dual drug resistance to hematopoietic cells, recombinant retrovirus was used to transduce the human CEM T lymphoblastic cell line as well as primary murine myeloid progenitors. Growth suppression assays, using polyclonal transduced CEM cells, demonstrated increased resistance to taxol (13-fold), trimetrexate (8.9-fold), vinblastine (5.6-fold), methotrexate (2.5-fold), and etoposide (1.5-fold) when used as single agents. HaMID-transduced cells also grew at a logarithmic rate in the simultaneous presence of 25 nM taxol and 100 nM trimetrexate while control cells were entirely growth inhibited by this drug combination. Similarly, HaMID-transduced murine myeloid progenitors acquired increased resistance to taxol (2.9-fold) and trimetrexate (140-fold), and were able to form colonies in the simultaneous presence of both drugs. Our results suggest that retroviral transfer of HaMID into primary hematopoietic cells should reduce the myelosuppression associated with the combined use of antifolates and P-glycoprotein-effluxed drugs.
化学抗性基因转移是一种保护造血细胞免受抗癌药物毒性影响的实验方法。由于在癌症治疗中通常会联合使用多种药物，因此该策略最终将需要表达多种化学抗性基因的载体。出于这个原因，我们设计了一种双顺反子逆转录病毒载体（HaMID），它包含一个修饰的人类多药耐药-1 cDNA和一个在密码子22处由亮氨酸替换为酪氨酸的突变型人类二氢叶酸还原酶cDNA（L22Y）。为了确定该载体是否能赋予造血细胞双重耐药性，重组逆转录病毒被用于转导人CEM T淋巴细胞系以及原代小鼠骨髓祖细胞。使用多克隆转导的CEM细胞进行的生长抑制试验表明，当作为单一药物使用时，对紫杉醇（13倍）、三甲曲沙（8.9倍）、长春碱（5.6倍）、甲氨蝶呤（2.5倍）和依托泊苷（1.5倍）的耐药性增加。当同时存在25 nM紫杉醇和100 nM三甲曲沙时，HaMID转导的细胞也以对数速率生长，而对照细胞则被这种药物组合完全抑制生长。同样，HaMID转导的小鼠骨髓祖细胞对紫杉醇（2.9倍）和三甲曲沙（140倍）的耐药性增加，并且在两种药物同时存在的情况下能够形成集落。我们的结果表明，将HaMID逆转录病毒转移到原代造血细胞中应能减少与联合使用抗叶酸药物和P-糖蛋白外排药物相关的骨髓抑制。
