Zhu Q, Sun H, Center M S
Division of Biology, Kansas State University, Manhattan 66506, USA.
Oncol Res. 1997;9(5):229-36.
HeLa cells were transfected with full-length multidrug resistance protein (MRP) cDNA and with MRP cDNAs that had been mutated at certain nucleotide binding domains. Stable transfectants were isolated and those producing equivalent amounts of P190 were tested in cytotoxicity assays using a variety of chemotherapeutic agents. The results demonstrate that deletions in the C-motif of NBD1 or the A-motif of NBD2 have a pronounced effect in reducing resistance levels to adriamycin, vincristine, or etoposide (VP-16). Single-site mutations of lysine in these same motifs reduce IC50 values but less than that observed with the deletion mutants. Additional studies have demonstrated an increase in drug accumulation and reduction in drug efflux in NBD deletion and single-site mutants. The results of this study therefore identify two lysines of the NBD A- and C-motifs that are critical for MRP-mediated multidrug resistance. The results also provide definitive evidence that resistance occurring as a result of MRP overexpression is related to enhanced levels of an ATP-dependent efflux pump.
将全长多药耐药蛋白（MRP）cDNA以及在某些核苷酸结合结构域发生突变的MRP cDNA转染到人宫颈癌细胞系（HeLa细胞）中。分离出稳定转染子，并使用多种化疗药物在细胞毒性试验中对产生等量P190的转染子进行测试。结果表明，核苷结合域1（NBD1）的C-基序或核苷结合域2（NBD2）的A-基序缺失对降低对阿霉素、长春新碱或依托泊苷（VP-16）的耐药水平有显著影响。这些相同基序中赖氨酸的单点突变会降低半数抑制浓度（IC50）值，但降低程度小于缺失突变体。进一步的研究表明，NBD缺失和单点突变体中药物积累增加，药物外排减少。因此，本研究结果确定了NBD A-基序和C-基序中的两个赖氨酸对于MRP介导的多药耐药至关重要。结果还提供了确凿证据，表明MRP过表达导致的耐药与ATP依赖性外排泵水平升高有关。
