McGrath T, Latoud C, Arnold S T, Safa A R, Felsted R L, Center M S
Division of Biology, Kansas State University, Manhattan 66506.
Biochem Pharmacol. 1989 Oct 15;38(20):3611-9. doi: 10.1016/0006-2952(89)90134-2.
HL60 cells isolated for resistance to Adriamycin do not contain P-glycoprotein, as determined with immunological probes. These cells, however, are multidrug resistant and defective in the cellular accumulation of drug. In view of these findings, we have examined in greater detail certain properties of the HL60/Adr cells and have compared these properties to an HL60 drug-resistant isolate (HL60/Vinc) which contains high levels of P-glycoprotein. The results of these studies demonstrated that verapamil induces a major increase in cellular drug accumulation in both HL60/Adr and HL60/Vinc isolates. An 125I-labeled photoaffinity analog of verapamil labeled P-glycoprotein contained in membranes of HL60/Vinc cells. In contrast, this agent did not label any protein selectively associated with drug resistance in membranes of the HL60/Adr isolate. The photoactive dihydropyridine calcium channel blocker [3H]azidopine and [125I]NASV, a photoaffinity analog of vinblastine, labelled P-glycoprotein in membranes from HL60/Vinc cells, whereas in experiments with the HL60/Adr isolate there was no detectable labeling of a drug resistance associated membrane protein. Additional studies have been carried out to analyze membrane proteins of HL60/Adr cells labeled with the photoaffinity agent 8-azido-alpha-[32P]ATP (AzATP32). The results demonstrate that this agent labeled a resistance associated membrane protein of 190 kilodaltons (P190). P190 is essentially absent in membranes of drug-sensitive cells. Labeling of P190 with AzATP32 in membranes of resistant cells was blocked completely when incubations were carried out in the presence of excess unlabeled ATP. Additional studies were carried out to analyze mdr gene amplification and expression in sensitive and resistant cells. Experiments carried out with human 5',mdr1 (1.1 kb) and mdr3 (1.0 kb) cDNAs demonstrate that both of these sequences were highly amplified in the HL60/Vinc isolate. Only the mrd1 gene sequence however, was overexpressed. In contrast, there was no detectable amplification or overexpression of mdr1 or mdr3 sequences in HL60/Adr cells. The results of this study thus identify a new nucleotide binding protein which is overexpressed in membranes of HL60 cells isolated for resistance to Adriamycin. P190, which exhibits properties distinct from P-glycoprotein, possibly functions in the energy-dependent drug efflux system contained in the HL60/Adr resistant isolate.
用免疫探针测定发现，分离出的对阿霉素耐药的HL60细胞不含有P - 糖蛋白。然而，这些细胞具有多药耐药性且在药物的细胞蓄积方面存在缺陷。鉴于这些发现，我们更详细地研究了HL60/Adr细胞的某些特性，并将这些特性与含有高水平P - 糖蛋白的HL60耐药分离株（HL60/Vinc）进行了比较。这些研究结果表明，维拉帕米可使HL60/Adr和HL60/Vinc分离株中的细胞药物蓄积大幅增加。维拉帕米的一种125I标记的光亲和类似物标记了HL60/Vinc细胞膜中所含的P - 糖蛋白。相比之下，该试剂未标记HL60/Adr分离株细胞膜中与耐药性选择性相关的任何蛋白质。光活性二氢吡啶钙通道阻滞剂[3H]叠氮平以及长春碱的光亲和类似物[125I]NASV标记了HL60/Vinc细胞膜中的P - 糖蛋白，而在对HL60/Adr分离株进行的实验中，未检测到与耐药性相关的膜蛋白有标记。已开展了更多研究来分析用8 - 叠氮基 - α - [32P]ATP（AzATP32）这种光亲和试剂标记的HL60/Adr细胞的膜蛋白。结果表明，该试剂标记了一种190千道尔顿的与耐药性相关的膜蛋白（P190）。在药物敏感细胞的膜中基本不存在P190。当在过量未标记ATP存在的情况下进行孵育时，耐药细胞的膜中用AzATP32对P190的标记被完全阻断。还开展了更多研究来分析敏感细胞和耐药细胞中mdr基因的扩增及表达情况。用人5'，mdr1（1.1 kb）和mdr3（1.0 kb）cDNA进行的实验表明，这两个序列在HL60/Vinc分离株中均高度扩增。然而，只有mrd1基因序列过度表达。相比之下，在HL60/Adr细胞中未检测到mdr1或mdr3序列的扩增或过度表达。因此，本研究结果鉴定出一种新的核苷酸结合蛋白，它在为抵抗阿霉素而分离出的HL60细胞的膜中过度表达。P190表现出与P - 糖蛋白不同的特性，可能在HL60/Adr耐药分离株所含的能量依赖性药物外排系统中发挥作用。
