Zhou D C, Ramond S, Viguie F, Faussat A M, Zittoun R, Marie J P
Laboratoire de Cinétique et de Cultures Cellulaires, Formation de Recherche Associée Claude Bernard, Paris, France.
Int J Cancer. 1996 Jan 26;65(3):365-71. doi: 10.1002/(SICI)1097-0215(19960126)65:3<365::AID-IJC15>3.0.CO;2-9.
To investigate the mechanism of resistance to an antineoplastic natural product homoharringtonine (HHT) in leukemic cells, we have established 5 sub-lines of human myeloid leukemia K562 cells, designated as K-H30, K-H100, K-H200, K-H300 and K-H400, which showed progressive resistance to different concentrations of HHT. These sub-lines were cross-resistant to daunorubicin, vincristine, etoposide and mitoxantrone, but not to melphalan. Immunofluorescence with monoclonal anti-Pgp antibody MRK16 and Northern-blot analysis demonstrated that resistance to HHT is related to the sequential emergence of MRP- and MDR1-gene over-expression. In the low-level-resistant K-H30 sub-line, the MDR1 gene was not over-expressed, but the MRP gene was over-expressed 2.1-fold. In the intermediate-level-resistant K-H100 and K-H200 sublines, both the MRP and the MDR1 genes were over-expressed. However, in the high-level-resistant K-H300 and K-H400 sublines, MDR1-gene over-expression predominated (20- and 21-fold respectively). On the other hand, GST pi-gene expression was decreased in all 5 sub-lines. Southern-blot analysis revealed no MRP-gene amplification in any of the 5 sub-lines, whereas the MDR1 gene was amplified in the high-level-resistant K-H300 and K-H400 sub-lines. The most interesting observation is a homogeneously staining region (HSR) found in chromosome 2 of the K-H300 and K-H400 sub-lines. Chromosome painting and in situ hybridization demonstrated that this HSR was translocated from chromosome 7 and consisted of the amplified MDR1 gene, suggesting that there is a relationship between MDR1-gene, translocation and MDR1-gene amplification.
为研究白血病细胞对抗肿瘤天然产物高三尖杉酯碱（HHT）耐药的机制，我们建立了5个人髓性白血病K562细胞亚系，命名为K-H30、K-H100、K-H200、K-H300和K-H400，它们对不同浓度的HHT呈现出逐渐增强的耐药性。这些亚系对柔红霉素、长春新碱、依托泊苷和米托蒽醌具有交叉耐药性，但对美法仑不耐药。用单克隆抗Pgp抗体MRK16进行免疫荧光和Northern印迹分析表明，对HHT的耐药性与MRP和MDR1基因的顺序过表达有关。在低水平耐药的K-H30亚系中，MDR1基因未过表达，但MRP基因过表达2.1倍。在中等水平耐药的K-H100和K-H200亚系中，MRP和MDR1基因均过表达。然而，在高水平耐药的K-H300和K-H400亚系中，MDR1基因的过表达占主导（分别为20倍和21倍）。另一方面，所有5个亚系中GST pi基因的表达均降低。Southern印迹分析显示，5个亚系中均未检测到MRP基因扩增，而在高水平耐药的K-H300和K-H400亚系中检测到MDR1基因扩增。最有趣的发现是在K-H300和K-H400亚系的2号染色体上发现了一个均匀染色区（HSR）。染色体描绘和原位杂交表明，这个HSR是从7号染色体易位而来的，由扩增的MDR1基因组成，提示MDR1基因、易位和MDR1基因扩增之间存在关联。
