Sequential emergence ofMRP- andMDR1-gene over-expression as well asMDR1-gene translocation in homoharringtonine-selected K562 human leukemia cell lines

Abstract

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.