Molecular mechanisms of drug resistance in K562 multidrug resistant leukaemic cell lines

Publication Type:
Thesis
Issue Date:
2005
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A major problem in chemotherapy is that many cancers are intrinsically drug resistant or later become resistant. Resistance to one drug is often accompanied by cross-resistance to many unrelated drugs and this is known as multidrug resistance (MDR). MDR is commonly associated with a 170kDa glycoprotein called P-glycoprotein (P-gp), which is thought to act as an ATP-dependant drug efflux pump and which is encoded for by the MDRI gene in humans. MDRI transcription can be initiated by various mechanisms such as demethylation of MDR1 promoter sequences or translocation of the MDR1 gene. In cells already expressing P-gp this expression can be increased by mechanisms such as amplification of the MDRI gene copy number or an increase in the rate of mRNA translation or stability. However the focus of many studies is on the regulation of MDR1 transcription through the binding of transcription factors to specific sequences in the MDR1 promoter region, in particular an inverted CCAAT element known as the Y-box and the -55GC box. The current study investigated the mechanisms for MDR in a series of K562 derived MDR cell lines demonstrating varying levels of low level MDR. Levels of resistance of each of the K562 MDR cell lines were all confirmed by performing vinblastine and paclitaxel cytotoxicity assays on the cell lines. Northern hybridisation of total RNA isolated from K562 MDR cells indicated a positive correlation existed between the level of MDR1 mRNA expressed in the MDR cells and the level of MDR displayed by the cells. Southern blot hybridisation of DNA from the cells with an MDRI probe indicated that increase of MDR1 mRNA in the K562 MDR cells was not due to amplification of the MDR1 gene. Bisulphite genomic sequencing of K562 and MDR cell lines revealed the MDRI promoter in both cell lines to be almost completely unmethylated apart from two distinct sites of methylation in K562 cells at two CpG sites downstream of the transcription start at +421 and +423bp respectively. Treatment of the K562 cells with the histone deacetylase inhibitor Trichostatin A (TSA) increased the cells resistance to epirubicin, however no effect was seen upon TSA treatment of the K562 MDR cells. This suggests there is a difference in the chromatin structure in the two cell lines. It was further demonstrated that resistance levels of K562 derived MDR cell lines declined with increasing time in drug free culture, but could be restimulated by short term drug exposure to P-gp substrate drugs, in some cases in as little as after 4 hours exposure, suggesting a transcriptional mechanism of MDRI upregulation is likely to be responsible for the induction. Consistent with this, mRNA stability studies indicated that the drug induction of K562 MDR cells that resulted in increased MDR and MDR1 mRNA levels was not mediated by an increase in the stability of MDR1 mRNA, as the rate of mRNA decay was the same in treated and untreated control cells. However, electrophoretic mobility shift assays (EMSA) of the two major transcription factors involved in MDR1 regulation, the Y-box binding protein (identified as NF-Y) and the - 55GC box binding protein indicated no difference in nuclear levels of these two proteins in untreated and drug induced cells. Thus the mechanism for up-regulation of MDR1 activity in K.562 MDR cell lines is most likely due to activation via alteration of other factors or via changes in chromatin structure regulating accessibility of transcription factors.
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