P-glycoprotein (MDR1) Expression in Leukemic Cells Is Regulated at Two Distinct Steps, mRNA Stabilization and Translational Initiation

Abstract

Multidrug resistance in acute myeloid leukemia is often conferred by overexpression of P-glycoprotein, encoded by the MDR1 gene. We have characterized the key regulatory steps in the development of multidrug resistance in K562 myelogenous leukemic cells. Unexpectedly, up-regulation of MDR1 levels was not due to transcriptional activation but was achieved at two distinct posttranscriptional steps, mRNA turnover and translational regulation. The short-lived (half-life 1 h) MDR1 mRNA of naïve cells (not exposed to drugs) was stabilized (halflife greater than 10 h) following short-term drug exposure. However, this stabilized mRNA was not associated with translating polyribosomes and did not direct Pglycoprotein synthesis. Selection for drug resistance, by long-term exposure to drug, led to resistant lines in which the translational block was overcome such that the stabilized mRNA was translated and P-glycoprotein expressed. The absence of a correlation between steadystate MDR1 mRNA and P-glycoprotein levels was not restricted to K562 cells but was found in other lymphoid cell lines. These findings have implications for the avoidance or reversal of multidrug resistance in the clinic. MDR1 is the most common impediment to successful chemotherapy for a variety of cancers (1). The most frequent form of drug resistance in relapsed acute leukemia is overexpression of P-glycoprotein (2, 3). P-glycoprotein is a member of the ATPbinding cassette superfamily of active transporters and functions as an energy-dependent efflux pump that reduces the intracellular concentration of cytotoxic compounds and, hence, their toxicity. P-glycoprotein has a broad substrate specificity and can confer resistance to a wide range of different cytotoxic compounds (4).Most pre-clinical and clinical efforts to overcome MDR aim to modulate P-glycoprotein activity. However, clinical trials of compounds that inhibit P-glycoprotein activity have had limited success and led to adverse pharmacokinetic side effects (1). It may, therefore, be more appropriate to target MDR1 expression. Indeed, MDR1 transcription has been targeted with Ecteinascidin 743 in pre-clinical studies (5) and more recently by modulation of the nuclear receptor SXR (6). Strategies involving antisense and transcriptional decoy (7) and the use of anti-MDR1 mRNA hammerhead ribozymes have also been suggested (8).Stresses such as short-term exposure to cytotoxic drugs results in the up-regulation of MDR1 mRNA levels in many cell lines (9 -13) and in human metastatic sarcomas in vivo (14). This is frequently due to transcriptional activation of the MDR1 gene and has been reported in many cell lines after different physical and chemical stimulations and in cells selected for resistance to a variety of cytotoxic drugs (5,9,10,15,16). In cell lines selected for drug resistance, increased MDR1 gene expression is also the result of amplification of the MDR1 locus and the appearance of self-replicating episomes (4). Gene rearrangements that constitutively activate MDR1 transcription have also...

The histone deacetylase inhibitor trichostatin A downregulates human MDR1 (ABCB1) gene expression by a transcription-dependent mechanism in a drug-resistant small cell lung carcinoma cell line model

“…Simultaneous treatments with the transcription inhibitor ActD suggested that these up-and downregulations occurred at the transcriptional level without any change in mRNA stability as reported in leukaemic cells (Yague et al, 2003). De novo protein synthesis inhibition with CHX did not suppress TSA-mediated ABCB1 modulations, suggesting that TSA influence mainly this gene transcription directly at the chromatin level.…”

The temporal relationship between ABCB1 promoter hypomethylation, ABCB1 expression and acquisition of drug resistance

“…Despite overexpression of ABCB1 mRNA, two independent studies identified a block in the translation of ABCB1 mRNA within K562 cells. 18,19 Although the possibility exists within our MCF-7 cell lines that all transcripts produced are not translated into protein, the high Abcb1 protein expression levels within MCF-7 EPI and MCF-7 TAX-2 indicates that a translational blockade was not present in these drug-resistant cell lines. Gene amplification is also a potential mechanism for increased ABCB1 transcription, given previously published findings using a variety of drug-resistant cancer cell lines.…”

“…A region with strong hypomethylation induced by selection for drug resistance spans CpG sites 49-66. Shared valleys of hypomethylation in drug-resistant and Azatreated cells span CpG sites [19][20][21][22], which correspond to the consensus Inr site, a site adjacent to the transcription start site (Table 3), and CpG sites 32-44 implicated in the regulation of ABCB1 expression by p53 (Table 4). Aza treatment did not result in dramatic reduction in the methylation of CpG sites 49-66 (Figure 5f).…”

Hypoxia upregulates hypoxia inducible factor (HIF)-3α expression in lung epithelial cells: characterization and comparison with HIF-1α

“…Amplification and detection were carried out by using an ABI PRISM 7700 detection system (Applied Biosystems) as fol- Results were normalized to the housekeeping gene β-actin for the study of transcriptional activity and mRNA half-life. To compare the expression levels among different samples, the relative expression of mRNA levels was calculated using the comparative delta C T (threshold cycle number) method as described previously [29]. Briefly, the following formula was used: 2 -DDC T , where DC T is the difference in C T between the gene of interest and β-actin, and DDC T for the sample=DC T for the actual sample-DC T of the lowest expression sample.…”