The role of intron 1 in MDR1 regulation in drug resistant cancer cells

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Multidrug resistance (MDR) is a major limitation in the successful treatment of cancer using chemotherapy. Cancers can be intrinsically resistant or develop resistance during treatment to a variety of structurally and functional unrelated drugs. Numerous cellular mechanisms which contribute to MDR have been identified, however, the most common mechanism is the over expression of a 170kDa glycoprotein referred to as P-glycoprotein (P-gp). P-gp acts as an ATP-dependant drug efflux pump and is encoded by the MDRI gene. An understanding of the regulation of the MDRI gene is essential if the clinical impact of MDR is to be reduced. However, at this stage how this gene is regulated in vivo remains elusive. Amplification of the MDRI gene or an increase in mRNA stability may increase expression in cells already expressing P-gp, though they do not account for activation and/or regulation of the MDRI gene itself. Hence the focus of many studies is on the regulation of MDRI transcription. The current study investigated the role of intron 1 in MDRI promoter regulation. The intron 1 region has been shown to contain a CpG island which is differentially methylated in the drug sensitive HL60 cells and completely unmethylated in MDR H/E8 cells. In vitro DNA:protein interactions were found to exist within this differentially methylated region. Thus the aim of the current study was to determine the functional role of the downstream CpG island in MDRI regulation in cells of varying origin and varying MDR levels. The MDR cells lines studied were the HL60 derived HlE8 leukaemic cell line, the K562 derived Kepru leukaemic cell line, the KB-3-J derived KB-8-5 cervical cancer cell lines and the Lo Vo and L/ ADR colon cancer cell lines. Reporter gene studies indicated an overall negative role in reporter activity for the +283 to +606bp region in the leukaemic MDR cell lines, whilst the same region was shown to increase reporter activity in the KB-8-5 cells. The colon LoVo and L/ADR cells also showed an increase in activity for the +283 to +606bp, however, the increase was not found to be significant. Further subdivision of the +283 to +606bp region suggested the existence of several different potential and repressor and enhancer regulatory elements in the H/E8, Kepru and KB-8-5 MDR cell lines. Similarly, reporter gene functional studies of the +65 to +282bp region resulted in an overall inhibitory effect in reporter gene activity in the HlE8 cells and a stimulatory effect in the KB-8-5, LoVo and L/ADR MDR cell lines. Further subdivision of the +65 to +282bp region revealed several different potential repressor and enhancer elements in the cell lines tested . This region had not been previously investigated for DNA:protein interactions. UV in vivo footprinting was used to investigate in vivo chromatin structure and potential DNA:protein interactions in intron 1 of the MDRI gene. Sites of hypersensitivity and protection were found in intron 1 in all cell lines, however, it is hypothesised that some of the sites observed relate directly to chromatin structure as opposed to specific transcription factor DNA bind ing. For example a region of concentrated protection, possibly due to a positioned nucleosome was fo und to exist in the drug sensitive cells, whilst this same region was less and less protected in the more drug resistant cells. Furthermore, an increase in the number of hypersensitive sites was observed in the more drug resistant cell lines which may be due to RNA polymerase II pausing. However, many of the protected sites were also found throughout the intron I region, including some with the previously observed in vitro DNA footprints, providing further evidence that intron 1 is involved in the regulation of the MDRI gene. A comparison of the locations of the in vivo footprints with those of transcription factor consensus binding sites suggests many possible candidates for intron 1 mediated MDRI regulation that can be followed up in future studies. This study is the first to provide in vivo evidence for specific and structural features and possible transcription factor mediated regulation in intron 1 of the MDRI gene.
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