Transcription of WTH3, a member of the Rab6 gene family, is suppressed in the multidrug resistant breast cancer cell line, MCF7/AdrR

Abstract

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The WTH3 gene was discovered by employing the Methylation Sensitive-Representational Difference Analysis (MS-RDA) technique to study DNA hypermethylation events in the drug sensitive cell line, MCF7/WT, as compared to its drug resistant counterpart, MCF7/AdrR. Similar to Rab6 and Rab6c, WTH3 binds to GTP. However, differing from those homologs that reside in the Golgi network, WTH3 is located mainly in the cytoplasm. Characterization of the WTH3 gene showed that its expression was down regulated in MCF7/AdrR and MES-SA/Dx5 (a human MDR uterine sarcoma cell line). Introducing the WTH3 gene into MDR cell lines reversed the MDR phenotype to various anti-cancer drugs. These results indicated that WTH3 could play an important role in MDR development. To understand the mechanisms that led to differential expression of WTH3 in MCF7/WT and MCF7/AdrR cell lines, its promoter region was analyzed. The PCR amplified promoter was cloned into the pGL3 luciferase reporter vector and its activity in MDR and non-MDR cell lines were measured. Analyses of the full length promoter showed 5 fold reduction of luciferase expression in the MCF7/AdrR cell line compared to the parental MCF7/WT, mimicking the in vivo expression of WTH3. Also, based on PATCH analysis for transcription protein binding sites, two deletion mutants of the wild type promoter were generated. One deletion was generated in a 28 bps repeat (RR28), while the other one was in the RRSp1 region (RR28 plus an adjacent Sp1 motif). omotll lenght The luciferase assays showed that both deleted sequences exerted a positive influence on the promoter’s function. Furthermore, these two cis-elements were subject to gel shift assays where MCF7/AdrR and MCF7/WT nuclear extracts were used. The results showed that different binding patterns with diverse quantities were created by the two extracts. It thus seemed that the drug treatment changed the transcriptional factors, in quantity and quality that drive the expression of WTH3 gene in MCF7/AdrR cells. Investigation of the possible characteristics of those DNA binding proteins is currently under way. Clearly, identification of those regulators could help us understand the role played by the WTH3 gene in MDR development, which may, in turn, provide future treatments of MDR in breast cancer.