WTH3, which Encodes a Small G Protein, Is Differentially Regulated in Multidrug-Resistant and Sensitive MCF7 Cells

Abstract

The WTH3 gene's biological characteristics and relationship to multidrug resistance (MDR) were investigated further. Results showed that WTH3 was mainly located in the cytosol and capable of binding to GTP. In addition, WTH3's promoter function was significantly attenuated in MDR (MFC7/AdrR) relative to non-MDR (MCF7/WT) cells. Advanced analyses indicated that two mechanisms could be involved in WTH3's down-regulation: DNA methylation and trans-element modulations. It was found that the 5Vend portion of a CpG island in WTH3's promoter was hypermethylated in MCF7/AdrR but not MCF7/WT cells, which could have a negative effect on the WTH3 promoter. This idea was supported by the observation that a 45-bp sequence (DMR45) in this differentially methylated region positively influenced promoter activity. We also discovered that different nuclear proteins in MCF7/AdrR and MCF7/WT cells bound to methylated or nonmethylated DMR45. Moreover, a sequence containing a unique repeat that was also a positive cis-element for the promoter was attached by different transcription factors depending on whether they were prepared from MCF7/AdrR or MCF7/WT cells. These molecular changes, apparently induced by drug treatment, resulted in WTH3's down regulation in MDR cells. Therefore, present studies support previous observations that WTH3, as a negative regulator, participates in MDR development in MCF7/AdrR cells. (Cancer Res 2005; 65(16): 7421-8)

WTH3 is a direct target of the p53 protein

“…Earlier studies suggested that it was a negative regulator for MDR development (Shan et al, 2002a;Tian et al, 2005a, b), which made it extremely interesting as most MDRrelated genes discovered so far exert a positive effect. To understand the mechanisms involved in its downregulation in MDR cells the gene's promoter was identified and analysed (Tian et al, 2005b). We found that it was differentially regulated in MCF7/AdrR and MCF7/WT cells.…”

“…Detailed information about generating the pcDNA3.1/WTH3 and pGL/WTH3P constructs containing the coding region and WTH3 promoter, respectively, were described previously (Shan et al, 2002a;Tian et al, 2005b). Six deletion mutants of the WTH3 promoter were created by polymerase chain reaction (PCR) amplification using pGL/WTH3P as the template.…”

“…A total of 10 mg nuclear extract and 50 ng of p53 protein were applied to perform EMSAs. The detailed procedure for carrying out EMSAs was described previously (Tian et al, 2005b). Super-EMSA experiments were performed in the presence or absence of 1 mg of monoclonal p53 antibody (p53-(DO)-1, Santa Cruz) for 30 min at room temperature and analysed on a 4% nondenaturing-PAGE gel.…”

“…The gene product is homologous to the Rab6 and Rab6c genes that belong to the ras super family and encode small G proteins (Zahraoui et al, 1989;Goud et al, 1990;Echard et al, 1998Echard et al, , 2000Shan et al, 2000Shan et al, , 2002a. Similar to the Rab6s, WTH3 is a housekeeping gene and its product is capable of binding to GTP molecules (Tian et al, 2005b). However, unlike the Rab6s that reside in the Golgi network, most of WTH3 locates in the cytoplasm and to a less degree in the nuclei.…”

“…This disparity could be due to WTH3's lack of a cysteine at its C terminus for geranyl -geranylation, a necessary posttranslational modification for membrane attachment (Barbacid, 1987). Previous studies found that the WTH3 gene was downregulated in MDR cell lines, MCF7/AdrR and MES-SA/Dx5 (a human uterine sarcoma cell line), and by introducing it back into those lines caused downregulation of MDR1 gene expression and reversed their MDR phenotypes to various anticancer drugs (Shan et al, 2002a;Tian et al, 2005b). In addition, our research also revealed that hypermethylation (an epigenetic modification event in mammals) of the WTH3 promoter and transcription factor modulation were involved in its differential expression in MCF7/ AdrR vs MCF7/WT cells (Tian et al, 2005b).…”