Methylation of WTH3, a possible drug resistant gene, inhibits p53 regulated expression.
BACKGROUND
Previous results showed that over-expression of the WTH3 gene in MDR cells reduced MDR1 gene expression and converted their resistance to sensitivity to various anticancer drugs. In addition, the WTH3 gene promoter was hypermethylated in the MCF7/AdrR cell line and primary drug resistant breast cancer epithelial cells. WTH3 was also found to be directly targeted and up regulated by the p53 gene. Furthermore, over expression of the WTH3 gene promoted the apoptotic phenotype in various host cells.


METHODS
To further confirm WTH3's drug resistant related characteristics, we recently employed the small hairpin RNA (shRNA) strategy to knockdown its expression in HEK293 cells. In addition, since the WTH3 promoter's p53-binding site was located in a CpG island that was targeted by methylation, we were interested in testing the possible effect this epigenetic modification had on the p53 transcription factor relative to WTH3 expression. To do so, the in vitro methylation method was utilized to examine the p53 transgene's influence on either the methylated or non-methylated WTH3 promoter.


RESULTS
The results generated from the gene knockdown strategy showed that reduction of WTH3 expression increased MDR1 expression and elevated resistance to Doxorubicin as compared to the original control cells. Data produced from the methylation studies demonstrated that DNA methylation adversely affected the positive impact of p53 on WTH3 promoter activity.


CONCLUSIONS
Taken together, our studies provided further evidence that WTH3 played an important role in MDR development and revealed one of its transcription regulatory mechanisms, DNA methylation, which antagonized p53's positive impact on WTH3 expression.
Tian
Kegui
K
Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, NY 11794, USA. ktian@ms.cc.sunysb.edu
Wang
Yuezeng
Y
Huang
Yu
Y
Sun
Boqiao
B
Li
Yuxin
Y
Xu
Haopeng
H
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism*; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D045744:Cell Line, Tumor; D019175:DNA Methylation; D000077209:Decitabine; D004317:Doxorubicin / Q000494:pharmacology; D018432:Drug Resistance, Multiple / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques; D016158:Genes, p53 / Q000235:genetics*; D006801:Humans; D011401:Promoter Regions, Genetic / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D019076:Transgenes / Q000235:genetics*; D020691:rab GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D034741:RNA, Small Interfering; D000077209:Decitabine; D004317:Doxorubicin; C417211:RAB6C protein, human; D020691:rab GTP-Binding Proteins; D001374:Azacitidine