Epigenetic Regulation of Organic Anion Transporting Polypeptide 1B3 in Cancer Cell Lines
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The expression of a multispecific organic anion transporter, OATP1B3/SLCO1B3, is associated with clinical prognosis and survival of cancer cells. The aims of present study were to investigate the involvement of epigenetic regulation in mRNA expression of a cancer-type variant of OATP1B3 (Ct-OATP1B3) in cancer cell lines.
The membrane localization and transport functions of Ct-OATP1B3 were investigated in HEK293 cells transiently expressing Ct-OATP1B3. DNA methylation profiles around the transcriptional start site of Ct-OATP1B3 in cancer cell lines were determined. The effects of a DNA methyltransferase inhibitor and siRNA knockdown of methyl-DNA binding proteins (MBDs) on the expression of Ct-OATP1B3 mRNA were investigated.
5′-RACE identified the TSS of Ct-OATP1B3 in PK-8 cells. Ct-OATP1B3 was localized on the plasma membrane, and showed the transport activities of E217βG, fluvastatin, rifampicin, and Gd-EOB-DTPA. The CpG dinucleotides were hypomethylated in Ct-OATP1B3-positive cell lines (DLD-1, TFK-1, PK-8, and PK-45P) but were hypermethylated in Ct-OATP1B3-negative cell lines (HepG2 and Caco-2). Treatment with a DNA methyltransferase inhibitor and siRNA knockdown of MBD2 significantly increased the expression of Ct-OATP1B3 mRNA in HepG2 and Caco-2.
Ct-OATP1B3 is capable of transporting its substrates into cancer cells. Its mRNA expression is regulated by DNA methylation-dependent gene silencing involving MBD2.
KEY WORDScancer cell line DNA methylation drug transporter epigenetics OATP1B3
5′-rapid amplification cDNA ends
gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid
inductively coupled plasma mass spectrometry
liquid chromatography-tandem mass spectrometry
methyl-DNA binding protein
organic anion transporting polypeptide
open reading frame
polymerase chain reaction
tissue-dependent differentially methylated region
transcriptional start site
ACKNOWLEDGMENTS AND DISCLOSURES
We thank Yuko Shiono and Yuta Shibue for their excellent technical assistance.
This study was supported by the Japan Society for the Promotion of Science [Grant-in-Aid for Scientific Research (S) 24229002, Scientific Research (B) 23390034, and Grant-in-Aid for Challenging Exploratory Research 21659037].
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