title: Methylation-dependent silencing of the reduced folate carrier gene in inherently methotrexate-resistant human breast cancer cells.
abstract: The molecular basis of methotrexate resistance was studied in human MDA-MB-231 breast cancer cells, which are inherently defective in methotrexate uptake and lack expression of the reduced folate carrier (RFC). Transfection of MDA-MB-231 cells with RFC cDNA restored methotrexate uptake and increased methotrexate sensitivity by approximately 50-fold. A CpG island in the promoter region of RFC was found to be methylated in MDA-MB-231 cells, but was unmethylated in RFC expressing, methotrexate-sensitive MCF-7 breast cancer cells. Chromatin immunoprecipitation with antibodies against acetylated histones H3 and H4 showed that the RFC promoter was enriched for acetylated histones on expressed, unmethylated alleles only. Treatment of MDA-MB-231 cells with 5-aza-2'-deoxycytidine restored RFC expression but also led to increased methotrexate efflux and did not reverse methotrexate resistance. This suggests that 5-aza-2'-deoxycytidine up-regulates both methotrexate uptake and some methotrexate-resistance mechanism(s). Reverse transcription-polymerase chain reaction analysis showed increased expression levels of several ATP-dependent efflux pumps in response to 5-aza-2'-deoxycytidine treatment, including P-glycoprotein and members of the multidrug resistance-associated protein family. Up-regulation of P-glycoprotein in response to 5-aza-2'-deoxycytidine was associated with demethylation of a CpG island in the MDR1 promoter, whereas the mechanism(s) for 5-aza-2'-deoxycytidine-induced up-regulation of multidrug resistance-associated proteins is probably indirect. Dipyridamole inhibited methotrexate efflux and reversed methotrexate resistance in 5-aza-2'-deoxycytidine-treated MDA-MB-231 cells.
authors.affiliation: Institute of Cancer Biology, Danish Cancer Society, Strandboulevarden 49, DK-2100 Copenhagen, Denmark.
authors.forename: J
authors.initials: J
authors.lastname: Worm
authors.forename: A F
authors.initials: AF
authors.lastname: Kirkin
authors.forename: K N
authors.initials: KN
authors.lastname: Dzhandzhugazyan
authors.forename: P
authors.initials: P
authors.lastname: Guldberg
mesh_terms: D000107:Acetylation; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D001692:Biological Transport; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000235:genetics*; D002352:Carrier Proteins / Q000235:genetics*; D018899:CpG Islands; D019175:DNA Methylation; D000077209:Decitabine; D004176:Dipyridamole / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D004357:Drug Synergism; D005260:Female; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D020868:Gene Silencing; D006655:Histone Deacetylases; D006657:Histones / Q000378:metabolism; D006801:Humans; D016623:Ion Pumps / Q000378:metabolism; D026901:Membrane Transport Proteins; D008727:Methotrexate / Q000378:metabolism / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D058978:Reduced Folate Carrier Protein
chemical_list: D000964:Antimetabolites, Antineoplastic; D002352:Carrier Proteins; D005493:Folic Acid Antagonists; D006657:Histones; D016623:Ion Pumps; D026901:Membrane Transport Proteins; D058978:Reduced Folate Carrier Protein; C548699:SLC19A1 protein, human; D004176:Dipyridamole; D000077209:Decitabine; D006655:Histone Deacetylases; D001374:Azacitidine; D008727:Methotrexate