Down-regulation of reduced folate carrier gene (RFC1) expression after exposure to methotrexate in ZR-75-1 breast cancer cells.
Methotrexate (MTX) is administered in intervals of one week or longer in the treatment of cancer and autoimmune disease. Early studies suggested that daily MTX administration was associated with decreased effectiveness and increased toxicity, leading to schedules of administration that include periodic intervals of rest during chronic MTX therapy. We hypothesized that these observations may be the result of the down-regulation of the reduced folate carrier, the major route of cellular uptake of both MTX and the endogenous folates, after MTX exposure. We exposed folate-depleted ZR-75-1 breast cancer cells to low-dose MTX in the presence of hypoxanthine, adenosine and thymidine. After 72 h, the initial rate of MTX uptake had decreased to 22% of the Day 0 value. Western blot analysis showed down-regulation of RFC1 protein expression, and Northern blot analysis showed a corresponding decrease in RFC1 RNA levels. Using an RT-PCR assay, we found that levels of RNA transcripts containing each of the three RFC1 5' noncoding exons were decreased after exposure to MTX, suggesting that MTX exposure causes transcriptional down-regulation of RFC1. Promoter-reporter construct assays demonstrated decreased activity of RFC1 promoter elements upstream of these exons after MTX exposure. Preexposure of the ZR-75-1 cells to 5-azacytidine, a DNA methylation inhibitor, further decreased MTX uptake rather than reverse the inhibition of RFC1 activity, indicating that RFC1 down-regulation after MTX exposure is not the result of methylation of the RFC1 promoter. In summary, these studies demonstrate that MTX exposure can down-regulate RFC1 expression and activity. These acute, inducible, epigenetic changes in RFC1 expression may ultimately be molded into the more permanent genetic changes that result in the transport-mediated MTX resistance that have been observed in MTX-resistant cell lines.
Ma
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Department of Pediatrics, University of Kentucky, Lexington 40502, USA.
Huang
H
H
Moscow
J A
JA
D000964:Antimetabolites, Antineoplastic / Q000378:metabolism / Q000494:pharmacology*; D001692:Biological Transport / Q000187:drug effects; D001943:Breast Neoplasms; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D015536:Down-Regulation; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D026901:Membrane Transport Proteins; D008727:Methotrexate / Q000378:metabolism / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D000964:Antimetabolites, Antineoplastic; D002352:Carrier Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C120043:SLC19A2 protein, human; D008727:Methotrexate