| Abstract: |
Background: Methotrexate (MTX) uptake is mediated by the reduced folate carrier (RFC). Defective drug uptake in association with decreased RFC expression is a common mechanism of MTX resistance in many tumor types. Heavy promoter methylation was previously identified as a basis for the complete silencing of RFC in MDA-MB-231 breast cancer cells, its role and prevalence in RFC transcription regulation are, however, not widely studied. Methods: In the current study, RFC promoter methylation was assessed using methylation specific PCR in a panel of malignant cell lines (n = 8), including MDA-MB-231, and M805, a MTX resistant cell line directly established from the specimen of a patient with malignant fibrohistocytoma, whom received multiple doses of MTX. A quantitative approach of real-time PCR for measuring the extent of RFC promoter methylation was developed, and was validated by direct bisulfite genomic sequencing. RFC mRNA levels were determined by quantitative real-time RT-PCR and were related to the extent of promoter methylation in these cell lines. Results: A partial promoter methylation and RFC mRNA down-regulation were observed in M805. Using the quantitative approach, a reverse correlation (correlation coefficient = -0.59, p < 0.05) was identified between the promoter methylation and RFC mRNA levels in this a panel of malignant cell lines. Conclusion: This study further suggests that promoter methylation is a potential basis for MTX resistance. The quantitative correlation identified in this study implies that promoter methylation is possibly a mechanism involved in the fine regulation of RFC transcription. © 2008 Yang et al; licensee BioMed Central Ltd. |
| Keywords: |
osteosarcoma; controlled study; carrier protein; gene sequence; human cell; promoter region; genetics; validation process; antineoplastic agents; methotrexate; antineoplastic agent; reverse transcription polymerase chain reaction; breast cancer; down-regulation; cancer cell culture; drug effect; drug resistance; enzymology; pathology; drug resistance, neoplasm; cell line, tumor; breast neoplasms; dna methylation; cancer resistance; gene expression regulation; gene expression regulation, neoplastic; biosynthesis; correlation analysis; correlation coefficient; statistical significance; transcription regulation; messenger rna; rna, messenger; promoter regions, genetic; quantitative analysis; nucleotide sequence; breast tumor; tumor cell line; down regulation; real time polymerase chain reaction; drug therapy; ic 50; leukemia cell line; bisulfite; process development; trimetrexate; membrane transport proteins; reduced folate carrier; multiple drug dose; fibrohistiocytoma
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