Quantitative correlation between promoter methylation and messenger RNA levels of the reduced folate carrier.
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.


CONCLUSIONS
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.
Yang
Rui
R
Department of Pediatrics and Molecular Pharmacology (R,Y,, R,G,), The Albert Einstein College of Medicine, The Children's Hospital at Montefiore, Bronx, NY 10461, USA. ruiyang@aecom.yu.edu
Li
Wei-Wei
WW
Hoang
Bang H
BH
Kim
Hansoo
H
Banerjee
Debabrata
D
Kheradpour
Albert
A
Healey
John H
JH
Meyers
Paul A
PA
Bertino
Joseph R
JR
Gorlick
Richard
R
D000970:Antineoplastic Agents / Q000008:administration & dosage; D001943:Breast Neoplasms / Q000188:drug therapy / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D045744:Cell Line, Tumor; D019175:DNA Methylation; D015536:Down-Regulation; D019008:Drug Resistance, Neoplasm / Q000187:drug effects; D004358:Drug Therapy; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D026901:Membrane Transport Proteins / Q000096:biosynthesis* / Q000235:genetics*; D008727:Methotrexate / Q000008:administration & dosage; D012516:Osteosarcoma / Q000188:drug therapy / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics*; D058978:Reduced Folate Carrier Protein
D000970:Antineoplastic Agents; D026901:Membrane Transport Proteins; D012333:RNA, Messenger; D058978:Reduced Folate Carrier Protein; C548699:SLC19A1 protein, human; D008727:Methotrexate