Research article

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

Rui Yang¹ , Wei-Wei Li² , Bang H Hoang^3,5 , Hansoo Kim^3,6 , Debabrata Banerjee^2,7 , Albert Kheradpour^2,8 , John H Healey³ , Paul A Meyers⁴ , Joseph R Bertino^2,7 and Richard Gorlick¹

¹Department 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

²Department of Molecular Pharmacology and Therapeutics Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA

³Orthopedic Surgery Service, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA

⁴Department of Pediatrics (P.A.M.), Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA

⁵Department of Orthopaedic Surgery, University of California, Irvine, CA 92868, USA

⁶Department of Orthopaedic Surgery, Seoul National University Hospital, Chongno-gu, Seoul 110-744, Korea

⁷The Cancer Institute of New Jersey and Departments of Medicine and Pharmacology, University of Medicine and Dentistry of New Jersey, New Brunswick, NJ 08901, USA

⁸Department of Pediatric Oncology & Hematology, Loma Linda University Medical Center, Loma Linda, CA 92354, USA

author email corresponding author email

BMC Cancer 2008, 8:124doi:10.1186/1471-2407-8-124

Published:	1 May 2008

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.