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

BMC Cancer Quantitative correlation between promoter methylation and messenger RNA levels of the reduced folate carrier Rui Yang 2 Wei-Wei Li 1 Bang H Hoang 0 5 Hansoo Kim 0 4 Debabrata Banerjee 1 3 Albert Kheradpour 1 7 John H Healey 0 Paul A Meyers 6 Joseph R Bertino 1 3 Richard Gorlick 2 0 Orthopedic Surgery Service, Memorial Sloan-Kettering Cancer Center , New York, NY 10021 , USA 1 Department of Molecular Pharmacology and Therapeutics Program, Memorial Sloan-Kettering Cancer Center , New York, NY 10021 , USA 2 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 3 The Cancer Institute of New Jersey and Departments of Medicine and Pharmacology, University of Medicine and Dentistry of New Jersey , New Brunswick, NJ 08901 , USA 4 Department of Orthopaedic Surgery, Seoul National University Hospital , Chongno-gu, Seoul 110-744 , Korea 5 Department of Orthopaedic Surgery, University of California , Irvine, CA 92868 , USA 6 Department of Pediatrics (P.A.M.), Memorial Sloan-Kettering Cancer Center , New York, NY 10021 , USA 7 Department of Pediatric Oncology & Hematology, Loma Linda University Medical Center , Loma Linda, CA 92354 , USA 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. - Background Methotrexate (MTX) remains an important drug in the treatment of a variety of malignancies, such as acute lymphocytic leukemia (ALL), choriocarcinoma, non-Hodgkin's lymphoma, osteosarcoma, breast cancer, and head and neck cancer. The mechanisms of MTX resistance include: 1) impaired drug transport; 2) reduced drug accumulation because of decreased MTX polyglutamylation or increased drug hydrolysis; 3) increased drug efflux possibly mediated by multiple drug resistance associated proteins (MRPs); 4) alterations in the structure or expression of the target enzyme dihydrofolate reductase (DHFR) and some novel mechanisms proposed recently [1-3]. MTX is delivered into cells predominantly via the reduced folate carrier (RFC), a bi-directional anion exchanger with 12 putative transmembrane domains [4]. To generate sufficient intracellular MTX, RFC transport of MTX is critical to its efficacy. Since the human RFC cDNA was cloned in 1995 [5-8], intensive efforts have been made to explore its clinical relevance in different tumor models. MTX resistance has been associated with decreased expression of RFC or loss of function of the carrier in many tumor types [9-12]. The RFC is ubiquitously expressed in normal tissues [13]; its regulatory mechanisms are, however, not clearly understood. Transcription of the RFC starts from at least four distinct promoters, (designated A, B, C, and D) [14], and is complicated by multiple 5'-non-coding exons resulting from alternative splicing [13,15-20]. The RFC promoter B appeared to be most potent in activity, and was predominantly utilized in tumor cells [16,19]. At least 18 different RFC transcripts have been reported, the functions of which are not clear, although links to tissue specific expression were suggested [13]. Further information on RFC regulation is of importance for understanding the mechanisms of MTX resistance in these diseases. DNA methylation plays an important role in embryonic development and gene imprinting [21]. In cancer cells, DNA methylation in the promoter region is often involved in gene silencing, particularly for some tumor suppressor genes [22]. Recently, it was reported that a ~1400 bp region, including RFC promoter B and A, was identified as a CpG island [23]. Moreover, heavy promoter methylation was the underlying mechanism for the complete lack of RFC expression in MDA-MB-231 breast cancer cell line [24], associated with MTX resistance [23]. However, the role and prevalence of promoter methylation in RFC are not widely studied. A study was therefore initiated to further investigate the role of promoter methylation in a panel of malignant cell lines. Methods Cell Culture The M805 cell line was directly established from a patient with malignant fibrohistocytoma (MFH), who was treated at Memorial Sloan-Kettering Cancer Center (MSKCC) and received multiple courses of chemotherapy, including MTX [25]. The fibrosarcoma cell line, HT1080; breast cancer cell lines MDA-MB-231, MCF-7; and leukemia cell line CCRF-CEM were purchased from ATCC (Rockville, MD). The MTX resistant leukemia cell line CEM-T and HL60R have been described previously [26,27]. The MTX resistant cell line, M316, was established from a relapsed pre-B ALL patient at MSKCC. Cells were maintained as monolayer or suspension in MEM- media, supplemented with 10% fetal calf serum (Life technologies, Bethesda, MD), 100 units/ml penicillin and 3 mg/ml streptomycin at 37C in a humidified atmosphere with 5% CO2. MTX Uptake, Polyglutamylation and Cytotoxicity Assays [3',5',7-3H] MTX was purchased from Moravek (Brea, CA). MTX polyglutamate standards containing one to five additional glutamate residues were obtained from B. Schircks Labs (Jona, Switzerland). MTX uptake and intracellular MTX polyglutamtes were measured as described previously [27]. Non-labeled MTX was purchased from Sigma (St. Louis, MO) and trimetrexate (TMTX) was obtained from Warner Lambert/Parke-Davis (Ann Arbor, MI). Cell growth inhibition studies with MTX and TMTX were performed as described previously [27] (...truncated)