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Transcriptional Regulation of the Human Reduced Folate Carrier in Childhood Acute Lymphoblastic Leukemia Cells

Authors' Affiliations: ¹ Developmental Therapeutics Program, Barbara Ann Karmanos Cancer Institute, and Departments of ² Pharmacology, ³ Medicine, and ⁴ Pediatrics, Wayne State University School of Medicine, and ⁵ Children's Hospital of Michigan, Detroit, Michigan

Requests for reprints: Larry H. Matherly, Developmental Therapeutics Program, Barbara Ann Karmanos Cancer Institute, 110 East Warren Avenue, Detroit, MI 48201. Phone: 313-833-0715, ext. 2407; Fax: 313-832-7294; E-mail: matherly{at}karmanos.org.

Purpose: The transcriptional regulation of the human reduced folate carrier (hRFC), involved in cellular uptake of methotrexate and reduced folates, was studied in childhood acute lymphoblastic leukemia (ALL). The hRFC gene is regulated by six noncoding exons (A1/A2 and A to E) and multiple promoters. In ALL, hRFC-A1/A2 and hRFC-B are the major transcript forms.

Experimental Design: RNAs from 18 ALL lymphoblast specimens and 10 nonobese diabetic/severe combined immunodeficient ALL xenografts were assayed by real-time reverse transcription-PCR for hRFC-A1/A2 and hRFC-B transcripts and for transcripts encoding USF1, GATA1, Sp1, and Ikaros transcription factors. For the xenografts, gel shift and chromatin immunoprecipitation assays assessed transcription factor binding to the hRFC-A1/A2 and hRFC-B promoters. CpG methylation density within a 334-bp region, including the core hRFC-B promoter, was established by bisulfite sequencing. hRFC-A1/A2 and hRFC-B promoter polymorphisms were assayed by DNA sequencing.

Results: For the 28 ALLs, hRFC-A1/A2 and hRFC-B transcripts spanned a 546-fold range. By chromatin immunoprecipitation and gel shift assays, binding was confirmed for USF1 and GATA1 for hRFC-A1/A2, and for Sp1, USF1, and Ikaros for hRFC-B. hRFC transcript levels correlated with those for GATA1 and USF1 for hRFC-A1/A2 and with Sp1 and USF1 transcripts for hRFC-B. CpG methylation in ALL did not correlate with hRFC-B transcripts. In 40 ALL and 17 non-ALL specimens, 2 cosegregating high-frequency polymorphisms (T-1309/C-1217 and C-1309/T-1217; allelic frequencies of 36% and 64%, respectively) were detected in the A1/A2 promoter; none were detected in promoter B. The hRFC-A1/A2 polymorphisms only slightly affected promoter activity.

Conclusions: Our results show a complex regulation of hRFC in ALL involving the hRFC-A1/A2 and hRFC-B promoters and noncoding exons. Although Sp1, USF1, and GATA1 levels are critical determinants of hRFC transcription in ALL, neither DNA methylation nor promoter polymorphisms contribute to differences in hRFC expression.

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