Our previous study identified two alternate non-coding upstream exons (A and B) in the human reduced folate carrier (hRFC) gene, each controlled by a separate promoter. Each minimal promoter was regulated by unique cis -elements and transcription factors, including stimulating protein (Sp) 1 and Sp3 and the basic leucine zipper family of proteins, suggesting opportunities for cell- and tissue-specific regulation. Studies were performed to explore the expression patterns of hRFC in human tissues and cell lines. Levels of hRFC transcripts were measured on a multi-tissue mRNA array from 76 human tissues and tumour cell lines and on a multi-tissue Northern blot of representative tissues, each probed with full-length hRFC cDNA. hRFC transcripts were ubiquitously expressed, with the highest level in placenta and the lowest level in skeletal muscle. By rapid amplification of cDNA 5'-ends assay from nine tissues and two cell lines, hRFC transcripts containing both A and B 5'-untranslated regions (UTRs) were identified. However, five additional 5'-UTRs (designated A1, A2, C, D and E) were detected, mapping over 35 kb upstream from the hRFC translation start site. The 5'-UTRs were characterized by multiple transcription start sites and/or alternative splice forms. At least 18 unique hRFC transcripts were detected. A novel promoter was localized to a 453 bp fragment, including 442 upstream of exon C and 11 bp of exon C. A 346 bp repressor flanked the 3'-end of this promoter. Our results suggest an intricate regulation of hRFC gene expression involving multiple promoters and non-coding exons. Moreover, they provide a transcriptional framework for understanding the role of hRFC in the pathophysiology of folate deficiency and antifolate drug selectivity.

Nucleotide efflux (especially cyclic nucleotides) from a variety of mammalian tissues, bacteria, and lower eukaryotes has been studied for several decades. However, the molecular identity of these nucleotide efflux transporters remained elusive, despite extensive knowledge of their kinetic properties and inhibitor profiles. Identification of the subfamily of adenosine triphosphate (ATP) binding cassette transporters, multidrug resistance protein (MRP) subfamily, permitted rapid advances because some recently identified MRP family members transport modified nucleotide analogs (ie, chemotherapeutic agents). We first identified, MRP4, based on its ability to efflux antiretroviral compounds, such as azidothymidine monophosphate (AZT-MP) and 9-(2-phosphonyl methoxyethyl) adenine (PMEA), in drug-resistant and also in transfected cell lines. MRP5, a close structural homologue of MRP4 also transported PMEA. MRP4 and MRP5 confer resistance to cytotoxic thiopurine nucleotides, and we demonstrate MRP4 expression varies among acute lymphoblastic leukemias, suggesting this as a factor in response to chemotherapy with these agents. The ability of MRP4 and MRP5 to transport 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP) suggests they may play a biological role in cellular signaling by these nucleotides. Finally, we propose that MRP4 may also play a role in hepatic bile acid homeostasis because loss of the main bile acid efflux transporter, sister of P-glycoprotein (SPGP) aka bile-salt export pump (BSEP), leads to a strong compensatory upregulation in MRP4 expression. Cumulatively, these studies reveal that the ATP-binding cassette (ABC) transporters MRP4 and MRP5 have a unique role in biology and in chemotherapeutic response.

The multidrug resistance protein MRP4, a member of the ATP-binding cassette superfamily, confers resistance to purine-based antiretroviral agents. However, the antiviral agent ganciclovir (GCV) has not been shown to be a substrate of MRP4. GCV is important not only in antiviral therapy, but also in the selective killing of tumor cells modified to express herpes simplex virus thymidine kinase (HSV-TK). We therefore tested the effect of MRP4 on the cytotoxicity of GCV, on the ability of GCV to kill cells genetically modified to express HSV-TK, and on the bystander effect in which unmodified target cells are killed by GCV. Cells overexpressing MRP4 had markedly increased resistance to the cytotoxicity of GCV. Although, expression of recombinant HSV-TK increased the intracellular concentration of GCV nucleotide, cells were rescued by the cytoprotective effect of MRP4. In cells that overexpressed MRP4, intracellular accumulation of GCV metabolites was reduced, efflux of these metabolites was increased, and resistance to bystander killing was increased. Therefore, MRP4 can strongly reduce the susceptibility of HSV-TK-expressing cells to GCV, and its overexpression in adjacent cells protects them from bystander cell death. These findings indicate that a nucleotide transporter, such as MRP4, modulates the cellular response to GCV and thus may influence not only the efficacy of antiviral therapy, but also prodrug-based gene therapy, which is critically dependent upon bystander cell killing.The multidrug resistance proteins (MRPs) 1 are a family of ATP-binding cassette transporters (ABC transporters; for an overview, see //nutrigene.4t.com/humanabc.html) that mediates drug efflux and multidrug resistance (1). We previously demonstrated that MRP4 (also known as ABCC4) severely reduces the antiviral efficacy of several nucleoside reverse transcriptase inhibitors, such as zidovudine (3Ј-azido-3Ј-deoxythymidine) (AZT)) and 9-(2-(phosphonomethoxy)ethyl)-adenine (PMEA), in mammalian cells and that this effect corresponds with increased ATP-dependent efflux of their nucleotide derivatives (2). These findings were, in part, replicated by Lee et al. (3). A better understanding of the role of MRP4 as a nucleotide efflux transporter may lead to improved nucleoside-based therapies for HIV, herpes viruses, and cancer. Ganciclovir (9-(1,3-dihydroxy-2-propoxymethyl)guanine (GCV)) is widely used against cytomegalovirus infection in patients with AIDS (4 -6) but is poorly tolerated in combination with AZT (7). Although the molecular basis of this interaction is unknown, in vitro studies indicate that the combination is extremely cytotoxic (8, 9). We therefore postulated that MRP4 might be involved in the cytotoxicity induced by the AZT-GCV combination. If so, such an interaction could have important therapeutic potential because of the widespread use of GCV as a prodrug in gene therapies for cancer (10,11). Transduction of tumor cells with the herpes simplex virus thymidine kinase (HSV-TK) gene and treatment with GCV is a common antica...