BMC Cancer 2006, 6:132 http://www.biomedcentral.com/1471-2407/6/132 Background Folate antimetabolites play a central role as anticancer agents. In mammalian tissues, intracellular folates and antifolates exist as poly-γ-glutamates with typical chains ranging from five to nine residues [1-3]. Polyglutamation is catalyzed by folylpoly-γ-glutamate synthetase (FPGS) and results in increased intracellular concentration and cytoxicity of classical antifolates [4]. Furthermore, when polyglutamated, some antifolates (e.g., raltitrexed, lome- trexol) increase their Ki against targeted enzymes by over 100-fold [5,6]. In childhood acute lymphoblastic leuke- mia (ALL) a strong correlation exists between FPGS expression, intracellular methotrexate (MTX) polygluta- mate accumulation and treatment outcome [4,7]. The FPGS gene is controlled by at least two mechanisms: one tissue/lineage-specific and a second proliferation- dependent [8-11]. FPGS activity is distributed to both cytosolic and mitochondrial compartments of mamma- lian cells. In humans, these two isoforms are encoded by a single locus in chromosome region 9q (34.1–34.2) [12], and differ by the use of two alternative translational start sites within exon 1 [13]. Use of these alternative start sites translates the FPGS protein with or without the addition of a mitochondrial leader sequence. Alternative FPGS exon 1 variants (exons 1, 1A, 1B, 1C, and 2A), all spliced to exon 2, have been described [12,14]. We have demon- strated no lineage-specific differences in the expression of these alternative transcripts in human leukemia and nor- mal tissues [15]. In mice, two promoters spaced by 10 kb were shown to express distinct functional tissue-specific FPGS isoen- zymes [16]. The upstream transcript (exon A1a) was expressed only in few differentiated tissues such as liver, whereas the downstream transcript (exon 1) was expressed in dividing normal and neoplastic tissues. This dual promoter mechanism directing expression of murine isoenzymes is not conserved in humans. In contrast, the human FPGS exon 1 transcript is present in both dividing and differentiated tissues [14]. In human leukemia cells, the enzyme translated from exon 1 transcript was reported as the only catalytically active form. Transcription of the human FPGS gene appears to be controlled by a TATA-less promoter driven by a set of 8 concatameric Sp1 sites spaced within a 150 bp region upstream of exon 1 [17]. Several additional transcription factors, including NFY (Y- box) and E-box motifs have been identified within the human minimal FPGS promoter region [13,17]. Our laboratory first demonstrated that constitutive levels of FPGS mRNA, protein, and enzyme activity are two- to three-fold higher in B-precursor (Bp) ALL cells compared to T-lineage ALL [9,11]. We now report studies investigat- ing the molecular mechanisms for this differential FPGS gene expression. Our results demonstrate that FPGS tran- scriptional start sites (+1) are the same in all hematopoi- etic lineages studied. To analyze lineage differences in FPGS promoter activity we used a FPGS promoter-luci- ferase gene reporter assay. All DNA fragments examined for promoter/enhancer activity exhibited higher levels of luciferase activity in CCRF-CEM vs. NALM6 cells. Finally, we determined the role of the putative NFY and E-box transcription factor binding sites on FPGS gene transcrip- tion using the same reporter gene assay with substitution mutants and EMSA analysis. Methods Leukemia cell lines The human leukemia cell lines CCRF-CEM (T-ALL) and REH (Bp-ALL t(12;21)) were obtained from the American Type Culture Collection. NALM6 (Bp-ALL) cell line was obtained from DSMZ (Germany). RCH-ACV (Bp- ALL t(1;19)) was kindly provided by Dr. Stephen Hunger (UFL, Gainesville, FL). All cell lines were grown in RPMI 1640 (Sigma) supplemented with 10% FBS at 37°C and 5% CO2. Normal bone marrow (BM) was extracted from normal volunteers. We obtained institutional review board approval, and IRB approved informed consent was obtained from normal volunteers prior to participation. RNA isolation and real-time RT-PCR Total RNA was isolated using the RNeasy kit (Qiagen Inc). FPGS exons 14 and 15 were RT-PCR amplified [15] and quantitated using pFPGS-cDNA, a pCR2.1-TOPO plasmid containing the human FPGS cDNA gene (1926 bp; Table 1). Briefly, the 1926 pb FPGS cDNA gene was amplified by PCR using primers 1exonF and 15exonR-c1934 (Table 1) from total RNA reverse transcribed with primer 15exonR- c2107 (5'-GGCCAGGCAGCGCACACAAT). Results were normalized to β-actin mRNA expression. All real-time PCR reactions (SYBR green) were performed using the BIO-RAD iCycler iQ system (Bio-Rad) [18]. Nuclear run-on assay Nuclear run-on assay was performed as described by Han- son et al. [19]. One µg of FPGS and 18S RNA cDNAs were blotted, UV cross-linked and hybridized with nascent labeled RNA transcripts. After washing, membranes were exposed to XAR-5 film (Kodak). Densitometric scan anal- ysis was performed using the GelPro Analyzer program. Results were normalized to the level of 18S RNA and sta- tistic achieved using one-tailed, Student paired t-test (GraphPad Prism, version 2.01). All data are expressed as mean ± S.E.M. 5'-Rapid amplification of cDNA ends (RACE) Amplification of the 5'-termini of FPGS mRNA was per- formed using the 5'-RACE system Version 2.0 (Invitrogen Corporation). Briefly, polyA+ RNA isolated from cells Page 2 of 12 (page number not for citation purposes)