Methotrexate (MTX) is a universal component of chidlhood ALL therapies and its conversion to long chain polyglutamates (PG) by folylpoly-γ-glutamate synthetase (FPGS) is essential for its antileukemic acitivity. Expression of FPGS appears to be controlled by tissue/lineage specific and proliferation-dependent mechanisms. Levels of FPGS mRNA, protein, and enzyme activity are 2-3 fold higher in B-precursor (Bp) ALL cells when compared to T-lineage ALL, and these differences correlate with intracellular accumulation of long chain MTX-PG and lymphoblast sensitivity to MTX. To characterize these lineage differences in FPGS expression between B and T lymphoblasts we examined its mRNA expression in Nalm6 (Bp-ALL) and CCRF-CEM (T-ALL) cells during cellular growth and cell cycle checkpoints. During early exponential growth (24 hrs), FPGS expression was 6-fold higher in Nalm6 when compared to CCRF-CEM but decreased significantly after 72 hrs while it was unchanged in CCRF-CEM cells. During G1/G0 phase we found that FPGS expression was 15-fold higher in Nalm6 when compared to CCRF-CEM cells. Taken together, these data suggest that during proliferation and cell cycle progression FPGS gene expression is regulated differently in Bp-ALL and T-ALL cells. To determine whether this lineage-specific regulation occurs at the transcriptional level we performed nuclear run-on assays. We found that FPGS mRNA transcription initiation rate was 1.7-fold higher in Nalm6 when compared to CCRF-CEM cells, indicating that differences in promoter regulation lead to the observed lineage differences in FPGS expression in Bp- vs. T-ALL. We then used a methylation specific PCR assay to investigate the methylation status of the FPGS proximal promoter region from both Nalm6 and CCRF-CEM cells. Our data indicate that the FPGS proximal promoter region is unmethylated in both cell lines and therefore can not explain the observed lineage-specific differences. 5′-RACE experiments demonstrated that Nalm6 and CCRF-CEM have the same FPGS transcriptional start sites, but a reporter gene assay indicated that the minimal promoter region that directs FPGS transcription in CCRF-CEM cells is insufficient to drive FPGS mRNA transcription in NALM6 cells. In order to identify potential regulatory regions directing FPGS transcription in Bp-lymphoblasts, we used DNaseI hypersensitivity assays. We identified a hypersensitive region located 8.5 kbp upstream to exon 1 in Nalm6 cells suggesting that tissue-specific regulatory elements responsible for lineage-specific FPGS expression in Bp-ALL cells may be localized within this region. Finally, we detected reduced levels of FPGS mRNA expression in RCH-ACV (Bp-ALL, t(1:19)/E2A-PBX1) and REH (Bp-ALL, t(12:21)/TEL-AML1) cells expressing chromosomal translocated fusions when compared to control (Nalm6). To characterize the molecular basis of the E2A-PBX1 and TEL-AML1 interactions with FPGS mRNA expression in Bp lymphoblasts we used antisense and RNAi technology to downregulate these two genetic fusions. Our data lead us to hypothesize that in addition to lineage-specific regulatory differences in FPGS expression, molecular mechanisms associated with non-random translocations may alter FPGS mRNA expression and influence MTX sensitivity in Bp-ALL lymphoblasts.