BMC Cancer Research article Analysis of folylpoly-γ-glutamate synthetase gene expression in human B-precursor ALL and T-lineage ALL cells Guy J Leclerc1, Gilles M Leclerc2, Ting Ting Hsieh Kinser1 and Julio C Barredo*1 BioMed Central Open Access Address: 1Department of Pediatrics and Department of Biochemistry and Molecular Biology, Medical University of South Carolina, PO Box 250558, Charleston SC, 29425, USA and 2Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston SC, 29425, USA Email: Guy J Leclerc - leclergj@musc.edu; Gilles M Leclerc - leclergm@musc.edu; Ting Ting Hsieh Kinser - kinser@musc.edu; Julio C Barredo* - barredjc@musc.edu * Corresponding author Published: 17 May 2006 BMC Cancer 2006, 6:132 doi:10.1186/1471-2407-6-132 This article is available from: http://www.biomedcentral.com/1471-2407/6/132 Received: 22 December 2005 Accepted: 17 May 2006 © 2006 Leclerc et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Expression of folylpoly-γ-glutamate synthetase (FPGS) gene is two- to three-fold higher in B-precursor ALL (Bp- ALL) than in T-lineage ALL (T-ALL) and correlates with intracellular accumulation of methotrexate (MTX) polyglutamates and lymphoblast sensitivity to MTX. In this report, we investigated the molecular regulatory mechanisms directing FPGS gene expression in Bp-ALL and T-ALL cells. Methods: To determine FPGS transcription rate in Bp-ALL and T-ALL we used nuclear run-on assays. 5'- RACE was used to uncover potential regulatory regions involved in the lineage differences. We developed a luciferase reporter gene assay to investigate FPGS promoter/enhancer activity. To further characterize the FPGS proximal promoter, we determined the role of the putative transcription binding sites NFY and E-box on FPGS expression using luciferase reporter gene assays with substitution mutants and EMSA. Results: FPGS transcription initiation rate was 1.6-fold higher in NALM6 vs. CCRF-CEM cells indicating that differences in transcription rate led to the observed lineage differences in FPGS expression between Bp-ALL and T-ALL blasts. Two major transcripts encoding the mitochondrial/cytosolic and cytosolic isoforms were detected in Bp-ALL (NALM6 and REH) whereas in T-ALL (CCRF-CEM) cells only the mitochondrial/cytosolic transcript was detected. In all DNA fragments examined for promoter/enhancer activity, we measured significantly lower luciferase activity in NALM6 vs. CCRF-CEM cells, suggesting the need for additional yet unidentified regulatory elements in Bp-ALL. Finally, we determined that the putative transcription factor binding site NFY, but not E-box, plays a role in FPGS transcription in both Bp- and T- lineage. Conclusion: We demonstrated that the minimal FPGS promoter region previously described in CCRF- CEM is not sufficient to effectively drive FPGS transcription in NALM6 cells, suggesting that different regulatory elements are required for FPGS gene expression in Bp-cells. Our data indicate that the control of FPGS expression in human hematopoietic cells is complex and involves lineage-specific differences in regulatory elements, transcription initiation rates, and mRNA processing. Understanding the lineage- specific mechanisms of FPGS expression should lead to improved therapeutic strategies aimed at overcoming MTX resistance or inducing apoptosis in leukemic cells. Page 1 of 12 (page number not for citation purposes)