Analysis of folylpoly-gamma-glutamate synthetase gene expression in human B-precursor ALL and T-lineage ALL cells.
BACKGROUND
Expression of folylpoly-gamma-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.


CONCLUSIONS
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.
Leclerc
Guy J
GJ
Department of Pediatrics, Medical University of South Carolina, PO Box 250558, Charleston SC 29425, USA. leclergj@musc.edu
Leclerc
Gilles M
GM
Kinser
Ting Ting Hsieh
TT
Barredo
Julio C
JC
D001665:Binding Sites; D023081:CCAAT-Binding Factor; D045744:Cell Line, Tumor; D019175:DNA Methylation; D024721:E-Box Elements; D004742:Enhancer Elements, Genetic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007945:Leukemia, Lymphoid / Q000235:genetics* / Q000378:metabolism; D010453:Peptide Synthases / Q000235:genetics* / Q000378:metabolism; D015452:Precursor B-Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D014157:Transcription Factors; D024363:Transcription Initiation Site; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D023081:CCAAT-Binding Factor; D014157:Transcription Factors; C402244:nuclear factor Y; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase