Analysis of folylpoly-γ-glutamate synthetase gene expression in human B-precursor ALL and T-lineage ALL cells
| Autor: | Guy J. Leclerc, Gilles M. Leclerc, Julio C. Barredo, Ting Ting Hsieh Kinser |
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| Jazyk: | angličtina |
| Předmět: |
Transcriptional Activation
Cancer Research Transcription Genetic Biology lcsh:RC254-282 E-Box Elements Cell Line Tumor Precursor B-Cell Lymphoblastic Leukemia-Lymphoma Gene expression Genetics Humans Binding site Peptide Synthases Promoter Regions Genetic Gene Transcription factor Binding Sites Lymphoblast DNA Methylation lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens Molecular biology Leukemia Lymphoid Gene Expression Regulation Neoplastic Enhancer Elements Genetic CCAAT-Binding Factor Oncology DNA methylation Stem cell Transcription Initiation Site Intracellular Research Article Transcription Factors |
| Zdroj: | BMC Cancer, Vol 6, Iss 1, p 132 (2006) BMC Cancer |
| ISSN: | 1471-2407 |
| DOI: | 10.1186/1471-2407-6-132 |
| Popis: | BackgroundExpression 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.MethodsTo 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.ResultsFPGS transcription initiation rate was 1.6-fold higher in NALM6vs. 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 NALM6vs. 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.ConclusionWe 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. |
| Databáze: | OpenAIRE |
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