Folylpoly-gamma-glutamate Synthetase Gene MRNA Splice Variants and Protein Expression in Primary Human Leukemia Cells, Cell Lines, and Normal Human Tissues

Folypoly-gamma-glutamate synthetase (FPGS) is essential for the cytotoxicity of "classical" antifolates and their efficacy in cancer chemotherapy. The expression of the FPGS gene appears controlled by both tissue/lineage-specific and proliferation-dependent mechanisms. Four alternative exon 1 splice variants of the FPGS gene have been described previously, but their significance in gene regulation has not been determined. Furthermore, alternative splicing of the human FPGS gene in normal or transformed cells in vivo has not been reported. We have examined the mRNA expression of these FPGS splice variants in primary human leukemia cells, cell lines, and normal human hematopoietic progenitors using reverse transcription-PCR. Specific primers were designed to amplify splice variants 1, 1A, 1B, and 1C; and full-length FPGS mRNA was amplified using primers for exons 14 and 15 at the 3' end of the gene. In this study, we demonstrate that all four alternative exon 1 variants are expressed in all primary leukemia cells and cell lines (ALL and AML), as well as in normal human hematopoietic progenitors. No significant differences in mRNA expression were detected in primary cells or cell lines for the four exon 1 splice variants. Normal circulating human lymphocytes (peripheral blood mononuclear cells) also expressed mRNA amplified from full-length FPGS and the four exon 1 splice variants, although no detectable FPGS activity was found. Using Western immunoblotting, we show that FPGS protein is expressed in these peripheral blood mononuclear cells; thus, we propose that posttranslational modification(s) is required for expression of a functional FPGS protein in human lymphohematopoietic cells. In addition, poly(A)(+) RNA from normal human adult and fetal tissues and leukemia cell lines was analyzed by Northern blot methodology. Total FPGS mRNA expression showed tissue-specificity, and higher levels were observed in human fetal tissues compared with adult tissues. The data presented herein demonstrate the existence of these FPGS mRNA splice variants in normal and transformed human hematopoietic cells and indicate that alternative splicing of the 5' end of the human FPGS gene does occur in primary cells in vivo. However, its role in regulating the expression of its mRNA remains to be determined.