Folylpoly-gamma-glutamate synthetase gene mRNA splice variants and proteinexpression in primary human leukemia cells, cell lines, and normal human tissues

Folypoly-gamma -glutamate synthetase (FPGS) is essential for the cytotoxici ty of "classical" antifolates and their efficacy in cancer chemotherapy. Th e expression of the FPGS gene appears controlled by both tissue/lineage-spe cific and proliferation-dependent mechanisms. Four alternative exon 1 splic e variants of the FPGS gene have been described previously, but their signi ficance in gene regulation has not been determined. Furthermore, alternativ e splicing of the human FPGS gene in normal or transformed cells in vivo ha s not been reported. We have examined the mRNA expression of these FPGS spl ice 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, IA, IB, and 1C; and full-lengt h FPGS mRNA was amplified using primers for exons 14 and 15 at the 3' end o f the gene. In this study, we demonstrate that all four alternative exon I variants are expressed in all primary leukemia cells and cell lines (ALL an d AML), as well as in normal human hematopoietic progenitors. No significan t differences in mRNA expression sere detected in primary cells or cell lin es for the four exon 1 splice variants. Normal circulating human lymphocyte s (peripheral blood mononuclear cells) also expressed mRNA amplified from f ull-length FPGS and the four exon 1 splice variants, although no detectable FPGS activity was found. Using Western immunoblotting, we show that FPGS p rotein is expressed in these peripheral blood mononuclear cells; thus, we p ropose that posttranslational modification(s) is required far 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 expressio n 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, i ts role in regulating the expression of its mRNA remains to be determined.