QUANTITATIVE-ANALYSIS OF FOLYLPOLYGLUTAMATE SYNTHETASE GENE-EXPRESSION IN TUMOR-TISSUES BY THE POLYMERASE CHAIN-REACTION - MARKED VARIATIONOF EXPRESSION AMONG LEUKEMIA PATIENTS

Evidence from previous in vitro studies indicates that the enzyme foly lpolyglutamate synthetase (FPGS) may be an important determinant of th e antitumor activity of antifolate drugs that are substrates for this enzyme. To facilitate investigations regarding the association between FPGS content of tumor tissues and the sensitivity of tumors to antifo lates, we developed a polymerase chain reaction (PCR)-based gene expre ssion quantitation assay for measuring relative amounts of FPGS mRNA i n tumor tissue specimens. From the known sequence of the human gene, F PGS-specific PCR primers were chosen that flanked a 263-base segment o f the FPGS gene. The PCR carried out with these primers was linear ove r at least a three orders of magnitude range of starting cDNA concentr ation. The amount of cDNA required per assay corresponded to the quant ity of RNA contained in nanogram to microgram amounts of tissue, depen ding on the level of gene expression. In CHO AUXB1 (FPGS) cell lines t ransfected with human DNA and expressing different levels of human FPG S, FPGS gene expression measured by this assay was linear with the FPG S enzyme activity in the cells. In human head and neck cell lines, whi ch contained naturally varying levels of FPGS enzyme activity, FPGS ge ne expressions were also linearly proportional to FPGS enzyme content as measured both by activity in cell-free extracts and by intracellula r methotrexate polyglutamate formation. Among leukemic cells from 11 a cute lymphocytic leukemia and acute myelogenous leukemia patients, FPG S expression varied by over 500-fold. This broad range of FPGS express ion in tumors from different patients coupled with the availability of a sensitive and accurate assay for gene expression should now make it possible to establish whether FPGS expression in tumors is predictive for response to therapy involving shortterm exposures to antifolates.