Rabbit Anti-Human FPGS Polyclonal Antibody

We investigated the expression of the folylpolyglutamate synthetase (FPGS) gene at the mRNA level in MOLT-3 and K562 human leukemia cell lines sensitive, or made resistant, to methotrexate (MTX) and/or trimetrexate (TMQ), or raltitrexed (ZD1694). Northern blot analysis demonstrated approximately 3-fold higher FPGS mRNA expression in K562 cells than that in MOLT-3 cells, being consistent with graded polyglutamation capacities of these cell lines. A slight increase in the expression of the FPGS gene was observed in the TMQ-resistant MOLT-3 cells (MOLT-3/TMQ(800)); moreover, sequential development of MTX resistance in the TMQ-resistant cells (MOLT-3/TMQ(800)-MTX10 (000)) resulted in a further enhancement of FPGS mRNA expression despite of decreased polyglutamation capacity in this subline. Another MTX-resistant subline with impaired reduced folate carrier (MOLT-3/MTX10 (000)) also showed overexpression of FPGS mRNA. Conversely, both raltitrexed-resistant sublines (MOLT-3/ZD1694 . C and K562/ZD1694 . C) displayed a moderately decreased expression of FPGS mRNA. These findings did not correspond to the virtual absence of ZD1694 polyglutamates inside the former cells nor to possibly intact polyglutamation capacity in the latter cells. These results indicate that FPGS mRNA expression may predict cellular ability to produce polyglutamate metabolites of antifolate drugs in the sensitive cells, but does not necessarily reflect FPGS function at the enzyme level in the antifolate-resistant tumor cells. [(C) 1999 Lippincott Williams & Wilkins.].

Pemetrexed is a newly developed multitargeted antifolate with promising clinical activity in many solid tumors including gastric cancer. The aim of the present study was to evaluate the cytotoxicity of pemetrexed and its mode of interaction with cisplatin in gastric cancer cell lines, and to identify genes associated with sensitivity to pemetrexed. The cytotoxic activity of pemetrexed was assessed by tetrazolium-based colorimetric assay (MTT assay) and the interaction between pemetrexed and cisplatin was evaluated by the isobologram method. Western immunoblotting and real time RT-PCR analysis of thymidylate synthase (TS), folylpoly-gamma-glutamate synthetase (FPGS) and reduced folate carrier (RFC1) were performed in order to determine whether sensitivity to pemetrexed would be predictable by protein or mRNA expression levels. Pemetrexed was more cytotoxic than 5-fluorouracil, with IC50 between 17 and 310 nM in most of the gastric cancer cell lines examined and the pemetrexed/cisplatin combination resulted in additive or synergistic interaction. The protein expressions of TS, FPGS, and RFC1 were significantly associated with IC50 for 5-fluorouracil, but no such association was found for pemetrexed chemosensitivity. The mRNA expressions of RFC1, FPGS and other target and resistance related genes revealed no significant association with pemetrexed sensitivity. In conclusion, pemetrexed is active against gastric cancer cell lines and the pemetrexed/cisplatin combination showed a synergistic or additive interaction, supporting its clinical use in gastric cancer. Drug sensitivity toward pemetrexed could not be predicted by the expressions of TS, RFC1, or FPGS and we suggest that it is determined by interactions between multiple genes.