CELL-CYCLE EFFECTS OF ANTIFOLATE ANTIMETABOLITES - IMPLICATIONS FOR CYTOTOXICITY AND CYTOSTASIS

Purpose: Cell cycle-related events in CCRF-CEM lymphocytic leukemia ce lls were examined subsequent to inhibition of thymidylate synthase (TS ) or GAR formyltransferase (GARFT) and prior to cell death or stasis. Methods: Cell populations were treated with the GARFT inhibitors 6R-5, 10-dideazatetrahydrofolate (lometrexol) or LY309887, the TS inhibitor ZD1694, or the multitargeted antifolate LY231514. DNA content, nucleo side precursor incorporation and proliferating cell nuclear antigen (P CNA) expression as functions of drug treatment were assessed by multip arameter flow cytometry. Cellular respiration was measured by MTT anal ysis and apoptosis was detected by extraction of DNA fragments. Result s. Cell populations treated for up to 96 h with lometrexol or LY309887 did not replicate and maintained a cell cycle distribution with disti nct G(1), S and G(2)/M regions. The number of S phase cells in treated populations was slightly elevated relative to control as measured by DNA content and PCNA. However, these cells were unable to incorporate 5-bromodeoxyuridine (BrdU). Throughout treatment, cells incubated with GARFT inhibitors maintained intact membranes and respired at a level comparable to untreated cells. In contrast, ZD1694 as well as LY231514 , induced synchronization of the treatment population at the G(1)/S in terface within 12 h of drug addition. This was followed by synchronous entry of the population into S phase. After 24 h of treatment, more t han 90% of the cells were capable of incorporating BrdU and stained po sitive for PCNA. DNA fragmentation occurred in cells treated with ZD16 94 or LY231514 but not in those treated with GARFT inhibitors. In addi tion, the viable cells remaining after 24-48 h of treatment with ZD169 4 or LY231514 were respiring at twice the level of untreated cells. Co nclusion: These results demonstrate that the distinct endpoints of GAR FT and TS inhibition are preceded by distinct cell cycle and metabolic alterations.