Related Experiment Videos
Cell cycle effects of antifolate antimetabolites: implications for cytotoxicity and cytostasis
J L Tonkinson1, P Marder, S L Andis
1Lilly Research Laboratories, Lilly Corporate Center, Eli Lilly and Co., Indianapolis, IN 46285, USA.
Purpose:
Cell cycle-related events in CCRF-CEM lymphocytic leukemia cells 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, nucleoside precursor incorporation and proliferating cell nuclear antigen (PCNA) expression as functions of drug treatment were assessed by multiparameter flow cytometry. Cellular respiration was measured by MTT analysis and apoptosis was detected by extraction of DNA fragments.
Results:
Cell populations treated for up to 96h with lometrexol or LY309887 did not replicate and maintained a cell cycle distribution with distinct G1, S and G2/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 G1/S interface within 12h of drug addition. This was followed by synchronous entry of the population into S phase. After 24 h of treatment, more than 90% of the cells were capable of incorporating BrdU and stained positive for PCNA. DNA fragmentation occurred in cells treated with ZD1694 or LY231514 but not in those treated with GARFT inhibitors. In addition, the viable cells remaining after 24-48 h of treatment with ZD1694 or LY231514 were respiring at twice the level of untreated cells.
Conclusion:
These results demonstrate that the distinct endpoints of GARFT and TS inhibition are preceded by distinct cell cycle and metabolic alterations.
Insights
Inhibition of thymidylate synthase (TS) or GAR formyltransferase (GARFT) in leukemia cells causes distinct cell cycle and metabolic changes. GARFT inhibition halts replication, while TS inhibition synchronizes cells and increases respiration before apoptosis.
Area of Science:
- Cell biology
- Cancer research
- Biochemistry
Background:
- Thymidylate synthase (TS) and GAR formyltransferase (GARFT) are critical enzymes in folate metabolism.
- Inhibition of these enzymes can disrupt DNA synthesis and cell proliferation.
- Understanding the distinct effects of inhibiting TS versus GARFT is crucial for developing targeted cancer therapies.
Purpose of the Study:
- To investigate the distinct cell cycle and metabolic alterations in CCRF-CEM lymphocytic leukemia cells following inhibition of TS or GARFT.
- To differentiate the cellular responses to specific inhibitors of TS (ZD1694), GARFT (Lometrexol, LY309887), and a multi-targeted antifolate (LY231514).
Main Methods:
- CCRF-CEM cells were treated with GARFT inhibitors (Lometrexol, LY309887), TS inhibitor (ZD1694), or a multi-targeted antifolate (LY231514).
- Cell cycle progression was analyzed using DNA content and PCNA expression via flow cytometry.
- Nucleoside precursor incorporation (BrdU) assessed DNA synthesis.
- Cellular respiration was measured by MTT assay, and apoptosis by DNA fragmentation.
Main Results:
- GARFT inhibitors (Lometrexol, LY309887) prevented cell replication, maintaining distinct G1, S, and G2/M phases, with no BrdU incorporation or DNA fragmentation.
- TS inhibitor (ZD1694) and multi-targeted antifolate (LY231514) induced G1/S synchronization, followed by S phase entry, increased BrdU incorporation, and DNA fragmentation.
- Cells treated with ZD1694 or LY231514 exhibited doubled respiration rates compared to controls.
Conclusions:
- Inhibition of GARFT and TS leads to distinct cell cycle arrest and metabolic profiles in leukemia cells.
- GARFT inhibition results in a non-replicative state without inducing apoptosis.
- TS inhibition triggers cell cycle synchronization, increased metabolic activity, and subsequent apoptosis.