Tunicamycin enhances the anticellular activity of interferon by inhibiting G1/S phase progression in 3T3 cells.
We have shown earlier that the cell growth inhibitory activity of interferon (IFN) is significantly enhanced by tunicamycin (TM) (Maheshwari et al., Science 219, 1339-1341, 1983). In this report, we investigated various regulatory points of synergistic action between TM and IFN-alpha/beta that inhibit cell growth in NIH 3T3 cells. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) viability assays showed a dose-dependent increase in percentage inhibition of the cells when treated with either TM or IFN. When doses of TM and IFN that had no significant inhibition on cell viability were used in combination, there was a pronounced suppression of DNA synthesis (tritiated thymidine incorporation). Flow cytometry studies revealed that individual treatments with either IFN or TM that did not alter the cell cycle profile, when combined, resulted in an impaired cell cycle by inhibiting G1/S progression. The blockage of G1/S transition was associated with reduction of cyclin-dependent kinase (CDK4) activity. The mRNA (analyzed by ribonuclease protection assay) and protein levels (assayed by Western blotting) of cyclins D1, D3, and CDK4 were downregulated by combined treatment with IFN and TM. An increase in the expression of p27/kipl, an inhibitor of CDK4, was observed in cells that were treated with both IFN and TM. These studies suggest that insufficient formation of the active cyclin/CDK complex could possibly be deferring the cells from normal cycling and may be responsible for the ability of TM to enhance cell growth inhibition induced by IFN.
Madhavan
S
S
Center for Combat Casualty and Life Sustainment Research, Department of Pathology, Uniformed Services University of Health Sciences, Bethesda, MD 20814, USA.
Singh
A K
AK
Maheshawari
R K
RK
D016475:3T3 Cells / Q000187:drug effects / Q000201:enzymology; D000818:Animals; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D019938:Cyclin D1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D056743:Cyclin D3; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D016213:Cyclins / Q000037:antagonists & inhibitors / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D004357:Drug Synergism; D016193:G1 Phase / Q000187:drug effects*; D006131:Growth Inhibitors / Q000502:physiology*; D007370:Interferon Type I / Q000494:pharmacology; D007372:Interferons / Q000502:physiology*; D051379:Mice; D011518:Proto-Oncogene Proteins; D016196:S Phase / Q000187:drug effects*; D013936:Thymidine / Q000378:metabolism; D014316:Tritium; D014415:Tunicamycin / Q000494:pharmacology*
C534697:Ccnd3 protein, mouse; D056743:Cyclin D3; D016213:Cyclins; D006131:Growth Inhibitors; D007370:Interferon Type I; D011518:Proto-Oncogene Proteins; D014316:Tritium; D014415:Tunicamycin; D019938:Cyclin D1; D007372:Interferons; C495902:Cdk4 protein, mouse; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases; D013936:Thymidine