Cell death mediated by alloreactive cytotoxic T cells via the granule exocytosis or the Fas pathway is independent of p34cdc2 kinase: Fas dependent killing of cells arrested in the cell cycle.
Inappropriate activation of p34cdc2 kinase has been shown to occur during apoptosis induced by cytotoxic T-cell derived perforin and fragmentin. We analysed the effect of two inhibitors of p34cdc2 kinase on alloreactive Tc-cell-mediated lysis and DNA fragmentation of P815 and L1210 target cells. Olomoucine, a specific inhibitor of cyclin dependent kinases, did not affect DNA fragmentation in the target cells. Lysis of olomoucine-treated target cells as assessed by 51Cr release over a typical 8-h period was also unaffected. We also examined the effects of thapsigargin on target cell death. This toxin causes increased intracellular calcium rises that then result in irreversible inhibition of cyclin dependent kinases, including p34cdc2 kinase. The same extent of specific cell lysis was induced by cytotoxic T cells from perforin(-/-), granzyme B(-/-), granzyme A(-/-), perforin(-/-) X granzymeB(-/-) X granzymeA(-/-) KO mice or normal mice in untreated target cells or target cells treated with either olomoucine or thapsigargin. Similarly DNA fragmentation measured by release of tritiated DNA was also unaffected. Thus inhibition of p34cdc2 kinase affects neither the Fas nor the perforin/granzyme pathways of alloreactive cytotoxic T-cell killing as measured by DNA fragmentation or chromium release. P815 cells treated with olomoucine were arrested in the cell cycle after 12-16 h exposure to the toxin. After cell cycle arrest, target cells now showed enhanced 51Cr release induced by effector cytotoxic T cells (CTL) derived from perforin(-/-) mice compared to untreated cells. This lysis was accompanied by an increase in cell surface Fas expression. Olomoucine induced cell cycle arrest and expression of Fas was reversible and when cells re-entered the cell cycle, surface expression of Fas was lost.
Waring
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Division of Immunology and Cell Biology, John Curtin School of Medical Research, Australian National University, Canberra, Australian Capital Territory, Australia. paul.waring@anu.edu.au
Müllbacher
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D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D017489:CDC28 Protein Kinase, S cerevisiae / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology; D002469:Cell Separation; D003601:Cytotoxicity Tests, Immunologic; D053938:DNA Fragmentation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005089:Exocytosis / Q000502:physiology; D005434:Flow Cytometry; D007694:Killer Cells, Natural / Q000378:metabolism; D007701:Kinetin; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D011687:Purines / Q000494:pharmacology*; D013602:T-Lymphocytes, Cytotoxic / Q000502:physiology*; D019284:Thapsigargin / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D019014:fas Receptor / Q000378:metabolism*
D004791:Enzyme Inhibitors; D011687:Purines; D019014:fas Receptor; D019284:Thapsigargin; C090046:olomoucine; D017489:CDC28 Protein Kinase, S cerevisiae; D007701:Kinetin