Abstract
Previously, we reported that 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced apoptosis of LNCaP human prostate cancer cells was accompanied by prolonged translocation of protein kinase C (PKC)α to non-nuclear membranes and that TPA-resistant LNCaP cells had down-regulated PKCα. Here we show that 10 nM bryostatin 1 induced transient membrane translocation and down-regulation of PKCα, prolonged translocation of PKCδ and ε to non-nuclear membranes, and did not induce cell death but blocked TPA-induced apoptosis. To test the hypothesis that inhibition of TPA-induced apoptosis by bryostatin 1 was due to down-regulation of PKCα, we inducibly overexpressed PKCα in LNCaP cells. Overexpression of PKCα alone did not induce apoptosis, even in clones that contained much more membrane-bound, active PKCα than was observed in TPA-treated untransfected LNCaP cells. However, the addition of 10 nM bryostatin 1 to PKCα-overexpressing LNCaP cells did not yield down-regulation of PKCα and induced extensive apoptosis. Immunoblot analysis revealed that TPA induced prolonged hyperphosphorylation of Raf-1 and activation of extracellular-regulated/mitogen-activated protein kinases 1 and 2 in untransfected LNCaP cells, as did bryostatin 1 in PKCα-overexpressing cells. On the other hand, bryostatin 1 induced only transient hyperphosphorylation of Raf-1 and activation of extracellular-regulated/mitogen-activated protein kinases 1 and 2 in untransfected LNCaP cells. These results confirm a role of prolonged membrane-associated PKCα in PKC activator-mediated LNCaP apoptosis and suggest involvement of the mitogen-activated protein kinase pathway.
Footnotes
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Send reprint requests to: C. Thomas Powell, Ph.D., Department of Cancer Biology, The Cleveland Clinic Foundation, 9500 Euclid Ave.-ND50, Cleveland, OH 44195.
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↵1 Current address: Department of Urology, University of Ulm, 89075 Ulm, Germany.
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This work was supported in part by National Institutes of Health Grant DK/CA47650. J.E.G. was supported by Deutsche Forschungsgemeinschaft (Bonn, Germany).
- Abbreviations:
- TPA
- 12-O-tetradecanoylphorbol-13-acetate
- ERK
- extracellular-regulated/mitogen-activated protein kinase
- FBS
- fetal bovine serum
- PKC
- protein kinase C
- tet
- tetracycline
- tTA
- tetracycline-repressible transactivator protein
- XTT
- 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt
- Received September 3, 1999.
- Accepted February 23, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
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