Carcinogenesis Advance Access published online on December 4, 2003
Carcinogenesis, doi:10.1093/carcin/bgh041
© 2003 by Oxford University Press
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MOLECULAR EPIDEMIOLOGY AND CANCER PREVENTION
1 Department of Cancer Biology, University of Texas M.D. Anderson Cancer Center, Houston, TX
* Corresponding author. E-mail: obrian{at}mdacc.tmc.edu.
Received 28 September 2003
; revised 20 November 2003
; accepted 22 November 2003
We previously reported that cystine produces regulatory responses in purified, recombinant human protein kinase C-
Cellular protein kinase C isozyme regulation by exogenously delivered physiological disulfides - implications of oxidative protein kinase C regulation to cancer prevention
(PKC) and PKC
via S-thiolation-triggered mechanisms that are consistent with a cancer preventive effect, i.e., stimulation of the pro-apoptotic, tumor-suppressive isozyme PKC and inactivation of the growth-stimulatory, oncogenic isozyme PKC, at S-cysteinylation stoichiometries that correspond to modification of a single redox-regulatory Cys switch in each isozyme. In this report, we show that the oxidative regulatory responses of purified PKC and PKC to cystine are recapitulated in disulfide-treated cells. We report that treatment of COS7-PKC transfectants with the cystine precursor cystine dimethyl ester (CDME) produced concentration- and time-dependent PKC inactivation that was associated with oxidative PKC modification manifested as attenuated band intensity in PKC immunoblot analyses, and that both PKC inactivation and modification were reversed by dithiothreitol (DTT) as well as by thioredoxin. We also show that CDME induced biphasic PKC regulation in COS7-PKC transfectants, with DTT-irreversible PKC stimulation at low and DTTreversible PKC inactivation at high CDME concentrations. The degrees of PKC versus PKC inactivation by CDME treatment of COS7-PKC transfectants indicate substantial resistance of PKC to inactivation. The PKC stimulatory response in COS7-PKC cells was triggered only by the disulfide agent and not by its reduced thiol counterpart, providing evidence for an oxidative mechanism. Also paralleling the oxidative stimulation of purified PKC by cystine, the stimulation of PKC elicited by CDME treatment of cells involved a stable structural change, which was evident from the stability of the stimulated form of PKC to immunoprecipitation. Demonstration of oxidative regulation of cellular PKC and PKC by disulfides in this report provides evidence that redox-regulatory sites in PKC and PKC may offer novel targets for development of cancer preventive or therapeutic agents that selectively inactivate PKC or stimulate PKC.
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