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Keratinocyte apoptosis induced by UV radiation is a major protective mechanism from skin photocarcinogenesis. The induction of apoptosis by UV radiation, as well as a variety of genotoxic stimuli, involves the activation of PKC-d by caspase-3-mediated cleavage in its hinge region, thus generating a constitutively active catalytic fragment. To determine the role of PKC-d cleavage in UV apoptosis signaling, we introduced a caspase-resistant PKC-d mutant (D330A) into human keratinocytes by retrovirus transduction. Overexpression of PKC-d(D330A) protected keratinocytes from UV-induced apoptosis and enhanced long-term survival. PKC-d(D330A) partially prevented the release of cytochrome c from the mitochondria and the loss of Mcl-1, a key antiapoptotic protein downregulated during UV apoptosis. Thus, the cleavage and activation of PKC-d are critical components of UV-induced apoptosis in human keratinocytes, and the inactivation of PKC-d can promote the survival of keratinocytes exposed to UV radiation.

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Purpose: This study aimed to identify and evaluate molecular targets for the development of a novel combination chemotherapy to treat refractory and recurrent diffuse large B-cell lymphoma (DLBCL). Experimental Design: Lymphoma samples from 38 cases of primary and recurrent DLBCL were analyzed using real-time quantitative PCR of the RPS6KB1 and CDC2 genes, and immunohistochemistry for their gene products p70S6K/p85S6K and cdc2/cdk1.The Farage, Karpas422, Pfeiffer, and Toledo DLBCL cell lines were subsequently treated with rapamycin and UCN-01 alone or in combination. Cell proliferation, apoptosis, and cell cycle progression were analyzed after the drug treatment. In addition, the levels of several key protein kinases involved in the phosphoinositide 3 ¶-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway, apoptosis, and cell cycle progression were analyzed in the presence and absence of the drugs. Results: Amplification of the RPS6KB1 and CDC2 genes was found in both primary and recurrent DLBCL. Moreover, the vast majority of these lymphomas (f94%) were strongly positive for phospho-p70S6K and cdc2/cdk1 proteins. The combination of rapamycin and UCN-01 synergistically inhibited the DLBCL cell proliferation by inducing G 1 arrest as well as apoptosis by suppressing the phosphorylation of p70S6K/p85S6K and CDC2 expression. Conclusion: RPS6KB1 and CDC2 overexpression is common in DLBCL. Simultaneously targeting the RPS6KB1 and CDC2 products phospho-p70S6K/p85S6K and cdc2/cdk1 is very effective in inhibiting DLBCL proliferation and overcoming drug resistance. This work suggests that multilevel inhibition of the PI3K/Akt/mTOR pathway and double-block of cell cycle progression are effective strategies for DLBCL therapy.