Individual protein kinase C (PKC) isozymes have been implicated in many cellular responses important in lung health and disease, including permeability, contraction, migration, hypertrophy, proliferation, apoptosis, and secretion. New ideas on mechanisms that regulate PKC activity, including the identification of a novel PKC kinase, 3-phosphoinositide-dependent kinase-1 (PDK-1), that regulates phosphorylation of PKC, have been advanced. The importance of targeted translocation of PKC and isozyme-specific binding proteins (like receptors for activated C-kinase and caveolins) is well established. Phosphorylation state and localization are now thought to be key determinants of isozyme activity and specificity. New concepts on the role of individual PKC isozymes in proliferation and apoptosis are emerging. Opposing roles for selected isozymes in the same cell system have been defined. Coupling to the Wnt signaling pathway has been described. Phenotypes for PKC knockout mice have recently been reported. More specific approaches for studying PKC isozymes and their role in cell responses have been developed. Strengths and weaknesses of different experimental strategies are reviewed. Future directions for investigation are identified.

We have previously shown that parotid C5 salivary acinar cells undergo apoptosis in response to etoposide treatment as indicated by alterations in cell morphology, caspase-3 activation, DNA fragmentation, sustained activation of c-Jun N-terminal kinase, and inactivation of extracellular regulated kinases 1 and 2. Here we report that apoptosis results in the caspase-dependent cleavage of protein kinase C-␦ (PKC␦) to a 40-kDa fragment, the appearance of which correlates with a 9-fold increase in PKC␦ activity. To understand the function of activated PKC␦ in apoptosis, we have used the PKC␦-specific inhibitor, rottlerin. Pretreatment of parotid C5 cells with rottlerin prior to the addition of etoposide blocks the appearance of the apoptotic morphology, the sustained activation of c-Jun N-terminal kinase, and inactivation of extracellular regulated kinases 1 and 2. Inhibition of PKC␦ also partially inhibits caspase-3 activation and DNA fragmentation. Immunoblot analysis shows that the PKC␦ cleavage product does not accumulate in parotid C5 cells treated with rottlerin and etoposide together, suggesting that the catalytic activity of PKC␦ may be required for cleavage. PKC␣ and PKC␤1 activities also increase during etoposide-induced apoptosis. Inhibition of these two isoforms with Gö 6976 slightly suppresses the apoptotic morphology, caspase-3 activation, and DNA fragmentation, but has no effect on the sustained activation of c-Jun N-terminal kinase or inactivation of extracellular regulated kinase 1 and 2. These data demonstrate that activation of PKC␦ is an integral and essential part of the apoptotic program in parotid C5 cells and that specific activated isoforms of PKC may have distinct functions in cell death.

The most significant long-term complication of radiotherapy in the head and neck region is hyposalivation and its related complaints, particularily xerostomia. This paper addresses the pathophysiology underlying irradiation damage to salivary gland tissue, the consequences of radiation injury, and issues contributing to the clinical management of salivary gland hypofunction and xerostomia. These include ways to: (1) prevent or minimize radiation injury of salivary gland tissue, (2) manage radiation-induced hyposalivation and xerostomia, and (3) restore the function of salivary gland tissue damaged by radiotherapy.

We have shown previously that protein kinase Cd (PKCd) is required for mitochondrial-dependent apoptosis. Here we show that PKCd is imported into the nucleus of etoposide-treated cells, that nuclear import is required for apoptosis and that it is mediated by a nuclear localization signal (NLS) in the C-terminus of PKCd. Mutation of the caspase cleavage site of PKCd inhibits nuclear accumulation in apoptotic cells, indicating that caspase cleavage facilitates this process. Expression of the PKCd catalytic fragment (CFd) in transfected cells results in nuclear localization and apoptosis. We show that the PKCd NLS is required for nuclear import of both full-length PKCd and CFd, and drives nuclear localization of a multimeric green¯uorescent protein. Mutations within the NLS of CFd prevent nuclear accumulation and block apoptosis. Conversely, nuclear expression of a kinase-negative catalytic fragment (KN-CFd) protects cells from etoposide-induced apoptosis. Mutation of the NLS blocks the ability of KN-CFd to protect against etoposide-induced apoptosis. These results indicate that PKCd regulates an essential nuclear event(s) that is required for initiation of the apoptotic pathway.

The protein kinase C (PKC) family consists of 10 related serine/threonine protein kinases some of which are critical regulators of cell proliferation, survival and cell death. While early studies relied on broad spectrum chemical activators or inhibitors of this family, the generation of isoform specific tools has greatly facilitated our understanding of the contribution of specific PKC isoforms to cell proliferation and apoptosis. These studies suggest that PKC-alpha, PKC-epsilon, and the atypical PKC’s, PKC-lambda/iota and PKC-zeta, preferentially function to promote cell proliferation and survival, while the novel isoform, PKC-delta is an important regulator of apoptosis. The essential role of this kinase family in both cell survival and apoptosis suggests that specific isoforms may function as molecular sensors, promoting cell survival or cell death depending on environmental cues. Given their central role in cell and tissue homeostasis, it is not surprising that the expression or activity of some of these kinases is altered in human diseases, particularly cancer.

Protein kinase C (PKC)␦

We report here that the novel protein kinase C isoform, PKC␦, is required at or prior to the level of the mitochondria for apoptosis induced by a diverse group of cell toxins. We have used adenoviral expression of a kinase-dead (KD) mutant of PKC␦ to explore the requirement for PKC␦ in the mitochondrial-dependent apoptotic pathway. Expression of PKC␦KD, but not PKC␣KD, in salivary epithelial cells resulted in a dosedependent inhibition of apoptosis induced by etoposide, UV-irradiation, brefeldin A, and paclitaxel. DNA fragmentation was blocked up to 71% in parotid C5 cells infected with the PKC␦KD adenovirus, whereas caspase-3 activity was inhibited up to 65%. The activation of caspase-9-like proteases by all agents was also inhibited in parotid C5 cells expressing PKC␦KD. The ability of PKC␦KD to block the loss of mitochondrial membrane potential was similarly determined. Expression of PKC␦KD blocked the decrease in mitochondrial membrane potential observed in cells treated with etoposide, UV, brefeldin A, or paclitaxel in a dose-dependent manner. In contrast to the protective function of PKC␦KD, expression of PKC␦WT resulted in a potent induction of apoptosis, which could be inhibited by coinfection with PKC␦KD. These results suggest that PKC␦ is a common intermediate in mitochondrial-dependent apoptosis in salivary epithelial cells.

Background The most manifest long-term consequences of radiation therapy in the head and neck cancer patient are salivary gland hypofunction and a sensation of oral dryness (xerostomia). Methods This critical review addresses the consequences of radiation injury to salivary gland tissue, the clinical management of salivary gland hypofunction and xerostomia, and current and potential strategies to prevent or reduce radiation injury to salivary gland tissue or restore the function of radiation-injured salivary gland tissue. Results Salivary gland hypofunction and xerostomia have severe implications for oral functioning, maintenance of oral and general health, and quality of life. Significant progress has been made to spare salivary gland function chiefly due to advances in radiation techniques. Other strategies have also been developed, e.g., radioprotectors, identification and preservation/expansion of salivary stem cells by stimulation with cholinergic muscarinic agonists, and application of new lubricating or stimulatory agents, surgical transfer of submandibular glands, and acupuncture. Conclusion Many advances to manage salivary gland hypofunction and xerostomia induced by radiation therapy still only offer partial protection since they are often of short duration, lack the protective effects of saliva, or potentially have significant adverse effects. Intensity-modulated radiation therapy (IMRT), and its next step, proton therapy, have the greatest potential as a management strategy for permanently preserving salivary gland function in head and neck cancer patients. Presently, gene transfer to supplement fluid formation and stem cell transfer to increase the regenerative potential in radiation-damaged salivary glands are promising approaches for regaining function and/or regeneration of radiation-damaged salivary gland tissue.