Persistent membrane translocation of protein kinase C alpha during 12-0-tetradecanoylphorbol-13-acetate-induced apoptosis of LNCaP human prostate cancer cells (original) (raw)
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Journal of Biological Chemistry, 2000
Phorbol esters, the activators of protein kinase C (PKC), induce apoptosis in androgen-sensitive LNCaP prostate cancer cells. The role of individual PKC isozymes as mediators of this effect has not been thoroughly examined to date. To study the involvement of the novel isozyme PKC␦, we used a replication-deficient adenovirus (PKC␦AdV), which allowed for a tightly controlled expression of PKC␦ in LNCaP cells. A significant reduction in cell number was observed after infection of LNCaP cells with PKC␦AdV. Overexpression of PKC␦ markedly enhanced the apoptotic effect of phorbol 12myristate 13-acetate in LNCaP cells. PKC␦-mediated apoptosis was substantially reduced by the pan-caspase inhibitor z-VAD and by Bcl-2 overexpression. Importantly, and contrary to other cell types, PKC␦-mediated apoptosis does not involve its proteolytic cleavage by caspase-3, suggesting that allosteric activation of PKC␦ is sufficient to trigger apoptosis in LNCaP cells. In addition, phorbol ester-induced apoptosis was blocked by a kinase-deficient mutant of PKC␦, supporting the concept that PKC␦ plays an important role in the regulation of apoptotic cell death in LNCaP prostate cancer cells.
Biochemical and Biophysical Research Communications, 1998
AR gene , other cellular pathways may be involved. The protein kinase C (PKC) activator 12-O-tetrade-Growth factors that are recognized to have an imcanoyl-phorbol-13-acetate (TPA) activated cell death portant role in prostatic tumor biology (2, 7, 8) signal in androgen-sensitive LNCaP cells but not in androthrough protein kinase C (PKC) (9-11) to elicits a plethgen-independent DU-145 or PC-3 cells, whose growth ora of cellular responses on cell growth and differentiawas significantly decreased by PKC inhibitors staurotion (12, 13). Furthermore, androgenic regulation of sporine and H7. All cell lines had similar levels of total prostatic genes, including the androgen receptor, is dis-PKC activities which, however, differed on their derupted when cells are treated with protein kinase C pendency on Ca 2/ ions and lipid and were regulated activators (14). Here we show that the PKC activator differently by TPA. Furthermore, expression of the im-TPA elicits disparate responses in androgen-sensitive mediate early genes c-fos and c-jun was up-regulated and insensitive prostatic cancer cells suggesting that by TPA only in LNCaP and DU-145 cells, whereas PCalterations in PKC-isozymes may contribute to andro-3 cells failed to express c-fos mRNA. The regulation gen insensitivity in prostatic cancer. of the c-myc mRNA by TPA correlated inversely with activation of cell death being down-regulated in LNCaP cells, and slightly increased in the androgen-MATERIALS AND METHODS independent cell lines. These results suggest that the PKC signal transduction pathway functions differ-Probes. The glyceraldehyde-3-phosphate dehydrogenase (GADPH) 2 Corresponding
Protein Kinase C Isoenzyme Patterns Characteristically Modulated in Early Prostate Cancer
The American Journal of Pathology, 1999
and -, and of their common receptor for activated C-kinase (RACK)-1 , was determined immunohistochemically using specific antibodies in formalin-fixed and paraffin-embedded specimens of early prostatic adenocarcinomas (n ؍ 23) obtained at radical prostatectomy. Expression of each isoenzyme by malignant tissues was compared with nonneoplastic prostate tissues removed at radical cystectomy (n ؍ 10). The most significant findings were decreased PKC- expression in early neoplasia when compared to benign epithelium (P < 0.0001), together with a reciprocal increase in expression of PKC-⑀ (P < 0.0001). Detectable levels of PKC-␣ and PKC-were also significantly increased in the cancers (P ؍ 0.045 and P ؍ 0.015 respectively) but did not correlate with either PKC- or PKC-⑀ for individual cases. Alterations in the levels of the four PKC isoenzymes occurred specifically and consistently during the genesis and progression of human prostate cancer. PKC-␦, -␥, and -were not expressed in the epithelium of either the benign prostates or the cancers. Levels of expression for PKC-, -, -, and RACK-1 were not significantly different between the benign and malignant groups. Although changes in PKC isoenzyme expression may assist in explaining an altered balance between proliferation and apoptosis, it is likely that changes in activity or concentrations of these isoenzymes exert important modulating influences on particular pathways regulating cellular homeostasis. The findings of this study raise an exciting possibility of novel therapeutic intervention to regulate homeostatic mechanisms controlling proliferation and/or apoptosis , including expression of the p170 drug-resistance glycoprotein , intracellular Ca 2؉ concentrations , and enhanced cellular mobility resulting in the metastatic dissemination of human prostate cancer cells. Attenuation of PKC- expres-sion is currently being assessed as a reliable objective adjunct to morphological appearance for the diagnosis of early progressive neoplasia in human prostatic tissues.
Journal of Biological Chemistry, 2001
Phorbol esters, the archetypical (PKC) activators, induce apoptosis in androgen-sensitive LNCaP prostate cancer cells. In this study we evaluate the effect of a novel class of PKC ligands, the diacylglycerol (DAG)-lactones, as inducers of apoptosis in LNCaP cells. These unique ligands were designed using novel pharmacophore-and receptorguided approaches to achieve highly potent DAG surrogates. Two of these compounds, HK434 and HK654, induced apoptosis in LNCaP cells with much higher potency than oleoyl-acetyl-glycerol or phorbol 12,13dibutyrate. Moreover, different PKC isozymes were found to mediate the apoptotic effect of phorbol 12-myristate 13-acetate (PMA) and HK654 in LNCaP cells. Using PKC inhibitors and dominant negative PKC isoforms, we found that both PKC␣ and PKC␦ mediated the apoptotic effect of PMA, whereas only PKC␣ was involved in the effect of the DAG-lactone. The PKC␣ selectivity of HK654 in LNCaP cells contrasts with similar potencies in vitro for binding and activation of PKC␣ and PKC␦. Consistent with the differences in isoform dependence in intact cells, PMA and HK654 show marked differences in their abilities to translocate PKC isozymes. Both PMA and HK654 induce a marked redistribution of PKC␣ to the plasma membrane. On the other hand, unlike PMA, HK654 translocates PKC␦ predominantly to the nuclear membrane. Thus, DAG-lactones have a unique profile of activation of PKC isozymes for inducing apoptosis in LNCaP cells and represent the first example of a selective activator of a classical PKC in cellular models. An attractive hypothesis is that selective activation of PKC isozymes by pharmacological agents in cells can be achieved by differential intracellular targeting of each PKC.
Protein Kinase C Has the Potential to Advance the Recurrence of Human Prostate Cancer1
2002
Prostatic epithelial cells that are capable of surviving in the absence of androgenic steroids were found to express protein kinase C (PKC), an oncogenic protein capable of promoting autocrine cell-signaling events. Gene transfer experiments demonstrated that PKC overexpression was sufficient to transform androgen-dependent LNCaP cells into an andro- gen-independent variant that rapidly initiated tumor growth in vivo in both intact
European Journal of Biochemistry, 1997
Protein kinase C (PKC) has been implicated in signaling induced by diverse sets of stimuli regulating growth, differentiation, and apoptosis. The present study focused on the fate of PKC isotype proteins during Fas-mediated apoptosis of human leukemic cell lines. Among the PKC isotypes expressed in different cell types, such as Jurkat, HPB-ALL, U937, and HL60, all the nPKC isotypes including nPKC6, nPKCE, and nPKCB, but not cPKCa and fl1 and aPKCC (n, c, and a represent novel, conventional and atypical, respectively), showed limited proteolytic cleavage during Fas-mediated apoptosis. The limited proteolysis of nPKC isotypes means the disappearance of the intact protein band concomitant with the appearance of two fragments, most likely containing the kinase and regulatory domains, in contrast to the so-called down-regulation known for both cPKC and nPKC isotypes following exposure to stimuli such as 12-0-tetradecanoyl-phorbol 13-acetate (TPA). The time course of Fas-mediated apoptosis in Jurkat cells parallels that of the activation of a 32-kDa cysteine protease (CPP32)-like protease and also closely parallels the proteolytic cleavage of nPKC isotypes. A peptide inhibitor of the CPP32-like protease, Ac-DEVD-CHO, blocked the proteolytic cleavage of nPKC isotypes as well as apoptosis mediated by Fas. Transfection of recombinant protein coding for the catalytic fragment of nPKCG to COSl cells resulted in the apoptotic morphology of cells and nuclei. The effect of TPA on apoptosis depends on the cell type. TPA significantly suppressed Fas-mediated apoptosis in Jurkat, whereas TPA alone caused apoptosis in HPB-ALL, U937, and HL60, only slight apoptosis in Jurkat. The proteolytic fragmentation of nPKC isotypes again closely correlated with the degree of apoptosis even in apoptosis induced by TPA. Separation of TPA-treated cells into apoptotic and non-apoptotic differentiating cells revealed that the proteolytic fragmentation of nPKC isotypes occurs only in apoptotic cells and, in adherent differentiating cells, nPKC isotypes as well as cPKCa were downregulated without the generation of nPKC fragments. These results are consistent with the idea that nPKC isotypes meet two different fates, down-regulation and proteolytic cleavage generating kinase and regulatory fragments, and that the proteolytic cleavage of nPKC isotypes is a step in the signaling pathway involved in Fas-mediated and TPA-induced apoptosis.
Protein kinase Cɛ interacts with Bax and promotes survival of human prostate cancer cells
Oncogene, 2003
Prostatic glandular epithelial cells express protein kinase Ce (PKCe), an oncoprotein that coordinately disrupts the reactivation of the tumor suppressor Rb, derepressess transcriptional elongation of the c-myc oncogene, and propagates survival signals in LNCaP cells. Since the activation of such a program may contribute to the progression of human prostate cancer, a proteomic analysis was performed to gain a more global perspective on the signaling network that PKCe might be capable of engaging in prostate cancer cells. Using CWR22 xenografts, we identified at least 18 different structural, signaling, and stress-related proteins that associated with PKCe, including an interaction with the proapoptotic protein Bax that was novel to recurrent CWR22 tumors. An investigation into the biological significance of the PKCe association with Bax provided the first evidence of an inverse relationship between endogenous levels of PKCe and susceptibility of prostate cancer cells to the apoptotic effects of phorbol esters. Western blot and antisense experiments demonstrated that CWR-R1 cells expressed moderate levels of PKCe and relied on this protein to survive in the presence of phorbol esters, while the apoptosis normally induced by phorbol esters in PKCe-deficient LNCaP cells was dependent on the presence of Bax. Forced expression of PKCe in LNCaP cells was sufficient to confer a significant resistance to phorbol esters and this resistance was associated with an inhibition of phorbol ester-induced Bax conformational rearrangements that are important for Bax oligomerization, mitochondrial integration, and cytochrome c release. Considered in their entirety, our data suggest that an association of PKCe with Bax may neutralize apoptotic signals propagated through a mitochondrial death-signaling pathway.