Expression Of Protein Kinase C Zeta Research Articles

Inhibition of human androgen-independent PC-3 and DU-145 prostate cancers by antagonists of bombesin and growth hormone releasing hormone is linked to PKC, MAPK and c-jun intracellular signalling

Bombesin/gastrin-releasing peptide (BN/GRP) antagonists RC-3940-II and RC-3940-Et, and growth hormone-releasing hormone (GHRH) antagonists MZ-J-7-118 and RC-J-29-18 inhibit the growth of human androgen-independent PC-3 and DU-145 prostate cancers in nude mice. Additive inhibitory effects were observed after treatment with both classes of analogs. In the present study, we investigated the effects of these antagonists on intracellular signalling pathways of protein kinase C (PKC), mitogen activated protein kinases (MAPK) and c-fos and c-jun oncogenes that are involved in tumour cell proliferation. In PC-3 tumours, antagonists of BN/GRP and GHRH decreased significantly the expression of PKC isoforms alpha (α), eta (η) and zeta (ζ) and increased that of delta (δ) PKC protein. MAPK was not detectable. In DU-145 tumours, which constitutively express MAPK, all treatments strongly decreased the levels of p42/44 MAPK. Treatment with the antagonists tended to reduce m-RNA for c-jun in both tumour models. In proliferation assays in vitro, inhibitors of PKC and MAPK diminished growth of DU-145 and PC-3 cells. These findings suggest that antagonists of BN/GRP and GHRH inhibit the growth of androgen-independent prostate cancer by affecting intracellular signalling mechanisms of PKC, MAPK and c-jun.

Expression of protein kinase C isoenzymes in benign hyperplasia and carcinoma of prostate

Protein kinase C family consists of 11 isoforms, classified into 3 categories according to their structure and mechanisms of activation. These isoenzymes are involved in processes, which maintain intracellular homeostasis. Alterations in activity, amount or distribution of protein kinase C (PKC) isoenzymes may cause cellular proliferation or induce apoptosis. We have studied and compared the expression levels of several PKC isoforms in benign prostatic hyperplasia (BPH) and prostate cancer (PCa). These are PKCs alpha (alpha), beta (beta), delta (delta), epsilon (epsilon), zeta (zeta), eta (eta), which have been detected as major isoforms in prostate tissue. Paraffin sections of 25 benign prostatic hyperplasia (BPH) and 25 of prostatic carcinoma (PCa) were examined for expression of PKC alpha, beta, delta, epsilon, zeta, and eta. Expression of PKC beta was examined in additional 3 BPH and 3 PCa using Western blot analysis. We found a significant high level of expression of PKC isoforms alpha, beta, epsilon and eta in PCa compared to BPH (p<0.01). Using backward logistic regression, we found changes in PKC epsilon expression to be most significant between malignant compared to benign tumor tissue specimens. Immunostaining for PKCs alpha, beta and eta in addition to PKC epsilon may aid in distinguishing between benign and malignant prostatic disease.

Induction and subcellular localization of protein kinase C isozymes following renal ischemia

We have previously reported that the expression of the receptor for activated C kinase (RACK1) is induced post-ischemia/preperfusion injury to the kidney, and activation of protein kinase C (PKC) protects renal cells from hypoxic injury. This study was done to determine whether the induced expression of RACK1 is accompanied by changes in the level of expression and subcellular distribution of PKC isozymes. Ischemia/reperfusion injury resulting in acute renal failure was induced by 60 minutes of bilateral renal artery clamping in rats. The expression levels and translocation of various PKC isozymes between soluble and particulate fractions in whole kidney homogenates were demonstrated by immunoblot analysis. The expression pattern of the various PKC isozymes in the kidney postinjury was performed by immunohistochemistry. PKC alpha, beta II, and zeta were induced and translocated from the soluble fraction to the particulate fraction post-injury. Immunolocalization showed PKC alpha, beta II, and zeta expression to be induced in the proximal tubule epithelial cell (PTEC) at 0 to 30 minutes post-ischemia/reperfusion injury (IRI). At one-day postinjury, the alpha isozyme was translocated to the plasma membrane of the undamaged PTEC, while it was translocated to the nucleus in damaged PTEC. PKC beta II expression was along the basal and lateral side of the undamaged PTEC, while it was distributed in the cytoplasm of sloughed cells in the damaged PTEC. PKC zeta expression at one day was along the apical side of the damaged PTEC. At seven-days postinjury, the expressions of the alpha and zeta isozymes were localized to the plasma membrane of the regenerating PTEC and the expression of PKC beta II isozyme to certain interstitial cells. The induced expression, translocation, and the intracellular spatial distributions of the enzymes suggest that they may mediate multiple processes during IRI.

Differential expression of protein kinase C isoforms in streptozotocin-induced diabetic rats

The cellular effects of hyperglycemia are mediated by protein kinase C (PKC). However, PKC consists of several distinct isoforms, and their contribution to the pathogenesis of diabetic complications in different organs is not clear. We investigated the expression and translocation of PKC isoforms alpha, betaI, betaII, delta, epsilon, and zeta in kidney, heart, and aorta from diabetic rats. Hyperglycemia was induced with streptozotocin (70 mg/kg) in the rat. After four weeks, PKC isoform expression was assessed by Western blot after tissue fractionation and by immunohistochemistry. Streptozotocin increased blood glucose from 117.0 +/- 3.6 to 510.0 +/- 19.4 mg/dl (N = 8, P < 0.01) and induced albuminuria. PKC isoforms alpha, beta, delta, epsilon, and zeta were all detected in control animals. Western blot showed increased PKC alpha expression in kidney and heart (160% and 170%, respectively). PKC betaI, betaII, and delta expression was not influenced by hyperglycemia. PKC zeta was decreased in diabetic animals in both tissues by 60%. The membrane association of PKC alpha and PKC epsilon was increased; however, the relative amount of PKC in the particulate fraction was not influenced by hyperglycemia. Immunohistochemistry revealed a marked increase in PKC alpha immunoreactivity in renal glomeruli and interstitial capillaries, cardiac capillaries, and skeletal muscle, as well as in the endothelial cells of larger arteries. PKC beta showed a small decrease in the glomeruli. PKC epsilon was increased in renal tubules in diabetic rats but was decreased in the myocardium. PKC zeta was expressed in both myocardial and glomerular cells but was decreased during hyperglycemia. Our results demonstrate that PKC isoforms are differentially regulated in kidney and heart in diabetes. High glucose increases PKC alpha expression, whereas PKC zeta is down-regulated. The finding that PKC alpha is mostly increased in endothelial cells supports a role for PKC alpha in functional endothelial disturbances observed in diabetes.

Tissue-specific protein kinase C isoforms differentially mediate macrophage TNFalpha and IL-1beta production.

Macrophage subpopulations are differentially activated during sepsis, shock, or trauma; however, it is unknown whether inherent mechanistic and phenotypic differences exist between macrophage subpopulations that may account for region-specific inflammation. We hypothesized that macrophage expression/function of protein kinase C (PKC) isoforms is tissue specific (alveolar versus peritoneal). Rat alveolar and peritoneal macrophages were each probed for the expression of PKC isoforms alpha, beta1, beta2, gamma, delta, epsilon, zeta, and theta by immunoblot. PKC isoforms alpha, beta1, beta2, and zeta were detected in both populations; however, isoforms epsilon, gamma, and eta were found in alveolar macrophages only. To investigate the functional role of the Ca2+-dependent PKC (cPKC) versus Ca2+-independent PKC (nPKC) isoforms, pan-PKC isoform inhibition (cPKC and nPKC), or cPKC isoform selective inhibition (alpha, beta1, beta2, gamma) was performed before endotoxin (lipopolysaccharide, Salmonella minnesota, 100 ng/mL) stimulation in vitro. Pan-PKC isoform inhibition attenuated TNFalpha and IL-1beta production by each population; however, selective cPKC (alpha, beta1, beta2, gamma) inhibition decreased peritoneal, but not alveolar, macrophage TNFalpha production. IL-1beta production was not affected by cPKC inhibition in either population. 1) alveolar and peritoneal macrophages constitutively express different PKC isoforms; 2) alveolar macrophages uniquely express isoforms epsilon, gamma, eta; 3) TNFalpha production is regulated by cPKCs in peritoneal macrophages, but by nPKCs in alveolar macrophages; 4) nPKCs regulate IL-1beta production in both populations. These results suggest that tissue-specific PKC isoforms differentially mediate macrophage function, which may have important regulatory implications in the compartmentalization of immune function. Further understanding may allow region-specific manipulation of inflammation.

Non-invasive detection of fecal protein kinase C betaII and zeta messenger RNA: putative biomarkers for colon cancer.

We have developed a non-invasive method utilizing feces, containing sloughed colonocytes, as a sensitive technique for detecting diagnostic colonic biomarkers. In this study, we used the rat colon carcinogenesis model to determine if changes in fecal protein kinase C (PKC) expression have predictive value in monitoring the neoplastic process. Weanling rats were injected with saline or azoxymethane (AOM) and 36 weeks later fecal samples and mucosa were collected, poly A+ RNA isolated, and quantitative RT-PCR performed using primers to PKC betaII and zeta. Fecal PKC betaII and zeta mRNA levels were altered by the presence of a tumor, with tumor-bearing animals having a 3-fold higher (P < 0.05) PKC betaII expression as compared with animals without tumors. In addition, AOM-injection increased mucosal PKC betaII mRNA expression compared with saline controls. No effect of tumor incidence on mucosal PKC betaII expression was observed. In contrast, fecal PKC zeta expression was 2.5-fold lower (P < 0.05) in animals injected with azoxymethane versus saline. Since tumor incidence exerts a reciprocal effect on fecal PKC betaII and zeta mRNA expression, data were also expressed as the ratio between PKC betaII and zeta. The isozyme ratio was strongly related to tumor incidence, i.e. ratio for animals with tumors was 2.18 +/- 1.25, animals without tumors was 0.50 +/- 0.16, P = 0.025. We demonstrate that the expression of fecal PKC betaII and zeta may serve as a noninvasive marker for development of colon tumors. A sensitive technique for the detection of colon cancer is of importance since early diagnosis can substantially reduce mortality.

Protein kinase C in the developing kidney: Isoform expression and effects of ceramide and PKC inhibitors

Protein kinase C (PKC) is a serine/threonine kinase recognized as a key enzyme in signal transduction mechanisms in various biological processes. During development, PKC is involved in the regulation of growth and differentiation. In mature tissue PKC is important for homeostatic functions. We studied PKC with regard to expression and effects on differentiation, growth and apoptosis in the developing kidney. Using in situ hybridization, we demonstrate age-dependent expression of PKC alpha, PKC delta, PKC zeta and PKC lambda during fetal and postnatal kidney development. The endogenous sphingolipid product ceramide, as well as specific PKC inhibitors, disturbed nephron formation and induced apoptosis in organ cultures of E13 kidneys. In primary cell cultures of proximal tubule cells, ceramide and the specific PKC inhibitors induced apoptosis. In conclusion, PKC alpha, PKC delta, PKC zeta and PKC lambda are expressed in an age-dependent pattern during kidney development. Inhibition of PKC disturbs nephron formation, inhibits growth and induces apoptosis in the developing kidney. The findings suggest that PKC plays an important role in regulating normal kidney growth and differentiation.

Protein kinase C-epsilon is the likely mediator of mucin exocytosis in human colonic cell lines.

The phorbol ester, phorbol 12-myristate 13-acetate (PMA), induces mucin secretion in the colonic tumor cell line T84 in a Ca(2+)-independent manner. To determine whether a specific protein kinase C (PKC) isoform is involved in colonic cells, we compared PMA-dependent mucin secretion by three human colonic tumor cell lines (T84, HT-29/A1, and LS 180) with the expression of PKC isoforms alpha, beta, delta, epsilon, and zeta, previously identified in human colon (L. A. Davidson, Y. H. Jiang, J. D. Derr, H. Aukema, J. R. Lupton, and R. S. Chapkin. Arch. Biochem. Biophys. 312:547-553, 1994). In each cell line PMA (10(-7) M) caused mucin secretion within 30 min. PMA-dependent mucin secretion was three to four times greater from HT-29/A1 and T84 cells than from LS 180 cells. All three-cell lines contained mRNA for PKC-alpha, PKC-epsilon, and PKC-zeta but not PKC-beta or -delta. Each cell line also expressed PKC-alpha, -epsilon, and -zeta protein. PKC-epsilon expression (mRNA and protein) was three to four times greater in HT-29/A1 and T84 cells than in LS 180 cells, correlating with PMA-responsive mucin secretion, whereas all cell lines contained similar levels of PKC-alpha mRNA and protein. When cells were stimulated by PMA, only PKC-epsilon was translocated from cytosol to membrane fractions early enough to stimulate mucin secretion. Because PKC-epsilon is also a Ca(2+)-independent isoform, it is likely to mediate mucin exocytosis in colonic cells.

Differential growth regulation by all-trans retinoic acid is determined by protein kinase C alpha in human pancreatic carcinoma cells.

We have investigated the role of protein kinase C (PKC) isoenzymes in the differential growth regulation of human pancreatic carcinoma cell lines by all-trans retinoic acid (RA). RA treatment results in dose-dependent stimulation of anchorage-independent growth in AsPc1 cells and growth inhibition in Capan 2 cells. Both cell lines express an identical pattern of nuclear RA and retinoid X receptors as determined by RT-PCR. Western blotting using monospecific antibodies revealed that both cell lines express PKC isoenzymes alpha and zeta, whereas beta, gamma, delta, and epsilon were not detected. Incubation with RA in the growth-stimulated AsPc1 cell line resulted in induction of PKC alpha expression, whereas PKC alpha expression was decreased by RA in the growth-inhibited Capan 2 cell line. In contrast, PKC zeta expression was not affected by RA in either cell line. Incubation of AsPc1 cells with the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate resulted in a time- and dose-dependent selective down-regulation of PKC alpha but not zeta. The dose-dependent decrease of intracellular PKC alpha concentration correlated well with the anchorage-independent growth rate of AsPc1 cells. Furthermore, selective down-regulation of PKC alpha blocks subsequent growth stimulation by RA in AsPc1 cells. When PKC alpha concentration was decreased by stably transfecting AsPc1 cells with a PKC alpha complementary DNA antisense construct, RA-stimulated growth could also be partially blocked. These data, therefore, suggest that differential regulation of PKC alpha expression plays a central role in determining the bidirectional effects of RA on growth in pancreatic carcinoma cells.

Dexniguldipine hydrochloride inhibits growth of human pancreatic adenocarcinoma cells and expression of protein kinase C isoforms alpha and zeta

New therapeutic regimens are needed for the treatment of pancreatic cancer. In view of the importance of protein kinase C (PKC) in tumorigenesis, we evaluated the effects of dexniguldipine hydrochloride (DNIG) on the in vitro growth of CFPAC-1 human pancreatic adenocarcinoma cells and the expression of PKC isoforms. DNIG is a potent antineoplastic drug with well-established anti-PKC activity and the ability to reverse multidrug resistance. DNIG (1.6-25 mu M) decreased the number of cells in culture in a time- and dose-dependent manner with an EC(50) of 4.9 mu M on day 1 and 2.8 mu M on day 3. When PKC isoform expression in CFPAC-1 cells was analyzed by immunoblotting, the predominant isoforms were identified as alpha and zeta. The expression of PKC alpha and zeta was inhibited significantly by DNIG (0.63-2.5 mu M). These results suggest that DNIG suppresses the proliferation of CFPAC-1 pancreatic cancer cells, possibly by inhibiting the expression of specific PKC isoforms.

DEXNIGULDIPINE HYDROCHLORIDE INHIBITS GROWTH OF HUMAN HT-29 COLON-CARCINOMA CELLS AND EXPRESSION OF PROTEIN-KINASE-C-DELTA AND PROTEIN-KINASE-C-ZETA

Previous studies have implicated protein kinase C (PKC) in colon carcinogenesis, but a clear understanding of the role of PKC in colon cancer is lacking. The purpose of this study was to investigate the effects of dexniguldipine hydrochloride (DNIG) on the in vitro growth of HT-29 human colon carcinoma cells and the expression of PKC isoforms. DNIG is a selective inhibitor of PKC that binds specifically to the regulatory region and is also a potent antineoplastic drug with an ability to reverse multidrug resistance. DNIG (1.6-25 mu M) decreased the number of HT-29 cells in culture in a time- and dose-dependent manner with an EC(50) of 1.4 mu M on day 3. Predominant PKC isoforms expressed in HT-29 cells were identified as Delta and zeta by immunoblotting. The expression of PKC Delta and zeta was inhibited significantly by DNIG (0.16-1.25 mu M). These results suggest that the suppression of the growth of HT-29 colon carcinoma cells by DNIG involves the inhibition of the expression of PKC Delta and zeta.

Dietary energy restriction‐induced modulation of protein kinase C ζ isozyme in the hamster pancreas

Dietary restriction in experimental animals enhances life span, delays disease, inhibits immunological perturbations, and ameliorates cancer. Protein kinase C (PKC) isozymes mediate signals generated by hormones, growth factors, and neurotransmitters for cell proliferation and differentiation. The results of our study showed that a C-terminally directed anti-PKC zeta antibody detected an 81-kDa band in the pancreases of control and energy-restricted hamsters. Syrian golden hamsters were fed energy-restricted diets formulated such that the hamsters received 90% (10% energy restriction (ER)), 80% (20% ER), or 60% (40% ER) of the total energy consumed by control hamsters, with the energy reduced proportionally from fat and carbohydrate. ER decreased PKC zeta isozyme levels by 40-75% in hamsters fed 10, 20, and 40% ER diets for 8 wk. PKC zeta isozyme expression was decreased by 75-80% in hamsters fed ER diets for 15 wk. Although ER caused significant decreases in PKC zeta isozyme levels compared with those of control hamsters at both time points, the relative differences in PKC zeta levels between the dietary ER groups (10, 20, and 40%) were small and not significant. A significant decrease in the body weights of ER animals compared with those of controls was observed at both time points. No differences in tomato lectin and phytohemagglutinin reactivity were observed between control animals and animals fed 10, 20, and 40% ER diets. Furthermore, the cellular expression of PKC zeta in the hamster pancreas did not differ among hamsters fed the various ER diets. These observations may be important for understanding not only the role of dietary ER in pancreatic cancers but also PKC zeta signal transduction mechanisms in normal pancreatic physiology.

Inhibition of ras p21 membrane localization and modulation of protein kinase C isozyme expression during regression of chemical carcinogen-induced murine skin tumors by lovastatin.

We investigated the ras p21 membrane localization and the expression and activation of protein kinase C (PKC) isozymes in activated ras oncogene-containing tumors and assessed whether these events were related to tumor growth. We used 7,12-dimethylbenz[a]anthracene-initiated and 12-O-tetradecanoylphorbol-13-acetate-promoted SENCAR mouse skin tumors, which were shown to contain Ha-ras oncogene activated by point mutation at codon 61, as an in vivo model for these studies. Compared with levels in epidermis, highly elevated levels of membrane-bound Ha-ras p21 were observed in growing tumors, which also showed strong expression and membrane translocation of PKC zeta and beta II and weak expression of PCK alpha. However, when ras p21 membrane localization was blocked in vivo in growing tumors by lovastatin, opposite results were evident. Compared with saline-treated animals, in which tumor growth continued, lovastatin-treated animals had significantly inhibited tumor growth, which led to tumor regression with concomitant inhibition of Ha-ras p21 membrane localization. These regressing tumors from lovastatin-treated animals also showed a decrease in the expression and membrane translocation of PKC zeta and beta II but increased expression of PKC alpha. Taken together, our results indicate that ras p21 membrane localization and the expression and activation of PKC zeta, beta II, and alpha may be the critical events in the regulation of the growth of tumors that contain activated ras oncogenes.

Expression of protein kinase C isoenzymes in colorectal cancer tissue and their differential activation by different bile acids.

Expression of protein kinase C (PKC) isoenzymes was determined in paired samples of normal mucosa and colorectal cancer tissue from 13 patients. Total PKC activity in cancer tissue was significantly decreased compared to that in normal mucosa. Western blotting, using PKC isoenzyme-specific antibodies, showed that two PKC isoenzymes, PKC beta and PKC epsilon, were significantly decreased in cancer tissue. The level of PKC delta was increased in cancer tissue and the expression of PKC alpha and zeta was not altered significantly. Primary bile acids--cholic acid (CA) and chenodeoxycholic acid (CDCA)--and the principal secondary bile acids--deoxycholic acid (DCA), lithocholic acid (LCA) and ursodeoxycholic acid (UDCA)--were found to be potent and selective activators of partially purified PKC isoenzymes. PKC beta 1 was the isoenzyme most effectively activated by secondary bile acids. Our data provide a model for the involvement of secondary bile acids in colorectal carcinogenesis through specific PKC isoenzyme modulation in colorectal mucosa.

Regulation of non-classical protein kinase C isoenzymes in a human T cell line.

We have examined the expression and responses to activation, of novel/atypical protein kinase C (PKC) isoforms epsilon, zeta, and delta in the T cell lymphoma cell line K-4. The effects of 1-h phorbol 12-myristate 13-acetate (PMA) and OKT3 activation of K-4 cells on PKC isoform distribution were examined. In addition, the effects of PMA-mediated down-regulation on the expression of PKC epsilon and zeta were determined using high concentrations of PMA over 24- and 48-h time periods in these cells. PKC zeta expression was not altered by incubation of K-4 cells with up to 200 ng/ml PMA over a 24- or 48-h period. PKC epsilon was down-regulated in a concentration-dependent manner by PMA after both 24- and 48-h of activation. Expression of PKC epsilon was not completely depressed, however, even at the highest concentration of the phorbol ester after 48-h incubation with PMA. The presence of PKC epsilon, zeta, and delta was confirmed by immunohistochemistry with distinct patterns of expression observed. PMA-induced PKC activation for a 1-h period resulted in a translocation of PKC delta from resting cytoplasmic/nuclear staining to a cytoplasmic aggregate. Following 1-h activation through the T cell receptor-associated complex CD3, PKC delta translocated from a peri-nuclear/cytoplasmic compartment to a putative cytoskeletal location in K-4 cells. This translocation was time dependent and redistributed to a cytoplasmic aggregate prior to the cytoskeleton. Similarly, following 1-h activation through the T cell receptor, PKC zeta redistributed directly to what is possibly a cytoskeletal cell compartment. The cytoplasmic distribution of PKC zeta was unaltered following activation with PMA over a 1-h time period. There was no apparent redistribution of PKC epsilon cytoplasmic staining pattern following a 1-h direct or indirect activation. These results underline the differences in individual PKC isoform distribution, and responses to different stimuli, thereby providing additional evidence for the use of discrete PKC isoform signaling pathways in T cells. Furthermore, this data underlines the differences in PMA-mediated PKC activation and activation through the T cell receptor.

MRNA levels of Ca(2+)-independent forms of protein kinase C in postischemic gerbil brain by northern blot analysis.

To investigate the role of Ca(2+)-independent forms of protein kinase C (PKC) in ischemic neuronal injury, mRNA expression of PKC was studied by Northern blot analysis. Ischemia was produced in gerbils by 10-min bilateral carotid artery occlusion and was followed by recirculation for 15 min, 6 h, and 24 h. Brains of postischemic and sham-operated animals were removed, forebrains fresh frozen, and processed for Northern blot analysis. Three synthetic oligonucleotide probes based on published cDNA sequences of rat brain PKC for the isozymes delta, epsilon, and zeta were utilized for hybridization. Northern blot analysis showed increased hybridization signal for all three PKC isozymes examined in the 6- and 24-h postischemic groups. Of these, the twofold increases in the expression of PKC delta and zeta were statistically significant in comparison to the control. These results suggest that the mRNA levels of Ca(2+)-independent forms of PKC, in particular, delta and zeta, are temporally stimulated by ischemic injury in the brain and may imply an important role of the enzyme in postischemic neuronal damage. However, since the protein itself was not examined in this study, the significance of the increased expression cannot be ascertained. However, it may reflect a compensatory response to the loss of PKC reported to occur in the reperfusion phase.