Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Treatment of metaphase II-arrested hamster eggs with activators of protein kinase C has been reported to promote resumption of the cell cycle, second polar body emission, and pronucleus formation (G.I. Gallicano, S.M. Schwarz, R.W. McGaughey, and D.G. Capco, 1993, Dev. Biol. 156, 94-106). In contrast, we have not observed these responses in mouse eggs obtained from CF-1 mice treated with these activators. In this report, we evaluated if this difference was due to differences in the technique used for PKC stimulation in the two different laboratories or due to species differences. Metaphase II-arrested hamster or mouse eggs were treated with phorbol diesters for 5 min or with a membrane-permeable diacylglycerol for 1 hr. Treatment of hamster eggs resulted in (1) the formation of "second polar body-like structures" commencing 5 min after treatment and reaching a maximum by 20-40 min; (2) a remarkable increase in the staining of filamentous actin in the region of these polar body-like structures; and (3) the disassembly of spindle microtubules. A reduction in cdc2/cyclin B1 kinase activity, as assessed by a decrease in H1 kinase activity, as well as progression from metaphase to anaphase were not observed. Treatment of mouse eggs from either CF-1 or CD-1 mice with these activators of PKC did not result in the formation of these polar body-like structures, did not cause an increase in filamentous actin, and did not result in a reduction in histone H1 kinase activity. This treatment, however, did induce disassembly of the spindle microtubules and the formation of multiple "pronucleus-like structures" that were more discernible in eggs from CD-1 mice. We conclude that the "apparent" activation of hamster eggs by activators of PKC is due to the effect of these agents on the cytoskeleton, which gives rise to structures that appear similar to polar bodies, but without any evidence of cell cycle resumption. The different responses seen in mouse and hamster eggs are mainly due to differences in the sensitivity of the cytoskeleton to rearrangements induced by these agents.
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PMID:Differential effect of activators of protein kinase C on cytoskeletal changes in mouse and hamster eggs. 764 80

The thioether phospholipid derivative ilmofosine (BM41440), a selective inhibitor of protein kinase C, is a new anticancer drug presently undergoing Phase II clinical trials. We have examined the influence of the compound on cell cycle progression. Ilmofosine was found to induce a dose-dependent accumulation of CA46 cells in G2-phase of the cell cycle. G2-arrest correlated with suppression of cdc2 kinase activation. Ilmofosine did not affect cdc2 kinase activity in vitro, consistent with an indirect locus of action. Ilmofosine treated CA46 cells failed to accumulate hyperphosphorylated-cdc2/cyclin B1 complexes that are observed when G2-arrest is induced by either nitrogen mustard or ionizing radiation. Indeed, cdc2 became dephosphorylated and cyclin B1 protein levels decreased as ilmofosine treated cells became arrested in G2. Our findings suggest that ilmofosine down-regulates cdc2 kinase activation through a mechanism that affects the formation of cdc2/cyclin B1 complexes.
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PMID:The protein kinase C inhibitor ilmofosine (BM 41 440) arrests cells in G2 phase and suppresses CDC2 kinase activation through a mechanism different from that of DNA damaging agents. 813 43

The p34cdc2 kinase is a highly regulated serine-threonine kinase that, when complexed with cyclins A and B, controls cell entry into mitosis. Recently, premature activation of p34cdc2 was shown to be required for apoptosis induced by a wide variety of agents. Here, we show that Taxol induced p34cdc2 kinase activity with a peak at 6 h in human breast carcinoma MCF-7 cells. We subsequently observed that the activation of CPP32/Yama protease as well as the cleavage of its substrate poly(ADP-ribose) polymerase occurred 9 h after Taxol treatment. Olomoucine, a potent p34cdc2 inhibitor, effectively prevented Taxol-induced p34cdc2 kinase activation and subsequent apoptosis. Furthermore, the treatment of cells with cyclin B1-specific antisense oligonucleotide also blocked Taxol-induced apoptosis, suggesting that cyclin B1-associated p34cdc2 kinase plays an important role in the induction of apoptosis by Taxol. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a protein kinase C activator, was found to exert strong protection against Taxol-induced cell death in MCF-7 cells. TPA inhibited Taxol-mediated activation of p34cdc2 kinase by preventing the dephosphorylation of the Tyr-15 residue on p34cdc2 without altering the levels of Cdc2 and cyclin B1. In contrast, the ability of Taxol to enhance tubulin polymerization was not inhibited by TPA. These findings suggest that modulation of protein kinase C signaling can protect against Taxol-induced cell death by inhibiting p34cdc2 kinase activation.
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PMID:Taxol-induced p34cdc2 kinase activation and apoptosis inhibited by 12-O-tetradecanoylphorbol-13-acetate in human breast MCF-7 carcinoma cells. 943 85

12-O-Tetradecanoyl phorbol-13-acetate (TPA) inhibits the growth of most malignant melanoma cells but stimulates the growth of normal human melanocytes. We previously showed that addition of TPA inhibits the growth of the human metastatic melanoma cell line, Demel, by blocking cells at both the G1/S and G2/M cell cycle transitions (D. L. Coppock et al., 1992, Cell Growth Differ. 3, 485-494). To examine the G2/M transition, we developed a method to synchronize the cells in early S phase using Lovastatin and mevalonate, followed by treatment with hydroxyurea (HU). TPA (30 nM) was effective in blocking cells from entering mitosis and reentering G1 when added up to the end of G2. These cells arrested in G2. Examination of the levels of cyclins A and B1 demonstrated that the levels of these cyclins were not limiting for entrance into M. However, the addition of TPA blocked the increase in p34(cdc2)/cyclin B1 kinase activity. In cells treated with TPA, most p34(cdc2) was found in the slowly migrating forms on Western blots, which contained increased levels of phosphotyrosine. In addition, the level of the cyclin-dependent kinase inhibitor p21(Cip1/Waf1), but not of p27(Kip1), was increased. We examined the expression of protein kinase C (PKC) isoforms in Demel cells using Western blots to understand which types were involved in the G2 arrest. Demel cells expressed the PKC alpha, betaI, betaII, delta, epsilon, iota/lambda, zeta, and mu isozymes. PKC eta and PKC theta were not detected. Addition of TPA did not completely down regulate any PKC isozymes over a 12-h period in these synchronized cells. PKC alpha, betaI, betaII, delta, and epsilon isozymes were translocated to the membrane fraction from the cytosolic fraction when treated with TPA. PKC delta appeared as a doublet and the addition of TPA shifted a majority to the slower migrating form. The level of PKC mu was constant; however, a slow mobility form was observed in TPA-treated cells. This reduced mobility was at least partially due to phosphorylation. Thus, the arrest of growth in G2 appears to be due to the inhibition of the p34(cdc2) kinase activity which is associated with the increased expression of p21(Cip1/Waf1) and increased phosphorylation on tyrosine of p34(cdc2). This arrest, in turn, is associated with a shift of PKC isozymes PKC alpha, PKC betaI, PKC betaII, PKC delta, PKC epsilon, and PKC mu to the membrane fraction which is induced by addition of TPA.
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PMID:Regulation of the cell cycle at the G2/M boundary in metastatic melanoma cells by 12-O-tetradecanoyl phorbol-13-acetate (TPA) by blocking p34cdc2 kinase activity. 968 25

The development of a malignant tumor involves the progressive acquisition of mutations and epigenetic abnormalities in multiple genes that have highly diverse functions. Some of these genes code for pathways of signal transduction that mediate the action of growth factors. The enzyme protein kinase C plays an important role in these events and in the process of tumor promotion. Therefore, we examined the effects of three inhibitors of protein kinase C, CGP 41251, RO 31-8220, and calphostin C, on human glioblastoma cells. These compounds inhibited growth and induced apoptosis; these activities were associated with a decrease in the level of CDC2 and cyclin B1/CDC2-associated kinase activity. This may explain why the treated cells accumulated in G2-M. In a separate series of studies, we examined abnormalities in cell cycle control genes in human cancer. We have found that cyclin D1 is frequently overexpressed in a variety of human cancers. Mechanistic studies indicate that cyclin D1 can play a critical role in carcinogenesis because: overexpression enhances cell transformation and tumorigenesis; introduction of an antisense cyclin D1 cDNA into either human esophageal or colon cancer cells reverts their malignant phenotype; and overexpression of cyclin D1 can enhance the amplification of other genes. The latter finding suggests that cyclin D1 can enhance genomic instability and, thereby, the process of tumor progression. Therefore, inhibitors of the function of cyclin D1 may be useful in both cancer chemoprevention and therapy. We obtained evidence for the existence of homeostatic feedback loops between cyclins D1 or E and the cell cycle inhibitory protein p27Kip1. On the basis of these and other findings, we hypothesize that, because of their disordered circuitry, cancer cells suffer from "gene addiction" and "gene hypersensitivity," disorders that might be exploited in both cancer prevention and therapy.
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PMID:Disorders in cell circuitry associated with multistage carcinogenesis: exploitable targets for cancer prevention and therapy. 1006 76

UCN-01 (7-hydroxystaurosporine) and CGP 41 251 (4'-N-benzoyl staurosporine), both of which were discovered as protein kinase C selective inhibitors, have entered in phase 1 clinical trials as anti-cancer drugs. In this study, we have directly compared the effects of these drugs as well as staurosporine (STP) on cell cycle progression of A431 human epidermoid carcinoma cells synchronized at M phase by treatment with nocodazole. The nocodazole-synchronized cells progressed from M to G1 phase in the absence of the drug, which was accompanied by a decrease of cyclin B1 protein expression, disappearance of the complex formation of CDC2 with cyclin B1 and reduction of the kinase activity. Treatments of the M phase cells with UCN-01, STP and CGP 41 251 at 80% growth-inhibitory concentrations (IC80S) resulted in specific G1 block, G2M block and polyploidy, respectively. Decreases of cyclin B1 protein expression was partially prevented by treatments with STP and CGP 41 251 but not with UCN-01 at IC80S. Reductions of active complex and kinase activity of CDC2/cyclin B1 were also observed in the presence of the three drugs. In addition, augmentation of CDC2 protein tyrosine phosphorylation was induced only when the cells were treated with STP. These observations demonstrated that higher concentrations of UCN-01, STP and CGP 41 251 showed different effects on cell cycle progression as well as CDC2/cyclin B1 regulation in A431 cells synchronized at M phase. The data suggest that UCN-01 and CGP 41 251 may act at quite different points on the cell cycle.
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PMID:Differential effects of UCN-01, staurosporine and CGP 41 251 on cell cycle progression and CDC2/cyclin B1 regulation in A431 cells synchronized at M phase by nocodazole. 1019 49

UCN-01 is a hydroxylated derivative of staurosporine and a potent protein kinase C (PKC) inhibitor. Interest in the potential usefulness of this compound as an anticancer drug stems mainly from its unique anti-signaling, growth-arresting properties on tumor cells. This include activation of CDC2 kinase (CDK1) which interacts with either cyclin A or cyclin B1 at the G1 or G2/M border, suggeting that this event is one of the major consequences of the drug action on eukaryotic cells. Nonetheless, the antiproliferative activity of UCN-01 on normal rapidly dividing cells (intestinal epithelial and bone marrow cells) is not well documented. Thus, the main objective of this study was to investigate the in vivo antiproliferative activity of UCN-01 on these normal hyperproliferative cells and evaluate whether cellular response to UCN-01 could be modulated in the presence of DNA damage. Mice were injected i.m. with a single dose of UCN-01 (2.5 mg/kg-20 mg/kg) followed 3 and 24 h later by in vivo BrdU labeling for 1 h. At autopsy, bone marrow cells were collected and fixed for dual parameter BrdU/DNA flow cytometry. Different regions of the gut were also fixed for immunoperoxidase BrdU assays. Newly replicated cells were mainly located in the lower compartments of the crypt columns and were scored for BrdU stained nuclei using an image analysis system. A comparison between groups showed that 5 mg/kg UCN-01 induced inhibition in BrdU incorporation at 3 and 24 h, as compared to the other groups injected with various doses of UCN-01. Flow cytometric analysis of bone marrow cells stained with fluorescein tagged anti-BrdU (FITC) along with propidium iodide (PI) also showed inhibition in BrdU incorporation of S phase fraction cells in mice treated with 5 mg/kg UCN-01. These bone marrow cells were arrested primarily in the G1 phase of the cell cycle. The colony-forming unit (CFU) assay of the bone marrow cells was then used to determine the level of drug interaction of UCN-01 and, topotecan, a topoisomerase I inhibitor, at a fixed dose ratio. An antagonistic drug interaction (CI > 1) was observed as determined by the median-effect analysis. However, an additive interaction (CI = 1) was obtained with the use of camptothecin or 10,11-methylenedioxycamptothecin and UCN-01. The results of the in vitro drug interaction with UCN-01 may predict protection from topotecan-induced bone marrow toxicity.
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PMID:UCN-01 dose-dependent inhibition of normal hyperproliferative cells in mice. 1140 42

The role of Bcl-2 in photodynamic therapy (PDT) is controversial, and some photosensitizers have been shown to induce Bcl-2 degradation with loss of its protective function. Hypericin is a naturally occurring photosensitizer with promising properties for the PDT of cancer. Here we show that, in HeLa cells, photoactivated hypericin does not cause Bcl-2 degradation but induces Bcl-2 phosphorylation in a dose- and time-dependent manner. Bcl-2 phosphorylation is induced by sublethal PDT doses; increasing the photodynamic stress promptly leads to apoptosis, during which Bcl-2 is neither phosphorylated nor degraded. Bcl-2 phosphorylation involves mitochondrial Bcl-2 and correlates with the kinetics of a G(2)/M cell cycle arrest, preceding apoptosis. The co-localization of hypericin with alpha-tubulin and the aberrant mitotic spindles observed following sublethal PDT doses suggest that photodamage to the microtubule network provokes the G(2)/M phase arrest. PDT-induced Bcl-2 phosphorylation is not altered by either the overexpression or inhibition of p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun NH(2)-terminal protein kinase 1 (JNK1) nor by inhibiting the extracellular signal-regulated kinases (ERKs) or protein kinase C. By contrast, Bcl-2 phosphorylation is selectively suppressed by the cyclin-dependent protein kinase (CDK)-inhibitor roscovitine, completely blocked by the protein synthesis inhibitor cycloheximide and enhanced by the overexpression of CDK1, suggesting a role for this pathway. However, in an in vitro kinase assay, active CDK1/cyclin B1 complex failed to phosphorylate immunoprecipitated Bcl-2, suggesting that this protein kinase may not directly modify Bcl-2. Mutation of serine-70 to alanine in Bcl-2 abolishes PDT-induced phosphorylation and restores the caspase-3 activation to the same levels of the vector-transfected cells, indicating that Bcl-2 phosphorylation may be a signal to delay apoptosis in G(2)/M phase-arrested cells.
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PMID:Phosphorylation of Bcl-2 in G2/M phase-arrested cells following photodynamic therapy with hypericin involves a CDK1-mediated signal and delays the onset of apoptosis. 1210 Nov 83

Signal transduction pathway and a new function of TIS21/BTG2/PC3 were investigated in p53 null U937 cells; Expression of TIS21 by 12-O-tetradecanoyl phorbol-13-acetate (TPA) stimulation was mediated by PKC-delta activation, however, was strongly inhibited by cPKC isozymes. When U937 cells were treated with TPA+Go6976, but not TPA+Go6850, the level of TIS21 mRNA was maintained over that of TPA alone. When analyzed by FACS, TPA-induced G2/M arrest was significantly inhibited by Go6850, but not by Go6976, suggesting the involvement of TIS21 and nPKC isozymes. Indeed, PKC-delta was found to be a regulator of the G2/M arrest and TIS21 expression, confirmed by employing rottlerin and dnPKC-delta experiments. In vivo accumulation of TIS21 protein significantly induced cell death through caspase 3 activation, which was supported further by degradations of procaspase 3, full-length PKC-delta, pRB, and p21(WAF1) in TIS21DeltaC expresser. When the cells were synchronized by nocodazole, TIS21 overexpressers inhibited degradations of cyclin A and cyclin B1 in 3 h after release from the synchronization. Furthermore, TIS21 inhibited cyclin B1-Cdc2 binding and its kinase activity in vivo. In summary, TPA-induced TIS21 mRNA expression is mediated by PKC-delta, and TIS21 induces G2/M arrest and cell death by inhibiting cyclin B1-Cdc2 binding and the kinase activity through its binding to Cdc2.
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PMID:TIS21/BTG2/PC3 is expressed through PKC-delta pathway and inhibits binding of cyclin B1-Cdc2 and its activity, independent of p53 expression. 1530 83

Mouse eggs arrest at metaphase II following ovulation and are only triggered to complete meiosis when fertilized. Sperm break the cell-cycle arrest by a long-lasting series of Ca2+ spikes that lead to an activation of the anaphase-promoting complex/cyclosome. The signal transduction pathway is not fully resolved but both protein kinase C (PKC) and calmodulin-dependent protein kinase II (CamKII) activities increase at fertilization and previous pharmacological studies have implicated both in cell-cycle resumption. We have used a combination of pharmacological inhibitors and constitutively active cRNA constructs of PKCalpha and CamKIIalpha microinjected into mouse eggs to show that it is CamKII and not PKC that is the sufficient trigger for cell-cycle resumption from metaphase II arrest. Constitutively active PKC constructs had no effect on the resumption of meiosis but caused an immediate and persistent elevation in intracellular Ca2+ when store-operated Ca2+ entry was stimulated. With respect to resumption of meiosis, the effects of constitutively active CamKII on eggs were the same as sperm. Eggs underwent second polar body extrusion and pronucleus formation with normal timings; while both securin and cyclin B1 destruction, visualised by coupling to fluorescent protein tags, were complete by the time of polar body extrusion. Induction of a spindle checkpoint by overexpression of Mad2 or by spindle poisons blocked CamKII-induced resumption of meiosis, but the Ca2+ chelator BAPTA did not. Furthermore direct measurement of Ca2+ levels showed that CamKII did not induce exit from metaphase II arrest by raising Ca2+. Therefore, we conclude that PKCs may play an important role in maintaining Ca2+ spiking at fertilization by promoting store-operated Ca2+ entry, while CamKII transduces cell-cycle resumption, and lies downstream of sperm-induced Ca2+ release but upstream of a spindle checkpoint. These data, combined with the knowledge that CamKII activity increase at fertilization, suggest that mouse eggs undergo cell-cycle resumption through stimulation of CamKII.
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PMID:Calmodulin-dependent protein kinase II, and not protein kinase C, is sufficient for triggering cell-cycle resumption in mammalian eggs. 1609 25


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