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

We have examined the interaction between 1-beta-D-arabinofuranosylcytosine (ara-C) and the macrocyclic lactone protein kinase C activator bryostatin 1 in the human promyelocytic leukemia cell line HL-60. Preexposure of cells to 10 nM bryostatin 1 for 24 h, followed by an additional 24-h incubation with 10 microM ara-C, resulted in greater than additive inhibitory effects toward clonogenic HL-60 cells. In a series of alkaline elution assays, cells preincubated with bryostatin 1 and prelabeled with [3H]thymidine exhibited a significant increase in DNA fragmentation following exposure to ara-C in comparison to cells exposed to ara-C alone. This increase in DNA damage was apparent at both neutral and alkaline pH and was not protein associated. In contrast, studies using cells pulse-labeled with [3H]thymidine immediately before analysis suggested that bryostatin 1 pretreatment did not increase the ability of ara-C to interfere with DNA replicative intermediates. Additional studies demonstrated that the increase in DNA fragmentation induced by bryostatin 1 and ara-C preceded both loss of cell membrane integrity (as determined by trypan blue exclusion) as well as depletion of intracellular ATP and NAD pools. Furthermore, the enhanced inhibitory effects of bryostatin 1 and ara-C toward clonogenic HL-60 cells did not appear to result from the induction of cellular differentiation. Finally, agarose gel electrophoresis of DNA obtained from cells exposed to both bryostatin 1 and ara-C revealed a pattern of integer multiples of 180- to 200-base pair fragments commonly associated with endonucleolytic cleavage; the extent of this fragmentation was considerably greater than that observed in cells exposed to ara-C alone. Taken together, these findings suggest that exposure of HL-60 cells to bryostatin 1 renders them more susceptible to ara-C-related DNA damage and that this phenomenon contributes to the cytotoxic effects of this drug combination. They also raise the possibility that bryostatin 1, perhaps through modulation of intracellular signaling events in leukemic cells, has the capacity to potentiate ara-C-related apoptosis or programmed cell death.
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PMID:Potentiation of the activity of 1-beta-D-arabinofuranosylcytosine by the protein kinase C activator bryostatin 1 in HL-60 cells: association with enhanced fragmentation of mature DNA. 142 73

The effects of the protein kinase C activator bryostatin 1, either with or without recombinant granulocyte-macrophage colony stimulating factor (rGM-CSF) were examined with respect to the in vitro metabolism of ara-C in leukaemic myeloblasts obtained from 10 patients with acute myelogenous leukaemia (AML). Coincubation of cells with 12.5 x 10(-9) M bryostatin 1 and 10(-5) M ara-C for 4 h resulted in a significant increase in ara-CTP formation (compared to controls) in 6/10 specimens (mean increase 106%; range 38-255%), and no change in the remainder. In contrast, coincubation of cells with 1.25 ng/ml rGM-CSF resulted in a significant increase in only one specimen, and decreases in two. Bryostatin 1 also significantly increased ara-C DNA incorporation in 6/9 evaluable samples, including two which did not display an increase in ara-CTP formation. Coincubation of cells with both bryostatin 1 and rGM-CSF did not lead to a further increase in ara-CTP formation or ara-C DNA incorporation compared to values obtained with either agent alone. Finally, exposure of blasts to bryostatin 1 for 24 h before ara-C led to an increase in ara-CTP formation in 3/8 additional specimens, and a decrease in one sample displaying evidence of bryostatin 1-induced macrophage differentiation. Incubation of cells with both rGM-CSF and bryostatin 1 for this period resulted in ara-CTP levels equivalent to those obtained with bryostatin 1 alone. These studies indicate that while bryostatin 1 exerts a heterogeneous effect on ara-C metabolism in leukaemic myeloblasts, it is capable of potentiating ara-C phosphorylation in a subset of patient samples, including some that do not exhibit an increase in response to rGM-CSF. They also raise the possibility that bryostatin 1-induced potentiation of ara-C metabolism in some leukaemic cells may contribute, at least in part, to the antileukaemic efficacy of this drug combination.
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PMID:Effects of bryostatin 1 and rGM-CSF on the metabolism of 1-beta-D-arabinofuranosylcytosine in human leukaemic myeloblasts. 148 32

We have examined the effect of a combined 24 h exposure to cytosine arabinoside (ara-C) and the protein kinase C activator bryostatin 1, either alone or in conjunction with recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF), on the clonogenic growth of 14 primary samples from acute myelogenous leukemia (AML) patients, as well as normal human committed and early hematopoietic progenitors. Incubation of blasts with 1 microM ara-C and 12.5 nM bryostatin 1(+/- 1.25 ng/ml rGM-CSF) resulted in a heterogeneous pattern of inhibitory effects toward primary leukemic colonies, ranging from 32-98%, and subadditive to synergistic drug interactions. However, exposure of blasts to ara-C and bryostatin 1, either with or without rGM-CSF, eliminated leukemic cell self-renewal in 80-93% of samples, and very substantially reduced growth in the remainder. Exposure of normal human bone marrow mononuclear cells to identical concentrations of ara-C and byostatin 1 permitted the survival of 23% of committed myeloid progenitors (granulocyte-macrophage colony-forming units), and greater than 50% when rGM-CSF was included. Finally, exposure of bone marrow populations highly enriched for progenitor cells (CD34+, DR-, CD71-) to ara-C and bryostatin 1 +/- rGM-CSF for 24 h led to minimal reductions (e.g. 10-15%) in the survival of early hematopoietic progenitors (high proliferative potential colony-forming cells). Together, these findings indicate that combined exposure in vitro to ara-C and bryostatin 1, both with and without rGM-CSF, effectively inhibits the growth of leukemic cells with self-renewal capacity, while sparing a significant fraction of normal committed and primitive hematopoietic progenitors.
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PMID:Effect of a combined exposure to cytosine arabinoside, bryostatin 1, and recombinant granulocyte-macrophage colony-stimulating factor on the clonogenic growth in vitro of normal and leukemic human hematopoietic progenitor cells. 159 8

Bryostatin 1 is a macrocyclic lactone protein kinase C (PK-C) activator which has demonstrated promising antileukemic activity in preclinical studies. We have examined the effect of this agent on the metabolism and cytotoxicity of 1-beta-D-arabinofuranosylcytosine (ara-C) in both log phase and high-density human promyelocytic leukemia cells (HL-60). Exposure of low-density cells to 12.5 nM bryostatin 1 for 24 hr prior to a 4-hr incubation with 1 or 10 microM ara-C resulted in nearly a 2-fold increase in ara-CTP formation. When cells were maintained under high-cell density conditions (e.g. 5 x 10(6) cells/mL) for 24 hr prior to ara-C exposure, a 90% reduction in ara-CTP formation and ara-C DNA incorporation was observed. However, coincubation of high-density cells with bryostatin 1 for 24 hr increased ara-CTP formation 6- to 8-fold, yielding levels essentially equivalent to those achieved in low-density cells. Smaller (but still significant) increases in ara-C DNA incorporation were also noted. Enhancement of ara-CTP formation by bryostatin 1 occurred over a broad ara-C concentration range (0.1 to 100 microM), involved a temperature-dependent process, could not be mimicked by addition of hematopoietic growth factors, and was not related to neutralization of toxic or inhibitory substances in high-density medium. Exposure of cells to bryostatin 1 did not lead to morphologic or functional evidence of HL-60 cell maturation or an increase in cell viability, but did produce a decline in cellular proliferative activity as determined by thymidine and bromodeoxyuridine incorporation and cytofluorometric analysis. Bryostatin 1 did not exert its effects in high-density cells by inhibiting ara-C deamination or by interfering with ara-CTP dephosphorylation, but instead appeared to act by enhancing ara-C phosphorylation. Although cell-free extracts obtained from high-density cells exposed to bryostatin 1 exhibited levels of deoxycytidine kinase activity compared to controls, treated cells did display a significant decline in intracellular dCTP levels (e.g. 0.7 vs 1.3 pmol/10(6)), and nearly a 2-fold increase in ATP and UTP concentrations. Ara-CTP formation was also increased substantially by other PK-C activators including phorbol dibutyrate and mezerein (10-100 nM); this process was inhibited more than 70% by the PK-C inhibitor H-7 (50 microM), but not by the PK-C inhibitors staurosporine, tamoxifen, and HA1004. Finally, coadministration of ara-C and bryostatin 1 resulted in greater than expected inhibitory effects toward HL-60 cell clonogenic growth.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:In vitro effects of bryostatin 1 on the metabolism and cytotoxicity of 1-beta-D-arabinofuranosylcytosine in human leukemia cells. 186 41

1-beta-D-Arabinofuranosylcytosine (ara-C) is an effective chemotherapeutic agent that incorporates into DNA and results in DNA fragmentation. Recent work has demonstrated that ara-C transiently induces expression of the c-jun immediate early response gene. The present studies in HL-60 myeloid leukemia cells extend these findings by demonstrating that the increase in c-jun mRNA levels at 6 h of ara-C treatment is regulated by a transcriptional mechanism. In contrast, the subsequent down-regulation of c-jun expression is controlled by a posttranscriptional decrease in the stability of the c-jun transcripts. Previous work in phorbol ester treated cells has indicated that c-jun expression is regulated by the activation of protein kinase C. The present results demonstrate that protein kinase C activity is increased in ara-C-treated cells. This increase was maximal at 60 min and remained detectable through 6 h of ara-C exposure. Moreover, the induction of c-jun transcripts by ara-C was inhibited by the isoquinolinesulfonamide derivative H7, but not by HA1004, suggesting that this effect is mediated by protein kinase C. Ara-C-induced c-jun expression was also inhibited by staurosporine, another inhibitor of protein kinase C. Taken together, these results indicate that the cellular response to ara-C includes the activation of protein kinase C and that ara-C potentially induces c-jun transcription by a protein kinase C dependent signaling mechanism.
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PMID:Regulation of c-jun gene expression in HL-60 leukemia cells by 1-beta-D-arabinofuranosylcytosine. Potential involvement of a protein kinase C dependent mechanism. 190 49

We have examined the effects of both nonspecific and highly selective pharmacological inhibitors of protein kinase C (PKC) on the capacity of a 6-h exposure to 1-[beta-D-arabinofuranosyl]cytosine (ara-C; 10 microM) to induce apoptotic DNA fragmentation and cell death in the human myeloid leukemia cell lines HL-60 and U937. Staurosporine, a highly potent, nonspecific inhibitor of PKC (20-50 nM), uniquely potentiated ara-C-related degradation of DNA to oligonucleosomal fragments in both cell lines (i.e., 2- to 3-fold), but was ineffective when given alone at these concentrations. In contrast, co-administration of the nonspecific PKC inhibitor H7 and two highly selective PKC inhibitors, calphostin C and chelerythrine, also increased the extent of DNA fragmentation observed in ara-C-treated cells, but these effects were evident only at inhibitor concentrations that were by themselves sufficient to induce DNA damage. Agarose gel electrophoresis demonstrated that cells co-exposed to staurosporine and ara-C exhibited considerably more pronounced internucleosomal DNA cleavage than did cells exposed to ara-C alone; moreover, this effect was suppressed by Zn2+ (1 mM) and the permeant Ca2+ chelator BAPTA-AM (50 microM). Potentiation of ara-C-related DNA fragmentation by subeffective concentrations of staurosporine was accompanied by a pronounced increase in the morphological features characteristic of apoptosis. A synergistic interaction between staurosporine and ara-C with respect to inhibition of clonogenicity in both HL-60 and U937 cells was demonstrated by median dose-effect analysis. The actions of staurosporine did not result from enhanced ara-C metabolism, as preincubation of cells with concentrations of this agent that potentiated ara-C actions (e.g., 20-50 nM) did not increase intracellular levels of the lethal metabolite ara-CTP. Lastly, preexposure of HL-60 and U937 cells to staurosporine did not block ara-C-mediated upregulation of c-jun, an oncogene whose increased expression has been temporally associated with ara-C-induced apoptosis. Together, these findings indicate that staurosporine exhibits a unique pattern of potentiation of ara-C-related apoptosis in human myeloid leukemias, and provide a rationale for exploring the antileukemic potential of this combination regimen.
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PMID:Modulation of 1-[beta-D-arabinofuranosyl] cytosine-induced apoptosis in human myeloid leukemia cells by staurosporine and other pharmacological inhibitors of protein kinase C. 794 69

Deoxycytidine (dCyd) kinase was effectively phosphorylated by protein kinase C. The reaction was rapid, occurring at 4 degrees C as well as at 37 degrees C and approximately 0.7 mol of phosphate could be incorporated per mol of deoxycytidine kinase. Phosphoserine was the primary amino acid to be phosphorylated. Phosphorylation of deoxycytidine kinase resulted in a 100% increase in the Vmax using dCyd as a substrate (52.16 +/- 1.3 versus 104.47 +/- 11.4 nmol/min/mg protein), and an increase in the apparent Km (2.0 +/- 0.2 microM versus 6.9 +/- 1.2 microM). The inactive antimetabolite, ara-C, is activated within a cell by deoxycytidine kinase phosphorylation of the prodrug. Recent studies have shown that ara-C activates protein kinase C in vivo [1]. Furthermore, ara-C has been shown to be metabolized to ara-CDP-choline via reversal of the cholinephosphotransferase [2] producing diglyceride, a cellular activator of protein kinase C. Thus, in situ, deoxycytidine kinase may be phosphorylated by protein kinase C with the result that self-potentiation of ara-C toxicity may occur via increased activity of deoxycytidine kinase.
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PMID:Deoxycytidine kinase is phosphorylated in vitro by protein kinase C alpha. 798 Dec 28

1-beta-D-Arabinofuranosylcytosine (ara-C) is an effective antileukemic agent that misincorporates into DNA. Recent studies have demonstrated that ara-C treatment is associated with transient induction of the c-jun early response gene. The present studies have examined the effects of ara-C on c-jun expression in a phorbol ester-resistant variant of the HL-60 myeloid leukemia cell line, designated HL-525, that is deficient in protein kinase C (PKC)-mediated signal transduction and fails to respond to 12-O-tetradecanoylphorbol-13-acetate with induction of c-jun transcripts. The results demonstrate that treatment of HL-525 cells with ara-C is associated with transcriptional activation of the c-jun gene. We also demonstrate that ara-C treatment is associated with activation of a PKC-like activity. Partial purification of this Ca(2+)-independent activity has demonstrated phosphorylation of synthetic peptides derived from (a) amino acids 4-14 of myelin basic protein and (b) the pseudosubstrate region of PKC (amino acids 19-31), with substitution of Ala25 with serine. The finding that the ara-C-induced activity is inhibited by the pseudosubstrate PKC(19-36) supports the activation of a PKC-like enzyme. Because PKC can act upstream of the mitogen-activated protein (MAP) kinases, we studied the effects of ara-C treatment on MAP kinase activity. The results demonstrate that MAP kinase is activated in ara-C-treated cells and that the kinetics of this activation are similar to those of the PKC-like activity. Because 12-O-tetradecanoylphorbol-13-acetate has little, if any, effect on the PKC-like and MAP kinase activities in HL-525 cells, these findings suggest that ara-C activates a distinct signaling cascade that may contribute to induction of the c-jun gene.
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PMID:1-beta-D-arabinofuranosylcytosine activates serine/threonine protein kinases and c-jun gene expression in phorbol ester-resistant myeloid leukemia cells. 805 58

We have demonstrated previously that bryostatin 1, a macrocylic lactone with putative protein kinase C (PKC)-activating properties, synergistically augments the antileukemic actions of the deoxycytidine analog 1-[beta-D-arabinofuranosyl]cytosine (ara-C) in HL-60 human promyelocytic leukemia cells (Grant et al., Biochem Pharmacol 42: 853-867, 1991), and that this effect appears to be related to sensitization to ara-C-induced apoptosis (Grant et al., Cancer Res 52: 6270-6278, 1992). In the present studies, we have assessed the extent of this damage by quantitative spectrofluorophotometry of small molecular weight, double-stranded DNA fragments in order to provide: (a) a more complete characterization of the interaction between ara-C and bryostatin 1, and (b) a direct comparison of the relative effects of bryostatin 1 treatment with other pharmacological manipulations known to modulate protein kinase C activity. Exposure of cells to ara-C (10(-9) to 10(-4) M; 1-24 hr) induced time- and concentration-related increases in the extent of DNA fragmentation. Treatment with bryostatin 1 (10(-11) to 10(-7) M; 1-24 hr) alone failed to induce DNA damage, but promoted substantial time- and concentration-related increases in the extent of fragmentation induced by a subsequent 6-hr exposure to ara-C. Maximal potentiation of fragmentation (e.g. 2- to 3-fold greater than that obtained with ara-C alone) was observed following a 24-hr pretreatment with 10(-8) M or 10(-7) M bryostatin 1, and correlated closely with enhanced inhibition of HL-60 cell clonogenicity. The stage-1 tumor-promoter phorbol dibutyrate potentiated the effects of ara-C in a biphasic manner, maximally augmenting the response at 2.5 x 10(-8) M, but exerting no effect at 10(-7) M, whereas the stage-2 tumor-promoter mezerein failed to augment ara-C-related DNA fragmentation at low concentrations, and antagonized ara-C action at high concentrations. In contrast, ara-C-related DNA fragmentation was attenuated or abolished either by continual preexposure to synthetic diglyceride or by pretreatment with exogenous phospholipase C at all concentrations tested. Increased DNA fragmentation was not specifically related to recruitment of cells into S-phase or enhancement of ara-C-related cellular differentiation. Finally, concentrations of bryostatin 1 that maximally potentiated ara-C-related DNA fragmentation were associated with virtually complete down-regulation of total cellular PKC activity, whereas diglyceride and phospholipase C, which suppressed the response to ara-C, moderately increased total PKC activity.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Effects of bryostatin 1 and other pharmacological activators of protein kinase C on 1-[beta-D-arabinofuranosyl]cytosine-induced apoptosis in HL-60 human promyelocytic leukemia cells. 813 59

Previous studies demonstrated that arabinosylcytosine (ara-C) induced internucleosomal DNA fragmentation and cell death in mouse thymocytes and that those were inhibited by 1-(5-iso-quinoline-sulfonyl)-2-methylpiperazine hydrochloride, an inhibitor of protein kinases. In the present study, we examined the relationship between the DNA fragmentation induced by ara-C and that by agents which activate intracellular signaling and induce apoptosis in mouse thymocytes. 12-O-tetradecanoyl 13-acetate, a phorbol ester capable of activating protein kinase C or A23187, a calcium ionophore, had no effect on ara-C induced DNA fragmentation. However, ara-C induced DNA fragmentation was synergistically enhanced by cAMP and cAMP receptor agonists. Ara-C inhibited the incorporation of choline into the acid soluble and lipid fractions, and cAMP enhanced this inhibition, suggesting that ara-C metabolites interfere with membrane phospholipid metabolism, partly evoking a certain cellular signaling for apoptosis and interacting with cAMP-evoked signaling.
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PMID:Synergic stimulation of arabinosylcytosine induced apoptosis in mouse thymocytes by cyclic AMP. 828 44


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