Novel "nonkinase" phorbol ester receptors: the C1 domain connection

In recent years, there have been great advances in our understanding of the pharmacology and biology of the receptors for the phorbol ester tumor promoters and the second messenger diacylglycerol (DAG). The traditional view of protein kinase C (PKC) as the sole receptor for the phorbol esters has been challenged with the discovery of proteins unrelated to PKC that bind phorbol esters with high affinity, suggesting a high degree of complexity in the signaling pathways activated by DAG. These novel "nonkinase" phorbol ester receptors include chimaerins (a family of Rac GTPase activating proteins), RasGRPs (exchange factors for Ras/Rap1), and Munc13 isoforms (scaffolding proteins involved in exocytosis). In all cases, phorbol ester binding occurs at the single C1 domain present in these proteins and, as in PKC isozymes, ligand binding is a phospholipid-dependent event. Moreover, the novel phorbol ester receptors are also subject to subcellular redistribution or "translocation" by phorbol esters, leading to their association to different effector and/or regulatory molecules. Clearly, the use of phorbol esters as specific activators of PKC in cellular models is questionable. Alternative pharmacological and molecular approaches are therefore needed to dissect the involvement of each receptor class as a mediator of phorbol ester/DAG responses.     

Pharmacology of the receptors for the phorbol ester tumor promoters: multiple receptors with different biochemical properties

The phorbol ester tumor promoters and related analogs are widely used as potent activators of protein kinase C (PKC). The phorbol esters mimic the action of the lipid second messenger diacylglycerol (DAG). The aim of this commentary is to highlight a series of important and controversial concepts in the pharmacology and regulation of phorbol ester receptors. First, phorbol ester analogs have marked differences in their biological properties. This may be related to a differential regulation of PKC isozymes by distinct analogs. Moreover, it seems that marked differences exist in the ligand recognition properties of the C1 domains, the phorbol ester/DAG binding sites in PKC isozymes. Second, an emerging theme that we discuss here is that phorbol esters also target receptors unrelated to PKC isozymes, a concept that has been largely ignored. These novel receptors lacking kinase activity include chimaerins (a family of Rac-GTPase-activating proteins), RasGRP (a Ras exchange factor), and Unc-13/Munc-13 (a family of proteins involved in exocytosis). Unlike the classical and novel PKCs, these "non-kinase" phorbol ester receptors possess a single copy of the C1 domain. Interestingly, each receptor class has unique pharmacological properties and biochemical regulation. Lastly, it is well established that phorbol esters and related analogs can translocate each receptor to different intracellular compartments. The differential pharmacological properties of the phorbol ester receptors can be exploited to generate specific agonists and antagonists that will be helpful tools to dissect their cellular function.     

Phorbol ester impairs melanotropin receptor function and stimulates growth of cultured M2R melanoma cells

We have examined the effects of a biologically active tumor promoting phorbol ester (phorbol 12-myristate, 13-acetate (PMA] which activates protein kinase C (PKC) on melanotropin receptor function and cell growth in the M2R mouse melanoma cell clone. Treatment of M2R cells with PMA resulted in a significant loss of beta-MSH binding. The effect was both time- and concentration-dependent. The inhibition of beta-MSH binding resulted from a decrease (greater than 85%) in active membranal receptors available on the external cell surface and not from either enhanced internalization or change in the binding affinity. Agonist-stimulated cyclic AMP accumulation was profoundly increased in a non-selective manner following short-term incubation (3 h) with PMA. This effect was completely reversed during long-term (72-96 h) incubation with the tumor promoting agent. Long-term culturing of M2R cells with PMA resulted in enhanced (+50%) proliferation of the melanoma cells. This enhancement was blocked by the addition of agents which stimulate the production of cAMP. Hence, phorbol esters are powerful growth promoters in transformed melanocytes and our findings indicate that the effects of melanotropins are selectively impaired during the process of growth promotion.     

PKCdelta associates with and is involved in the phosphorylation of RasGRP3 in response to phorbol esters

RasGRP is a family of guanine nucleotide exchange factors that activate small GTPases and contain a C1 domain similar to the one present in protein kinase C (PKC). In this study, we examined the interaction of RasGRP3 and PKC in response to the phorbol ester PMA. In Chinese hamster ovary or LN-229 cells heterologously expressing RasGRP3, phorbol 12-myristate 13-acetate (PMA) induced translocation of RasGRP3 to the perinuclear region and a decrease in the electrophoretic mobility of RasGRP3. The mobility shift was associated with phosphorylation of RasGRP3 on serine residues and seemed to be PKCdelta-dependent because it was blocked by the PKCdelta inhibitor rottlerin as well as by a PKCdelta kinase-dead mutant. Using coimmunoprecipitation, we found that PMA induced the physical association of RasGRP3 with PKCdelta and, using in situ methods, we showed colocalization of PKCdelta and RasGRP3 in the perinuclear region. PKCdelta phosphorylated RasGRP3 in vitro. Previous studies suggest that ectopic expression of RasGRP3 increases activation of Erk1/2. We found that overexpression of either PKCdelta or RasGRP3 increased the activation of Erk1/2 by PMA. In contrast, coexpression of PKCdelta and RasGRP3 yielded a level of phosphorylation of Erk1/2 similar to that of control vector cells. Our results suggest that PKCdelta may act as an upstream kinase associating with and phosphorylating RasGRP3 in response to PMA. The interaction between RasGRP3 and PKCdelta points to the existence of complex cross-talk between various members of the phorbol ester receptors which can have important impact on major signal transduction pathways and cellular processes induced by phorbol esters or DAG     

Tumor cell adherence to cultured capillary endothelial cells is promoted by activators of protein kinase C

Stimulation of cells with protein kinase C (PKC)-specific activators such as phorbol esters increased in a reversible manner the rate of adherence of [3H]leucine-labelled L1210 cells to cultured bovine cerebral cortex capillary endothelial cells (CEC). This effect was not specific for L1210 cells since 12-O-tetradecanoyl phorbol 13-acetate (TPA) strongly increased the binding of various other tumor cell lines. Phorbol esters increased the rate of L1210 cell adhesion to CEC by enhancing their binding capacity without affecting the apparent affinity of L1210 cells for CEC. This stimulation was specific to the phorbol analogs which activate PKC since it was not effected by 4 alpha-phorbol didecanoate, known to be inactive for PKC. Down-regulation experiments showed that adhesion enhancement was entirely attributable to an effect on tumor cells without contribution of CEC intracellular PKC. PKC inhibitors like staurosporine, sphingosine and H-7 showed strong antagonistic activity towards TPA-induced L1210 cell adherence to CEC (IC50 = 0.5 nM, 160 nM and 10 microM, respectively). Adhesive proteins such as vitronectin, fibrinogen, fibronectin and the tetrapeptide RGDS, an active sequence from their cell-binding domains, exhibited potent, dose-dependent inhibition of PKC-induced tumor cell adhesion.     

The structural requirements for phorbol esters to enhance noradrenaline and dopamine release from rat brain cortex.

1. The effects of various protein kinase C (PKC) activators on the stimulation-induced (S-I) release of noradrenaline and dopamine was studied in rat cortical slices pre-incubated with [3H]-noradrenaline or [3H]-dopamine. The aim was to investigate a possible structure-activity relationship for these agents on transmitter release. 2. 4 beta-Phorbol 12,13-dibutyrate (4 beta PDB, 0.1-3.0 microM), enhanced S-I noradrenaline and dopamine release in a concentration-dependent manner whereas the structurally related inactive isomer 4 alpha-phorbol 12, 13-dibutyrate (4 alpha PDB, 0.1-3.0 microM) and phorbol 13-acetate (PA, 0.1-3.0microM) were without effect on noradrednaline release. Another group of phorbol 12, 13-diesters containing a common 13-ester substituent (phorbol 12, 13-diacetate, PDA, 0.1-3.0 microM; phorbol 12-myristate 13-acetate, PMA, 0.1-3.0 microM; phorbol 12-methylaminobenzoate 13-acetate, PMBA, 0.03-3.0 microM) also enhanced S-I noradrenaline and dopamine release in a concentration-dependent manner with PMA being the least potent. 3. The 12-deoxyphorbol 13-substituted monoesters, 12-deoxyphorbol 13-acetate (dPA, 0.1-3.0 microM), 12-deoxyphorbol 13-angelate (dPAng, 0.1-3.0 microM), 12-deoxyphorbol 13-isobutyrate (dPiB, 0.03-3.0 microM) and 12-deoxyphorbol 13-phenylacetate (dPPhen, 0.1-3.0 microM) enhanced S-I noradrenaline and dopamine release in a concentration-dependent manner. In contrast, 12-deoxyphorbol 13-tetradecanoate (dPT, 0.1-3.0 microM) was without effect. 4. The involvement of PKC in mediating the effects of the various phorbol esters was further investigated. PKC was down-regulated by 20 h exposure of the cortical slices to 4 beta-phorbol 12,13-dibutyrate (1 microM). In this case the facilitatory effect of 4 beta PDB and dPA was abolished whilst that of dPAng was significantly attenuated. This indicates that these agents were acting selectively at PKC. In support of this the PKC inhibitors, polymyxin B (21 microM) and bisindolylmaleimide I (3 microM), attenuated the facilitatory effect of 4 beta PDB and dPAng although that of dPA was not significantly altered. 5. The effects of these agents on transmitter release were not correlated with their in vitro affinity and isozyme selectivity for PKC. Short chain substituted mono- and diesters of phorbol were more potent enhancers of action-potential evoked noradrenaline and dopamine release than the long chain esters. Interestingly, these former agents are the least potent or non effective (e.g. dPA, PDA) tumour promoters. We suggest that the reason for the poor effects of lipophilic long chain phorbol esters (PMA, dPT) on transmitter release is that they are sequestered in the plasmalemma and do not access the cell cytoplasm where the PKC may be located.     

Involvement Of B-50 (GAP-43) Phosphorylation In The Modulation Of Transmitter Release By Protein Kinase C

1. Protein kinase C (PKC) is a family of enzymes that is activated by diacylglycerol (DAG) following phospholipase (PL) C activation. Protein kinase C may also be activated by metabolites and arachidonic acid generated by breakdown of membrane phospholipids by PLD and PLA2, respectively. Subsequent to PKC activation, key protein substrates are phosphorylated, resulting in the facilitation of transmitter release. 2. Phorbol esters are compounds that mimic the actions of DAG on PKC and have been shown to facilitate stimulation-induced (S-I) transmitter release in rat brain. However, some phorbol esters that have a high affinity for PKC have no effect on transmitter release, whereas others with a lower affinity for PKC markedly elevate S-I transmitter release. 3. The structure and, more importantly, the lipophilicity of the phorbol esters determines their ability to access and activate the intraneuronal pools of PKC that are involved with transmitter release. In studies in which cell membranes were intact, phorbol esters did not display the characteristics expected based on their affinities for PKC in contrast with studies in disrupted synaptosomes. This supports the hypothesis that the membrane plays a critical role in determining the effects of phorbol esters on PKC. 4. B-50, a PKC substrate thought to be involved in transmitter release, also appears to be differentially phosphorylated by various phorbol esters. The effects on B-50 phosphorylation in intact synaptosomes, but not disrupted synaptosomes, are well correlated with the effects of phorbol esters on S-I transmitter release. 5. B-50 is colocalized with actin, which has also been suggested to play an important role in facilitating the movement of reserve pools of transmitter vesicles to the readily releasable state. Therefore, it is possible that the phosphorylation status of B-50 directly influences the organization of actin filaments, thereby allowing transmitter output to be sustained under high levels of stimulation.     

Divergence and complexities in DAG signaling: looking beyond PKC

For many years protein kinase C (PKC) has been the subject of extensive studies as a molecular target for the treatment of cancer and other diseases. To better define the role of PKC isozymes in the control of cell proliferation, survival and transformation, the examination of PKC-mediated signal transduction pathways by isozyme-specific intervention has become essential. However, issues related to the selectivity of activators and inhibitors of PKC isozymes, in addition to convoluted cross-talks between phorbol ester-regulated pathways, have greatly complicated our understanding of PKC-mediated responses. An additional level of complexity is provided by the fact diacylglycerol (DAG) signals can be transduced by phorbol ester receptors other than PKC. These receptors include chimaerins, RasGRPs, MUNC13s, PKD (PKC mu) and DAG kinases beta and gamma. Thus, it is conceivable that some of the effects that were originally attributed to PKC isozymes in response to phorbol esters might be mediated by PKC-independent pathways. A key issue for the design of novel therapeutic strategies that target PKC isozymes is a comprehensive analysis of isozyme-specific signal transduction pathways in different cell types and the development of pharmacological and molecular tools that can distinguish between the various PKC and 'non-PKC' phorbol ester receptors.     

Protein kinase Cε as a cancer marker and target for anticancer therapy

Protein kinase Cε (PKCε) is a representative member of a family of novel PKC isoforms that are independent of calcium, but can be activated by phorbol esters, diacylglycerol (DAG) and phosphatidylserine (PS). This kinase is capable of modulating crucial cell functions, including proliferation, differentiation and survival. These activities depend on enzyme translocation to subcellular compartments upon binding DAG, PS or exogenous stimulators. PKCε initiates malignant transformation of cells through its effects on the Ras/Raf/MAPK pathway and displays the greatest carcinogenic potential of all PKC isoforms. PKCε also promotes tumor metastatic capacity and resistance to anti-cancer therapy. Overexpression of PKCε is found in numerous cancers including colon, breast, stomach, prostate, thyroid and lung and is considered an important marker of negative disease outcome. Although overexpression of PKCε is observed in tumors, it is not found in healthy tissues hence it has been suggested as a diagnostic marker or a putative target for specific inhibitors used for treatment of cancer. Research on selective inhibition of PKCε is under way and diverse approaches may become clinically applicable anti-tumor strategies. Suppression of the PKCε-encoding gene achieved through the antisense cDNA, suppression of PKCε with RNAi and inhibition achieved with translocation-inhibitory peptides may provide novel treatment strategies for cancer.     

Protein kinase C activity and contractile responsiveness in senescent blood vessels

To examine Ca2(+)-signaling receptor function in the aging vasculature, the status of the diacylglycerol/protein kinase C (DAG/PKC) arm of the signal transduction pathway was assessed. contractile responsiveness to the PKC activator, phorbol 12-myristate 13-acetate (PMA), is substantially reduced in aortae from 24-month-old Fischer 344 rats compared to 6-month-old rats. PKC activity is reduced in all senescent vessels yet studied including aorta, renal artery, iliac artery and vena cava. This may account for the reduced contractile responsiveness. Additionally, PMA-stimulated PKC translocation is substantially reduced in senescent aorta and this may also contribute to reduced contraction.     

Role of protein kinase Calpha in signaling from the histamine H(1) receptor to the nucleus

Stimulation of histamine H(1) receptors produced a marked activation of inositol phospholipid hydrolysis, intracellular calcium mobilization, and stimulation of the c-fos promoter in CHO-H1 cells expressing the H(1) receptor at a level of 3 pmol/mg protein. The latter response was determined using a luciferase-based reporter gene (pGL3). This response to histamine was not sensitive to inhibition by pertussis toxin but could be completely attenuated by the protein kinase C (PKC) inhibitor Ro-31-8220, or by 24-h pretreatment with the phorbol esters phorbol 12,13-dibutyrate or phorbol-12-myristate-13-acetate. Several isoforms of PKC can be detected in CHO-H1 cells (alpha, delta, epsilon, mu, iota, zeta) but only PKCalpha and PKCdelta were down-regulated by prolonged treatment with phorbol esters. Of the two isoforms that were down-regulated, only protein kinase Calpha was translocated to CHO-H1 cell membranes after stimulation with either histamine or phorbol esters. The PKC inhibitor G? 6976, which inhibits PKCalpha but not PKCdelta, was also able to significantly attenuate the c-fos-luciferase response to histamine. The mitogen-activated protein kinase kinase inhibitor PD 98059 markedly inhibited the response to histamine, suggesting that the likely major target for PKCalpha was the mitogen-activated protein kinase pathway. These data suggest that the histamine H(1) receptor can signal to the nucleus via PKCalpha after activation of phospholipase Cbeta.     

Protein kinase C ligands based on tetrahydrofuran templates containing a new set of phorbol ester pharmacophores.

A series of substituted tetrahydrofurans with an embedded glycerol backbone carrying additional tetrahydrofuranylideneacetate or tetrahydrofuranylacetate motifs were grouped into four distinct templates (I-IV) according to stereochemistry. The compounds were designed to mimic three essential pharmacophores (C(3)-C=O, C(20)-OH and C(13)-C=O) of the phorbol esters according to a new, revised model. The tetrahydrofuran ring was constructed from glycidyl 4-methoxyphenyl ether, and the structures of the isomeric templates were assigned by NMR spectroscopy, including NOE. The binding affinity for protein kinase C (PKC) was assessed in terms of the ability of the ligands to displace bound [(3)H-20]phorbol 12, 13-dibutyrate (PDBU) from a recombinant alpha isozyme of PKC. Geometric Z- and E-isomers (1 and 3, respectively) containing a tetrahydrofuranylideneacetate motif were the most potent ligands with identical K(i) values of 0.35 microM. Molecular modeling studies of the four templates showed that the rms values when fitted to a prototypical phorbol 12,13-diacetate ester correlated inversely with affinities in the following order: I approximately II > III > IV. These compounds represent the first generation of rigid glycerol templates seeking to mimic the binding of the C(13)-C=O of the phorbol esters. The binding affinities of the most potent compounds are in the same range of the diacylglycerols (DAGs) despite the lack of a phorbol ester C(9)-OH pharmacophore surrogate. This finding confirms that mimicking the binding of the C(13)-C=O pharmacophore of phorbol is a useful strategy. However, since the C(9)-OH and C(13)-C=O in the phorbol esters appear to form an intramolecular hydrogen bond that functions as a combined pharmacophore, it is possible the lack of this combined motif in the target templates restricts the compounds from reaching higher binding affinities.     

Phorbol esters inhibit smooth muscle contractions through activation of Na(+)-K(+)-ATPase.

1. The role of protein kinase C (PKC) in agonist-induced contractions of guinea-pig ileum longitudinal smooth muscle has been investigated. 2. The phorbol esters, phorbol 12,13-dibutyrate (PDBu), phorbol 12,13-diacetate (PDA) and phorbol 12-myristate 13-acetate (PMA), relaxed tissues precontracted by submaximal concentrations of carbachol, histamine or substance P. 3. This inhibitory action of the phorbol esters was reversed following the application of ouabain, a specific inhibitor of Na(+)-K(+)-ATPase. Similarly, pretreatment with ouabain inhibited the ability of phorbol esters to relax tissues precontracted by the above agonists. 4. The slow relaxation of the tonic component of contraction induced by submaximal concentrations of carbachol and histamine, and all concentrations of substance P, was abolished in the presence of ouabain. 5. In Na(+)-loaded tissues, PDBu and carbachol caused a concentration-dependent increase of Na(+)-K(+)-ATPase activity, assessed by ouabain-sensitive 86Rb(+)-uptake. Extrusion of Na+, assessed by the cellular content of the ion, was also stimulated by PDBu (the effect of carbachol was not investigated). 6. We conclude that phorbol esters inhibit the tonic component of contractions induced by submaximal concentrations of these agonists through activation of Na(+)-K(+)-ATPase. We suggest that PKC may exert feedback control over the tonic component of agonist contractions through stimulation of the pump.     

Phorbol esters: structure, biological activity, and toxicity in animals

Phorbol esters are the tetracyclic diterpenoids generally known for their tumor promoting activity. The phorbol esters mimic the action of diacyl glycerol (DAG), activator of protein kinase C, which regulates different signal transduction pathways and other cellular metabolic activities. They occur naturally in many plants of the family Euphorbiacaeae and Thymelaeaceae. The biological activities of the phorbol esters are highly structure specific. The phorbol esters, even at very low concentrations, show toxicological manifestations in animals fed diets containing them. This toxicity limits the use of many nutritive plants and agricultural by-products containing phorbol esters to be used as animal feed. Therefore, various chemical and physical treatments have been evaluated to extract or inactivate phorbol esters so that seed meals rich in proteins could be used as feed resources. However, not much progress has been reported so far. The detoxifying ability has also been reported in some molluscs and in liver homogenate of mice. Besides, possessing antinutritional and toxic effects, few derivatives of the phorbol esters are also known for their antimicrobial and antitumor activities. The molluscicidal and insecticidal properties of phorbol esters indicate its potential to be used as an effective biopesticide and insecticide.     

PROTEIN KINASE C AND TRANSMITTER RELEASE

1. Protein kinase C (PKC) is an important second messenger-activated enzyme. In noradrenergic nerves it appears to be tonically activated by diacylglycerol (DAG) to facilitate transmitter release and the steps in this involve activation of phospholipase C, generation of DAG and activation of PKC. It is suggested that the subsequent facilitation of transmitter release is due to the phosphorylation of proteins involved in the release process distal to Ca²⁺entry, presumably those involved in vesicle dynamics. 2. There are differences between central noradrenergic neurons and sympathetic nerves. In central neurons PKC appears to be tonically active and its inhibition results in a decrease in noradrenaline release under most, if not all, conditions. 3. In sympathetic nerves PKC inhibitors only decrease transmitter release during high-frequency stimulation and not during low-frequency stimulation. At high frequency there is a gradual increase in the effect of PKC inhibitors on transmitter release during the first 15 s of a stimulation train. It is suggested that this is due to a progressive rise in intracellular Ca²⁺ and a consequent activation of PKC. 4. Activation of PKC by phorbol esters produces a large enhancement in action potential-evoked noradrenaline release in both the central nervous system and in peripheral tissues. The structural requirements of the phorbol esters for maximal effect suggest that the phorbol esters must access the interior of the nerve terminal to activate PKC and the neural membrane acts as a barrier for highly lipophilic phorbol esters, thereby reducing their activity. Activation of PKC represents one of the most powerful ways to enhance transmitter release and may have therapeutic potential.     

The structural requirements for phorbol esters to enhance serotonin and acetylcholine release from rat brain cortex.

The effects of various phorbol-based protein kinase C (PKC) activators on the electrical stimulation-induced (S-I) release of serotonin and acetylcholine was studied in rat brain cortical slices pre-incubated with [3H]-serotonin or [3H]-choline to investigate possible structure-activity relationships. 4beta-phorbol 12,13-dibutyrate (4betaPDB, 0.1-3.0 microM), enhanced S-I release of serotonin in a concentration-dependent manner whereas the structurally related inactive isomer 4alpha-phorbol 12, 13-dibutyrate (4alphaPDB) and phorbol 13-acetate (PA) were without effect. Another group of phorbol esters containing a common 13-ester substituent (phorbol 12,13-diacetate, PDA; phorbol 12-myristate 13-acetate, PMA; phorbol 12-methylaminobenzoate 13-acetate, PMBA) also enhanced S-I serotonin release with PMA being least potent. The deoxyphorbol monoesters, 12-deoxyphorbol 13-acetate (dPA), 12-deoxyphorbol 13-angelate (dPAng), 12-deoxyphorbol 13-phenylacetate (dPPhen) and 12-deoxyphorbol 13-isobutyrate (dPiB) enhanced S-I serotonin release but 12-deoxyphorbol 13-tetradecanoate (dPT) was without effect. The 20-acetate derivatives of dPPhen and dPAng were less effective in enhancing S-I serotonin release compared to the parent compounds. With acetylcholine release all phorbol esters tested had a far lesser effect when compared to their facilitatory action on serotonin release with only 4betaPDB, PDA, dPA, dPAng and dPiB having significant effects. The effects of the phorbol esters on serotonin release were not correlated with their reported in vitro affinity and isozyme selectivity for PKC. A comparison across three transmitter systems (noradrenaline, dopamine, serotonin) suggests basic similarities in the structural requirements of phorbol esters to enhance transmitter release with short chain substituted mono- and diesters of phorbol being more potent facilitators of release than the long chain esters. Some compounds notably PDA, PMBA, dPPhen, dPPhenA had different potencies across noradrenaline, dopamine and serotonin.     

Protein kinase C as a drug target: implications for drug or diet prevention and treatment of cancer

Protein kinase C (PKC) isoforms are serine/threonine kinases involved in signal transduction pathways that govern a wide range of physiological processes including differentiation, proliferation, gene expression, brain function, membrane transport and the organization of cytoskeletal and extracellular matrix proteins. PKC isoforms are often overexpressed in disease states such as cancer. In this review, PKC in a variety of cancers is discussed along with some specific cell biological mechanisms by which PKC exerts its function(s). The PKC family consists of several isoforms comprising three groups: classical, novel and atypical. Although PKC has been investigated for around 2 decades, only recently has the specific function of each isoform started to be elucidated and the isoforms evaluated for use as targets of drug action. Phorbol esters such as the tumor-promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) or diacylglycerol (DAG) activate classical and novel PKC isoforms. Naturally occurring retinoids, antisense oligonucleotides against specific PKC isoforms and specific PKC inhibitors can block this activation. Beta carotene and retinoid derivatives act as anticarcinogenic agents and can antagonize some of the biological actions of phorbol esters and oxidants. Another important area of investigation is the use of antisense oligonucleotides to inhibit specific PKC isoforms. These compounds have proven effective in reducing specific types of cancer in rodents and humans and are currently used in clinical trials. This review examines PKC isoforms as a target of drug action with special emphasis on their use in cancer therapy.     

A comparison of the priming effect of phorbol myristate acetate and phorbol dibutyrate on fMet-Leu-Phe-induced oxidative burst in human neutrophils

Phorbol esters such as phorbol myristate acetate (PMA) and phorbol dibutyrate (PDBU) are generally considered to have similar effects through a similar mechanism, i.e. protein kinase C (PKC) activation. We recently suggested that this was not the case in human neutrophils. To identify further differences between the two phorbol esters, we compared their priming effects on fMet-Leu-Phe-induced superoxide anion (O2-) production, cytosolic PKC activity and binding of fMet-Leu-Phe. Priming could be initiated with a low (0.2 nM) concentration of both PDBU and PMA. Their effects on the pattern of fMet-Leu-Phe-induced superoxide production were similar in both Ca2(+)-containing and Ca2(+)-free medium. PDBU, like PMA, abolished the Ca2+ dependency of fMet-Leu-Phe-induced O2- production in a dose-dependent manner. In cytochalasin B-treated cells and in the presence of Ca2+, priming with PDBU or PMA did not alter the enhancing effect of cytochalasin B on fMet-Leu-Phe-induced O2- production. In Ca2(+)-free medium, priming abolished the Ca2+ dependency of fMet-Leu-Phe stimulation in cytochalasin B-treated cells. Cytochalasin B, however, enhanced the effect of PMA but not that of PDBU. Priming with PDBU was not associated under any experimental conditions with a decrease in cytosolic PKC activity, or an increase in PKM activity before or after fMet-Leu-Phe stimulation. Furthermore, priming effects were abolished by cell washing but not by H-7 or staurosporine, which are potent PKC inhibitors. PDBU, in contrast to PMA, increased fMet-Leu-Phe binding to PMNs through a decrease in the dissociation constant and induced degranulation of specific granules as measured by the release of vitamin B12 binding protein. These findings show that the priming effects of PDBU differ in certain respects from those of PMA, namely with regard to its synergism with cytochalasin B and the expression of fMet-Leu-Phe receptors. In addition, priming concentrations of PDBU, like PMA, did not alter cytosolic PKC activity in fMet-Leu-Phe-stimulated neutrophils.     

Lithium treatment inhibits protein kinase C translocation in rat brain cortex

RATIONALE: Lithium, an effective psychotropic agent, affects membrane phospholipid metabolism, interferes with phosphoinositide signal transduction, and antagonizes the biological activity of calcium, all major factors of protein kinase C (PKC) activation. Consequently, lithium may interfere with cellular functions requiring PKC. Supporting this hypothesis, lithium was found to inhibit increased neurotransmitter release upon PKC activation and to prevent phorbol ester-mediated PKC translocation. OBJECTIVES: The present study was undertaken to determine whether the frontal cortex of rats treated with lithium exhibits altered PKC activity and translocation in response to phorbol ester, K+, or serotonin (5-HT) receptor stimulation and to determine whether specific PKC isozymes are disproportionately affected. METHODS: Rats were fed either a normal diet or one enriched with LiCl. In cerebrocortical slices or synaptosomes, cytosolic and membranous PKC activity and translocation in response to stimuli were determined after partial purification with anion exchange chromatography. RESULTS: In brain slices, lithium treatment inhibited phorbol 12-myristate, 13-acetate (PMA)-, 5-HT-, or K+-induced PKC translocation from cytosol to membrane without affecting basal membrane or cytosolic PKC activity. In synaptosomes, lithium also attenuated PMA- or K+-evoked translocation of PKC. Immunoblotting with isozyme-specific PKC antibodies revealed that chronic lithium treatment reduced basal cytosolic alphaPKC and deltaPKC but increased membrane-associated zetaPKC immunoreactivities. Stimulation with PMA, 5-HT or K+ elicited translocation of alpha, beta and gammaPKC isozymes and PMA induced translocation of delta and epsilonPKC isozymes. Stimulus-mediated translocation of PKC isozymes was attenuated in cortical tissue obtained from animals that received lithium for 6 weeks. In synaptosomes, PMA- or K+-induced PKC translocation was attenuated by in vitro lithium or chronic lithium treatment. Neither rubidium nor cesium affected PKC activities or PMA-induced translocation. Suppression of PMA-elicited translocation by lithium was partially antagonized by Ca2+. CONCLUSIONS: Lithium treatment reduces PKC translocation induced by either stimulation of a cell surface receptor or by direct enzyme stimulation with phorbol ester. This effect leads to reduced PKC-mediated phosphorylation of cellular proteins and may be responsible for the pharmacotherapeutic action of lithium.     

Protein kinase C isozymes as potential targets for anticancer therapy

Protein kinase C (PKC) comprises a family of isozymes (alpha, betaI, betaII, gamma, delta, epsilon, theta, eta, lambda/iota [mouse/human], and zeta) which are involved in signal transduction from membrane receptors to the nucleus. Activation of PKC by phorbol esters promotes tumor formation, and from that it was concluded that inhibitors of PKC might prevent carcinogenesis or inhibit tumor proliferation. However, the situation is more complicated because the exact function of the different PKC isozymes is not known at present. They have been shown to be involved in synaptic transmissions, the activation of ion fluxes, secretion, cell cycle control, differentiation, proliferation, tumorigenesis, metastasis and apoptosis. Modulators such as bryostatin-1, phospholipid analogues, PKC-activating adriamycin derivatives, CGP41251, UCN-01, and antisense oligonucleotides directed against PKCalpha, have shown antitumor activity in cancer patients. PKC inhibitors are not specific to PKC, but also interact with other signaling molecules, which may contribute to the antitumor effects. Modulators of PKC have also been shown to influence non-MDR1-mediated and MDR1-mediated antitumor drug resistance. This review is focussed on the role of PKC isozymes in human cell proliferation, apoptosis and antitumor drug resistance, and on the use of PKC modulators as antitumor agents.