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.     

Regulation of cyclic AMP levels by phorbol esters in bovine adrenal medullary cells

Cyclic AMP responses to phorbol esters were studied in cultured bovine adrenal medullary cells. Phorbol esters that activate protein kinase C (PKC: phorbol 12,13-dibutyrate, phorbol 12,13-didecanoate) increased cellular cyclic AMP levels by up to 100% over 5 min, and this was maintained for up to 3 h. The effect was mimicked by 1,2-dioctanoyl-sn-glycerol but not by inactive phorbol esters. The effect of active phorbol esters was concentration dependent over the range 50–500 nM, and was abolished by the PKC inhibitor, Ro 31–8220 (10μM). The response was enhanced by 3-isobutyl-1-methylxanthine (1 mM) and by forskolin (0.3 μM), was enhanced following pertussis toxin pretreatment (100 ng/ml, 7.5 h) and was unaffected by removing extracellular Ca²⁺. The phorbol ester cyclic AMP response was additive with that to K⁺ depolarisation, and synergised with those to prostaglandin E₁ and dimaprit. The results indicate PKC activation increases cyclic AMP formation in bovine adrenal medullary cells, probably by a direct action on adenylate cyclase or G_s.     

Design and synthesis of 8-octyl-benzolactam-V9, a selective activator for protein kinase C epsilon and eta

Conventional (alpha, betaI, betaII, gamma) and novel (delta, epsilon, eta, theta) protein kinase C (PKC) isozymes are main targets of tumor promoters, such as phorbol esters and indolactam-V (ILV). We have recently found that 1-hexyl derivatives of indolinelactam-V (2, 3), in which the indole ring of ILV was replaced with the indoline ring, showed a binding preference for novel PKCs over conventional PKCs. To develop a new ILV analogue displaying increased synthetic accessibility and improved binding selectivity for novel PKCs, we have designed 8-octyl-benzolactam-V9 (4), a simple analogue without the pyrrolidine moiety of 2 and 3. Compound 4 showed significant binding selectivity for isolated C1B domains of novel PKCs. Moreover, 4 translocated PKC epsilon and eta from the cytoplasm to the plasma membrane of HeLa cells at 1 microM, whereas other PKC isozymes did not respond even at 10 microM. These results indicate that 4 could be a selective activator for PKC epsilon and eta.     

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.     

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.     

Binding of [3H]bryostatin 4 to protein kinase C.

The bryostatins represent a unique class of activators of protein kinase C (PKC) which induce only a subset of the responses typical of the phorbol esters and block those responses to the phorbol esters which they themselves do not induce. To better understand the interaction of the bryostatins with PKC, we have synthesized [26-3H]bryostatin 4 and characterized its binding to PKC. [3H]Bryostatin 4 and [3H]phorbol 12,13-dibutyrate ([3H]PDBu) differed markedly in their binding to PKC reconstituted with phosphatidylserine (PS). The binding affinity of [3H]bryostatin 4 under these conditions was too high to measure and the rate of release of bound bryostatin was much slower than that of the phorbol esters, with a half-time of several hours. These properties caused bryostatin 1 to appear to inhibit [3H]PDBu binding under these conditions in a non-competitive fashion. Both the high potency and the slow rate of release of the bryostatins may contribute to their unique pattern of biological activity. By reconstituting PKC in a mixture of 1.5% Triton X-100:0.3% PS, we were able to establish reversible conditions for [3H]bryostatin 4 binding. Under these latter conditions, binding of [3H]bryostatin 4 was competitively inhibited by PDBu, consistent with both the bryostatin and phorbol esters binding to PKC in a qualitatively similar fashion. Binding affinities to PKC isozymes alpha, beta, and gamma were compared and little difference was found, suggesting that differential recognition by these isozymes does not account for the unique biological activity of the bryostatins.     

Phorbols: chemical synthesis and chemical biology

The phorbol esters, such as phorbol 12- myristate 13-acetate (PMA), are known to be powerful tumor promoters and activators of protein kinase C (PKC). First discovered by Nishizuka et al., PKC is a phospholipid- and calcium-dependent serine/threonine kinase, phisiologically activated by 1,2-diacyl-sn-glycerol (DAG). PKC is also known to be an important target for other structurally diverse tumor promoters such as ingenols, teleocidins, and aplysiatoxins. Structure-activity analyses of a variety of analogs of DAG and these tumor promoters have been carried out. Although many pharmacophore models have been proposed from molecular modeling, no information about specific amino acid residues that interact with these ligands is available. Moreover it has been shown that the biological activity of 11-demethyl-13-deoxyphorbol esters 1, which were synthesized by our group, was not fully consistent with the pharmacophore models so far. Thus, we are now interested in determining the importance of the 13-acetoxy group in phorbol ester-PKC complexes. This has led us to design new photoaffinity probes 66 and 67 and to carry out previously unprecedented photoaffinity labeling of PKC. Photoaffinity labeling of protein kinase C isozymes by both the probes resulted in specific cross-linking. Although the cross-linking yield is not very high, we suppose that determination of the cross-linking site can be realized by taking advantage of subpicomole order analysis by mass spectrometry and other methodologies to clarify the role of individual cysteine rich domein (CRD) in native PKC. We have also designed a new phorbol ester-phosphatidylserine hybrid molecule 69. Because phosphatidylserines in phospholipid membranes are known to have specific interactions with phorbol ester-PKC complexes, such a hybrid molecule can be expected to act as a specific inhibitor of PKC by preventing PKC from interacting with phospholipid membranes. The hybrid molecule was synthesized and preliminary biological activities were examined to inhibit PKC. A catalytic asymmetric synthesis of phorbol PMA is also currently under investigation. Progress is discussed.     

Growth inhibition by homofolate in tumor cells utilizing a high-affinity folate binding protein as a means for folate internalization

A subline (JT-1) of L1210 mouse leukemia cells that contains elevated levels of a high-affinity folate binding protein is sensitive to growth inhibition by homofolate. Inhibition was observed at nanomolar concentrations of folate or 5-formyltetrahydrofolate where the high-affinity binding protein is the predominant uptake route for folate compounds. At 1.0 nM folate, inhibition of growth by 50% occurred at 0.7 nM homofolate, and maximal inhibition exceeded 90% at homofolate concentrations above 10 nM. Homofolate also inhibited the uptake of 1.0 nM [3H]folate by L1210/JT-1 cells in 72-hr cultures, and the extent of uptake inhibition by 1.0 and 20 nM homofolate was comparable to the inhibition of cell growth by the same concentrations of homofolate. At a growth-limiting concentration of 5-formyltetrahydrofolate (0.5 nM), half-maximal inhibition of L1210/JT-1 cell growth occurred at 1.0 nM homofolate. When excess concentrations of folate (5 microM) or 5-formyltetrahydrofolate (0.5 microM) were added to the medium, no growth inhibition was observed for homofolate at concentrations up to 100 microM. Parental cells lacking the folate binding protein did not respond to homofolate either at growth-limiting (0.5 microM) or excess (5.0 microM) levels of folate. Binding measurements showed that homofolate has a high affinity for the folate-binding protein (Ki = 0.03 nM) but interacts poorly with the reduced-folate transport system (Ki = 203 microM). These results indicate that homofolate inhibits the growth of L1210 cells when intracellular folates are acquired via the high-affinity folate binding protein. The basis for this inhibition appears to be competition by homofolate for substrate binding and internalization.     

Modulation of cellular processes by H7, a non-selective inhibitor of protein kinases.

H7 has been described as a potent inhibitor of protein kinase C (PKC) and has been widely used to investigate the regulatory role of this enzyme in intact cell systems. In this comparative study between H7 and the microbial alkaloid, staurosporine, we found that the former inhibited rat brain PKC and cAMP dependent protein kinase with IC50 values of 18 and 16 microM respectively whereas the latter was a much more potent inhibitor of both kinases with IC50 values of 9.5 nM and 42 nM respectively. H7, at concentrations up to 100 microM, failed to block cellular events induced by phorbol esters, agents which specifically stimulate PKC, yet was a potent inhibitor of IL-2 induced T cell proliferation with an IC50 value of 19 microM. In contrast, staurosporine was a potent inhibitor of both phorbol ester induced p47 phosphorylation in platelet (I50 value = 540 nM) and also CD3 and CD4 down-regulation in T cells (I50 values 200 nM and 50 nM respectively). Staurosporine was also a potent inhibitor of IL-2 induced T cell proliferation I50 value = 9 nM). These results provide a strong argument against the use of H7 to probe for PKC involvement in cellular processes.     

Human alpha1D-adrenoceptor phosphorylation and desensitization

Rat-1 fibroblast were transfected with a plasmid containing the cDNA of the human alpha(1D)-adrenoceptor. A cell line was isolated that stably expressed the receptor as evidenced by BMY 7378-sensitive noradrenaline-induced increases in intracellular calcium concentration. The effect of noradrenaline was blocked by active phorbol esters; such blockade was mediated by protein kinase C (PKC) as evidenced by its inhibition by staurosporine or the downregulation of this protein kinase. Radioligand binding experiments showed expression of receptors with high affinity for [3H]tamsulosin (K(D) 0.30 +/- 0.05 nM) but low density (B(max) 35 +/- 4 fmol/mg protein). The receptors had the expected orders of potency for agonists (adrenaline = noradrenaline > oxymetazoline) and antagonists (BMY 7378 > 5-methyl-urapidil = phentolamine). Photoaffinity labeling identified the receptor as a band of M(r) 70-80kDa, which could be immunoprecipitated with a selective anti-alpha(1D)-adrenoceptor antiserum. In cells metabolically labeled with radioactive phosphate the adrenoceptor was identified as a phosphoprotein whose phosphorylation state was increased by the agonist, noradrenaline, and by phorbol myristate acetate. The data indicate that the human alpha(1D)-adrenoceptor function was regulated through phosphorylation by PKC.     

Mechanism of the phorbol ester-mediated redistribution of asialoglycoprotein receptor: selective effects on receptor recycling pathways in Hep G2 cells

We have investigated the effect of phorbol dibutyrate on intracellular routing of the asialoglycoprotein receptor (ASGP-R) in a human hepatoma cell line, Hep G2. We have previously shown that this agent causes a net redistribution of 50% of cell surface receptors to the cell interior (Fallon, R.J., and A.L. Schwartz, J. Biol. Chem. 261: 15081-15089 (1986)). To explore the mechanism of this effect, we measured the rate constants of receptor and ligand movement during internalization, ligand-receptor uncoupling, sorting of ligand to degradative sites or return to the extracellular medium, and return of receptor to the plasma membrane. The rate of internalization of bound asialoorosomucoid (ASOR) is identical in phorbol ester-treated and control cells, over a range of ASOR concentrations from 5 to 125 nM. The pathway of ligand recycling returns approximately 30% of internalized ASOR undegraded to the extracellular medium; phorbol esters do not modify the extent of this pathway in Hep G2 cells nor the kinetics of recovery of undegraded ASOR in the medium (t1/2 = 20 min). The rate of ligand-receptor uncoupling is similarly unaltered by phorbol esters, as measured by the amount of free ASOR that accumulates intracellularly and exits the cell after saponin permeabilization. In contrast, phorbol esters cause a rapid (less than 5 min) 50% decrease in receptor return to the cell surface from internal sites. This suggests that 1) phorbol esters interfere with selected specific sites in receptor and ligand pathways of receptor-mediated endocytosis and 2) the apparent net "internalization" of ASGP-R by phorbol esters results from an inhibition of receptor recycling to the cell surface and not from a direct stimulation of the internalization process.     

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.     

重组L—门冬酰胺酶与其野生型在体外和体内的抗肿瘤作用比较

目的:研究重组L-门冬酰胺酶对肿瘤细胞体外生长及实验性肿瘤的抑制作用.方法:体外培养肿瘤细胞(K562、L1210和P815),分别用倒置显微镜和透射电镜观察肿瘤细胞的形态学,MTT比色法观察细胞增殖率及抑制率,流式细胞仪分析DNA含量.同时根据移植性肿瘤研究方法小鼠接种L1210、P388、Heps及S_(180)瘤株,观察给药后小鼠存活天数和瘤重.结果:体外细胞培养实验证明,重组L-门冬酰胺酶对K562、L1210和P815细胞生长具有显著抑制作用(P<0.01).体内实验表明,重组L-门冬酰胺酶ip能显著延长移植肿瘤小鼠(L1210和P388)的存活天数(P<0.01),并对小鼠移植性肝癌实体瘤(Heps)生长有明显的抑制作用(P<0.01)。重组L-门冬酰胺酶iv对小鼠Heps和S_(180)肿瘤生长有明显的抑制作用(P<0.01).结论:重组L-门冬酰胺酶对肿瘤细胞体外生长及受试肿瘤有明显抑制作用.本结果对重组L-门冬酰胺酶的临床应用提供实验依据.     

The guanine nucleotide exchange factor RasGRP is a high -affinity target for diacylglycerol and phorbol esters

RasGRP is a recently described guanine nucleotide exchange factor (GEF) that possesses a single C1 domain homologous to that of protein kinase C (PKC). The phorbol ester [(3)H]phorbol 12, 13-dibutyrate ([(3)H]PDBu) bound to this C1 domain (C1-RasGRP) with a dissociation constant of 0.58 +/- 0.08 nM, similar to that observed previously for PKC. Likewise, the potent PKC activator bryostatin 1, a compound currently in clinical trials, showed high affinity binding for C1-RasGRP. Structure activity analysis using several phorbol ester analogs showed both similarities and differences in ligand selectivity compared with PKC; the differences were comparable in magnitude to those between different PKC isoforms. Similarly, the potency of the PKC inhibitor calphostin C to inhibit [(3)H]PDBu binding to C1-RasGRP was similar to that observed for PKC. In contrast to the relative similarities in ligand recognition, the lipid cofactor requirements differed between RasGRP and PKC. The C1 domain plus the EF-hand motif of RasGRP (C1EF-RasGRP) was markedly less dependent on acidic phospholipids than was PKCalpha. The differences in lipid requirements were reflected in differential ligand selectivity under conditions of limiting lipid. Despite the presence of twin EF-hand like motifs, calcium did not affect the binding of [(3)H]PDBu to C1EF-RasGRP. We conclude that RasGRP is a high affinity receptor for phorbol esters and diacylglycerol. RasGRP thus provides a direct link between diacylglycerol generation or phorbol ester/bryostatin treatment and Ras activation.     

Targeting protein kinase C (PKC) in physiology and cancer of the gastric cell system

Protein kinase C (PKC) family members are multifunctional kinases that have been implicated in many cell biological and physiological tasks including acid, pepsinogen, and mucous production. Through the use of small-molecule PKC modulators, PKC has been found to be involved in gene expression, the control of cytoskeleton, membrane and secretagogue-dependent signal transduction for secretion of acid. Gastric carcinoma and adenocarcinoma cells often show dysregulated PKC-dependent cell signal transduction compared to normal gastric cells. Moreover, PKC was the first known target of tumor promoting phorbol esters. These findings support PKC as a potential chemotherapy target in gastric cancer. Various approaches have been launched in directly targeting PKC for chemotherapy of gastric cancer. The macrocyclic lactone bryostatin-1 is a promising agent that acts as a modulator of PKC activity, and enhances the effect of chemotherapeutic agents such as paclitaxel. This article provides an overview of the findings to date regarding the physiological role of PKC in the gastric cell system by various pharmacological approaches and examines PKC as a target in gastric (adeno-)carcinoma chemotherapy.     

Vascular protein kinase C in Wistar-Kyoto and spontaneously hypertensive rats.

Phorbol esters which activate protein kinase C (PKC) produced concentration-related force development in aorta from spontaneously hypertensive rat (SHR) and Wistar-Kyoto rat (WKY); all were 2-7 x more potent in SHR. However, total PKC activity in aortas, as well as carotid, caudal and renal arteries, was not different, when SHR was compared with WKY. Binding of phorbol dibutyrate to particulate aortic PKC was similar in SHR and WKY (same apparent Kd and Bmax values), as was potency for displacement of phorbol dibutyrate by phorbol myristate acetate. Furthermore, there was no difference in potency with staurosporine, H-7, and calmidazolium in inhibiting SHR and WKY aortic PKC. These data demonstrate enhanced contractile sensitivity to PKC-activating phorbol esters in SHR aortic smooth muscle that is not related to activity, phorbol ester binding, or sensitivity to inhibitors when SHR PKC is compared with WKY PKC. Thus, signal transduction events distal to PKC activation may be responsible for enhanced vascular contractile sensitivity to phorbol esters in SHR.     

Role of protein kinase C in bradykinin-induced prostaglandin formation in osteoblasts

Bradykinin (1 μM 5 min) induced translocation of protein kinase C (PKC) to the plasma membrane fraction in osteoblastic MC3T3-E1 cells. Bradykinin also enhanced the binding of phorbol 12,13-dibutyrate (PDBu) to intact cells, a measure of PKC activation. Addition of bradykinin (1 μM) to cells preincubated with [³H]PDBu (10 nM, 20 min) caused an increase in specific PDBu binding that was maximal after 5–10 min. The bradykinin-induced enhancement of PDBu binding was seen at 1 nM and was maximal at 10 nM. The bradykinin B1 receptor agonist des-Arg⁹-bradykinin (1 μM) did not enhance specific PDBu binding to intact MC3T3-E1 cells. PDBu at and above 3 nM stimulated the formation of prostaglandin E2 (PGE₂) in MC3T3-EI cells. This stimulatory effect was seen after 15–20 min incubation. The Ca²⁺ ionophore A23187 at and above 1 μM induced a rapid (within seconds) burst of PGE₂ formation in MC3T3-E1 cells. The effect of PDBu and A23187 on PGE₂ formation was synergistic. The PKC inhibitor staurosporine (200 nM) inhibited basal as well as bradykinin-induced prostaglandin-formation in MC3T3-E1 cells. In conclusion: bradykinin enhances PKC activation in osteoblastic MC3T3-E1 cells. This kinase activation may be involved in bradykinin-induced prostaglandin formation.     

Ligand structure-activity requirements and phospholipid dependence for the binding of phorbol esters to protein kinase D

Although protein kinase D (PKD), like protein kinase C (PKC), possesses a C1 domain that binds phorbol esters and diacylglycerol, the structural differences from PKC within this and other domains of PKD imply differential regulation by lipids and ligands. We characterized the phorbol ester and phospholipid binding properties of a glutathione S-transferase-tagged full-length PKD and compared them with those of PKC-alpha and -delta. We found that PKD is a high-affinity phorbol ester receptor for a range of structurally and functionally divergent phorbol esters and analogs and showed both similarities and differences in structure-activity relations compared with the PKCs examined. In particular, PKD had lower affinity than PKC for certain diacylglycerol analogs, which might be caused by a lysine residue at the 22 position of the PKD-C1b domain in place of the tryptophan residue at this position conserved in the PKCs. The membrane-targeting domains in PKD are largely different from those in PKC; among these differences, PKD contains a pleckstrin homology (PH) domain that is absent in PKC. However, phosphatidylinositol-4,5-bisphosphate PIP2, a lipid ligand for some PH domains, reconstitutes phorbol 12,13-dibutyrate (PDBu) binding to PKD similarly as it does to PKC-alpha and -delta, implying that the PH domain in PKD may not preferentially interact with PIP2. Overall, the requirement of anionic phospholipids for the reconstitution of [3H]PDBu binding to PKD was intermediate between those of PKC-alpha and -delta. We conclude that PKD is a high-affinity phorbol ester receptor; its lipid requirements for ligand binding are approximately comparable with those of PKC but may be differentially regulated in cells through the binding of diacylglycerol to the C1 domain.     

Indolactam and benzolactam compounds as new medicinal leads with binding selectivity for C1 domains of protein kinase C isozymes

Protein kinase C (PKC) isozymes (alpha, betaI, betaII, gamma, delta, epsilon, eta, theta) are major receptors of tumor promoters and also play a crucial role in cellular signal transduction via the second messenger, 1,2-diacyl-sn-glycerol (DG). Each isozyme of PKC is involved in diverse biological events, indicating that it serves as a novel therapeutic target. Since PKC isozymes contain two possible binding sites of tumor promoters and DG (C1A and C1B domains), the design of agents with binding selectivity for individual PKC C1 domains is a pressing need. We developed a synthetic C1 peptide library of all PKC isozymes for high-throughput screening of new ligands with such binding selectivity. This peptide library enabled us to determine that indolactam and benzolactam compounds bound to the C1B domains of novel PKC isozymes (delta, epsilon, eta, theta) in some selective manner, unlike phorbol esters and DG. Simpler in structure and higher in stability than the other potent tumor promoters, a number of indolactam and benzolactam derivatives have been synthesized to develop new PKC isozyme modulators by several groups. We focused on the amide function of these compounds because recent investigations revealed that both the amide hydrogen and carbonyl oxygen of indolactam-V (ILV) are involved in hydrogen bonding with the C1B domains of PKCdelta. Synthesis of several conformationally fixed analogues of ILV led to the conclusion that the trans-amide restricted analogues with a hydrophobic chain at an appropriate position (2,7) are promising leads with a high binding selectivity for novel PKC isozyme C1B domains. We also developed a new lactone analogue of benzolactam-V8 (17) which shows significant binding selectivity for the C1B domains of PKCepsilon and PKCeta. Furthermore, our synthetic approach with the PKC C1 homology domains clarified that diacylglycerol kinase beta and gamma are new targets of phorbol esters.     

Phorbol esters inhibit the proliferation of MCF-7 cells. Possible implication of protein kinase C

The effect of tumor promoter phorbol esters on cell proliferation was investigated in human breast cancer cell line MCF-7. During a 4-day culture period, the various phorbol ester derivatives TPA, PDD, PDBu, PDBz and PDA inhibited the proliferation of MCF-7 cells in a dose-dependent manner, with respective IC50 of 0.06, 0.75, 2.4, 3.6 and 15 X 10(-9) M. The 4-O-met-TPA, alpha PDD and alph PHR were ineffective at 2 X 10(-7) M, the highest concentration tested. Using a 3H-PDBu probe, we demonstrated the presence of specific, high affinity binding sites in intact cultured cells, with a Kd of about 9 X 10(-9) M. Unlabelled TPA, PDD, PDBU and PDBz competed with 3H-PDBu with respective IC50 of 35, 12.5, 150 and 220 X 10(-9) M. High concentrations of PDA, 4-O-met-TPA and alpha PDD slightly inhibited the 3H PDBu binding, whereas alpha PHR did not until 10(-5) M. The correlation that we observed between the relative potencies of the various phorbol derivatives for inhibiting both PDBu binding and cell proliferation, suggests that tumor promoter phorbol esters may induce growth arrest in MCF-7 cells by the mediation of protein kinase C.