Potent inactivation of representative members of each PKC isozyme subfamily and PKD via S-thiolation by the tumor-promotion/progression antagonist glutathione but not by its precursor cysteine.

We recently established that S-glutathiolation of cPKCalpha fully inactivates the isozyme, at a stoichiometry of approximately 1 mol GSH/mol cPKCalpha. In this report we demonstrate that, in addition to cPKCalpha, six other PKC isozymes that are representative of the three subfamilies within the PKC family (cPKCbeta1, cPKCbeta2 and cPKCgamma, nPKCdelta and nPKCepsilon and aPKC-zeta) are subject to inactivation by S-glutathiolation induced by the thiol-specific oxidant diamide, which induces disulfide bridge formation. Among PKD and the seven PKC isozymes examined in this report only nPKCdelta has been directly implicated as an antagonist of tumor promotion/progression, while several of the kinases have been implicated in the mediation of tumor promotion/progression. We report that of the kinases examined nPKCdelta was the most resistant to inactivation by diamide-induced S-glutathiolation. In the absence of GSH only nPKCdelta activity exhibited a biphasic response to diamide, with low diamide concentrations oxidatively enhancing nPKCdelta activity and higher concentrations inactivating the isozyme; the other seven kinases were subject to monophasic, concentration-dependent, oxidative inactivation by diamide to various extents. The results provide evidence that at least some pro-oxidant environments may support the potent inactivation of nPKCepsilon and other PKC isozymes implicated in tumor promotion/progression by the mechanisms of S-glutathiolation and, in some cases, disulfide bridge formation among the isozyme thiols, without inducing substantial nPKCdelta inactivation. The results also show that neither the seven PKC isozymes examined nor PKD are inactivated by S-cysteinylation under conditions that support potent inactivation by S-glutathiolation. This indicates that the protection that the tumor promotion/progression antagonist GSH may afford against oxidative tumor promotion/progression mechanisms by S-thiolating and inactivating PKC isozymes and PKD cannot be afforded by the metabolic GSH precursor cysteine. These observations support a role for PKC inactivation via S-glutathiolation in the mechanism of tumor promotion/progression antagonism by GSH in pro-oxidant environments.

Phase II evaluation of bryostatin-1 in metastatic melanoma.

In this phase II study we assessed the efficacy of bryostatin-1 (NSC 339555) in metastatic melanoma patients when given intravenously either once a week at a dose of 25 microg/m2 per day over 24 h for 3 weeks or at 40 microg/m2 per day over 72 h every 2 weeks. Treatment courses were repeated every 4 weeks. Patients who had received one prior chemotherapy regimen for advanced melanoma, with or without biotherapy, were randomized to one or the other bryostatin-1 dose schedules until 12 patients were registered to each arm. Because there was one confirmed response among the 12 patients who received the 72 h dose schedule, 25 more patients were added to that arm. No prophylactic medications were given. Objective tumour measurements were used to assess the efficacy of the regimen. The National Cancer Institutes common toxicity criteria were used to grade reactions. In total, 49 patients with metastatic melanoma, none having symptomatic brain metastasis, were studied. Of these, 12 patients received the 24 h bryostatin-1 regimen, while the remaining 37 received the 72 h regimen. One patient receiving the 72 h regimen had a partial response lasting over 7 months. Muscle pain occurred in over 90% of the patients and was the dose-limiting side effect of the 72 h regimen. Grade 3/4 nausea and vomiting were more common on the 24 h regimen than on the 72 h one (35% versus 5% of patients). There was no therapy-related thrombocytopenia. Neutropenia was mild and mainly limited to patients receiving the 72 h regimen. Bryostatin-1 has limited activity against melanoma when given by 72 h intravenous infusion.

Accelerated HER-2 degradation enhances ovarian tumor recognition by CTL. Implications for tumor immunogenicity.

We investigated the ubiquitination and degradation of a tumor antigen, the HER-2/neu (HER-2) protooncogene product which is overexpressed in epithelial cancers. HER-2 degradation was investigated in the ovarian tumor line, SKOV3.A2, that constitutively overexpressed long-life HER-2. We used as agonist geldanamycin (GA), which initiated downmodulation of HER-2 from the cell surface. HER-2 was polyubiquitinated and degraded faster in the presence than in the absence of GA. GA did not decrease HLA-A2 expression. Presentation of the immunodominant cytotoxic T lymphocyte (CTL) epitope, E75 (369-377) from SKOV.A2 was inhibited by proteasome inhibitors, such as LLnL but was enhanced by cysteine protease inhibitors such as E64, indicating that both the proteasome and cysteine proteases are involved in epitope formation but have different effects. Enhanced tumor recognition was not an immediate or early effect of GA treatment, but was evident after 20 h of GA treatment. In contrast, 20 h GA treatment did not increase tumor sensitivity to LAK cell lysis. Twenty hour GA-treated SKOV3.A2 cells expressed an unstable HER-2 protein synthesized in the presence of GA, of faster electrophoretic mobility than control HER-2. This suggested that the newly synthesized HER-2 in the presence of GA was the main source of epitopes recognized by CTL. Twenty hour GA-treated SKOV3.A2 cells were better inducers of CTL activity directed to a number of HER-2 CTL epitopes, in peripheral blood mononuclear cells compared with control untreated SKOV3.A2 cells. Thus, induction of HER-2 protein instability enhanced the sensitivity of tumor for CTL lysis. Increased HER-2 CTL epitopes presentation may have implications for overcoming the poor immuno-genicity of human tumors, and design of epitope precursors for cancer vaccination.

Heregulin-induced apoptosis is mediated by down-regulation of Bcl-2 and activation of caspase-7 and is potentiated by impairment of protein kinase C alpha activity.

Heregulins are a group of growth factors that play diverse and critical roles in the signaling network of the human epidermal growth factor receptor (HER or EGFR) superfamily. Our earlier studies have shown that recombinant heregulinbeta1 (HRG) induces apoptosis in SKBr3 breast cancer cells that overexpress HER2. Here we report molecular mechanisms of HRG-induced apoptosis. HRG treatment of SKBr3 cells for 72 h decreased the level of Bcl-2 protein. HRG treatment led to degradation of poly (ADP-ribose) polymerase (PARP) and activated both caspase-9 and caspase-7. No significant activation of caspase-3, -6, or -8 was detected. Expression of exogenous caspase-7 by adenovirus-caspase-7 (Ad-casp-7) in SKBr3 cells resulted in apoptosis, which mimicked the effect of HRG treatment. Expression of exogenous caspase-7 had no impact on Bcl-2 expression, but promoted PARP degradation. Two highly selective inhibitors of protein kinase C (PKC), GF109203X (GF) and Ro318425 (Ro), significantly enhanced HRG-induced apoptosis as determined by flow cytometric analysis and DNA fragmentation assay. Accordingly, the PKC inhibitor GF further decreased the level of Bcl-2 protein and further degraded PARP in HRG-treated cells. Assay of PKC activity indicated that HRG activated PKC in SKBr3 cells, predominantly affecting the PKCalpha isoform. To confirm which PKC isoform(s) mediated potentiation of HRG-induced apoptosis, the profile of PKC isoforms was measured in SKBr3 cells. Five PKC isoforms, PKCalpha, PKCiota, PKCzeta, PKClambda, and PKCdelta as well as their receptors (RACK1) were expressed in this cell line. Treatment with PKC inhibitors GF and Ro decreased protein levels of both PKCalpha and PKCdelta at 24 h. PKCalpha levels were still depressed at 72 h. GF and Ro had little effect on the expression of other PKC isoforms. An inhibitor of classical PKC isoforms (Go6976) enhanced HRG-induced apoptosis, whereas the PKCdelta selective inhibitor rottlerin did not. As PKCalpha was the only classical isoform expressed in SKBr3 cells, the effect of Go6976 on HRG-induced apoptosis largely related to inhibition of PKCalpha. Constitutive expression of wild-type PKCalpha attenuated the apoptosis produced by HRG and GF. Consequently, HRG-induced apoptosis in SKBr3 cells appeared to involve down-regulation of Bcl-2 protein, activation of caspase-9 and caspase-7, and degradation of PARP. Inhibition of PKC function enhanced HRG-induced apoptosis, leading to synergistic down-regulation of Bcl-2 expression. Impairment of the PKCalpha isoform alone was sufficient to potentiate HRG-induced apoptosis.

Prospects for targeting protein kinase C isozymes in the therapy of drug-resistant cancer--an evolving story.

The seminal discovery in 1988 that selective protein kinase C (PKC) activators induce multidrug resistance (MDR) in human cancer cells spawned several years of intensive investigations; these studies were primarily directed at the question of whether isozyme-selective PKC antagonism could reverse MDR phenotypes produced in cancer cells by P-glycoprotein and other ATP-binding cassette (ABC) transporters. The first section of this commentary provides a succinct overview of those studies. In the second section, we evaluate why the enthusiasm for studies of the involvement of PKC in transport-related drug resistance is currently diminished, and we offer an assessment of whether the PKC/MDR field should be revisited. The final section of the commentary highlights recent developments in studies of PKC targeting in experimental cancer therapeutics, which continues to be a vibrant field. Highlights include the sensitization of cancer cells to radiation- and drug-induced apoptosis by PKC inhibition.

Effects of resveratrol on the autophosphorylation of phorbol ester-responsive protein kinases: inhibition of protein kinase D but not protein kinase C isozyme autophosphorylation.

The natural product resveratrol is a potent antagonist of phorbol ester-mediated tumor promotion and in vitro cellular responses to phorbol-ester tumor promoters, but it is only weakly inhibitory against the phosphorylation of conventional exogenous substrates by phorbol ester-responsive protein kinase C (PKC) isozymes. In this report, we compare the effects of resveratrol against the autophosphorylation reactions of PKC isozymes versus the novel phorbol ester-responsive kinase, protein kinase D (PKD). We found that resveratrol inhibits PKD autophosphorylation in a concentration-dependent manner, but has only negligible effects against the autophosphorylation reactions of representative members of each PKC isozyme subfamily (cPKC-alpha, -beta(1), and -gamma, nPKC-delta and -epsilon, and aPKC-zeta). Resveratrol was comparably effective against PKD autophosphorylation (IC(50) = 52 microM) and PKD phosphorylation of the exogenous substrate syntide-2 (IC(50) = 36 microM). The inhibitory potency of resveratrol against PKD is in line with the potency of resveratrol observed in cellular systems and with its potency against other purified enzymes and binding proteins that are implicated in the cancer chemopreventive activity of the polyphenol. Thus, PKD inhibition may contribute to the cancer chemopreventive action of resveratrol.

Oxidant-induced S-glutathiolation inactivates protein kinase C-alpha (PKC-alpha): a potential mechanism of PKC isozyme regulation.

Protein kinase C (PKC) isozymes are subject to inactivation by reactive oxygen species (ROS) through as yet undefined oxidative modifications of the isozyme structure. We previously reported that Cys-containing, Arg-rich peptide-substrate analogues spontaneously form disulfide-linked complexes with PKC isozymes, resulting in isozyme inactivation. This suggested that PKC might be inactivated by oxidant-induced S-glutathiolation, i.e., disulfide linkage of the endogenous molecule glutathione (GSH) to PKC. Protein S-glutathiolation is a reversible oxidative modification that has profound effects on the activity of certain enzymes and binding proteins. To directly examine whether PKC could be inactivated by S-glutathiolation, we used the thiol-specific oxidant diamide because its oxidant activity is restricted to induction of disulfide bridge formation. Diamide weakly inactivated purified recombinant cPKC-alpha, and this was markedly potentiated to nearly full inactivation by 100 microM GSH, which by itself was without effect on cPKC-alpha activity. Diamide inactivation of cPKC-alpha and its potentiation by GSH were both fully reversed by DTT. Likewise, GSH markedly potentiated diamide inactivation of a PKC isozyme mixture purified from rat brain (alpha, beta, gamma, epsilon, zeta) in a DTT-reversible manner. GSH potentiation of diamide-induced cPKC-alpha inactivation was associated with S-glutathiolation of the isozyme. cPKC-alpha S-glutathiolation was demonstrated by the DTT-reversible incorporation of [(35)S]GSH into the isozyme structure and by an associated change in the migration position of cPKC-alpha in nonreducing SDS-PAGE. Diamide treatment of NIH3T3 cells likewise induced potent, DTT-reversible inactivation of cPKC-alpha in association with [(35)S] S-thiolation of the isozyme. Taken together, the results indicate that PKC isozymes can be oxidatively inactivated by S-thiolation reactions involving endogenous thiols such as GSH.

Resveratrol preferentially inhibits protein kinase C-catalyzed phosphorylation of a cofactor-independent, arginine-rich protein substrate by a novel mechanism.

Resveratrol, a polyphenolic natural product abundantly present in grape skins, is a candidate cancer chemopreventive agent that antagonizes each stage of carcinogenesis and inhibits protein kinase C (PKC), a key mediator of tumor promotion. While resveratrol has been shown to antagonize both isolated and cellular forms of PKC, the weak inhibitory potency observed against isolated PKC cannot account for the reported efficacy of the polyphenol against PKC in cells. In this report, we analyze the mechanism of PKC inhibition by resveratrol. Our results indicate that resveratrol has a broad range of inhibitory potencies against purified PKC that depend on the nature of the substrate and the cofactor dependence of the phosphotransferase reaction. Resveratrol weakly inhibited the Ca2+/phosphatidylserine-stimulated activity of a purified rat brain PKC isozyme mixture (IC(50) = 90 microM) by competition with ATP (K(i) = 55 microM). Consistent with the kinetic evidence for a catalytic domain-directed mechanism, resveratrol inhibited the lipid-dependent activity of PKC isozymes with divergent regulatory domains similarly, and it was even more effective in inhibiting a cofactor-independent catalytic domain fragment (CDF) of PKC generated by limited proteolysis. This suggested that regulatory features of PKC might impede resveratrol inhibition of the enzyme. To explore this, we examined the effects of resveratrol on PKC-catalyzed phosphorylation of the cofactor-independent substrate protamine sulfate, which is a polybasic protein that activates PKC by a novel mechanism. Resveratrol potently inhibited protamine sulfate phosphorylation (IC(50) = 10 microM) by a mechanism that entailed antagonism of the activation of PKC by protamine sulfate and did not involve competition with either substrate. On the basis of the presence of PKC isozymes at subcellular sites rich in polybasic proteins, it has been proposed that certain endogenous polybasic PKC substrates may activate PKC in cells by the same mechanism as protamine sulfate. Our results suggest that antagonism by resveratrol of the phosphorylation of cellular PKC substrates that resemble protamine sulfate in their interactions with PKC may contribute to the efficacy of resveratrol against PKC in cells.

A peptide substrate-based affinity label blocks protein kinase C-catalyzed ATP hydrolysis and peptide-substrate phosphorylation.

Studies focused on the cAMP-dependent protein kinase (PKA) have led to the identification of conserved active-site residues involved in Ser/Thr protein kinase catalysis and have ruled out a role for Cys residues in the catalytic mechanism. Protein kinase C (PKC) is a Ser/Thr protein kinase isozyme family. We recently reported that the peptide-substrate analog N-biotinyl-Arg-Arg-Arg-Cys-Leu-Arg-Arg-Leu (N-biotinyl-RRRCLRRL) spontaneously forms intermolecular disulfide bridges with the active-site region of PKC isozymes concomitant with inactivation of histone kinase catalysis. Because Cys does not participate in PKC catalysis, one can analyze the active-site topology of PKC by examining which catalytic reactions are sterically hindered when the inactivator peptide is tethered to Cys in the active-site region of the enzyme. In this report, we show that N-biotinyl-RRRCLRRL inactivates the bulky PKC-catalyzed histone phosphorylation reaction, the comparatively less bulky PKC-catalyzed phosphorylation of a series of octapeptide, hexapeptide, and pentapeptide substrates, the intramolecular autophosphorylation reaction of PKC, and the least bulky PKC-catalyzed reaction, ATP hydrolysis, in a dithiothreitol-sensitive manner with comparable efficacy. Our results provide evidence that the covalent linkage of N-biotinyl-RRRCLRRL to the active-site region of PKC sterically hinders PKC catalysis, even in the absence of peptide and protein substrates.

Differential non-redox inhibitory effects of glutathione against protein kinase C isozyme family members.

Glutathione (GSH) and the GSH metabolic precursor N-acetylcysteine (NAC) are potent antioxidants that have clear potential either as cancer chemopreventive agents or as lead compounds for new cancer chemopreventive agents. The potential efficacy of GSH and NAC in clinical cancer chemoprevention is suggested by their antagonism of tumor promotion in animal models. Protein kinase C (PKC) is an isozyme family that plays a critical role in phorbol ester-mediated tumor promotion. We recently found that GSH and NAC exert direct inhibitory effects against a purified PKC isozyme mixture by a mechanism that did not involve their antioxidant properties. In this report, we characterize non-redox inhibitory effects of glutathiones on PKC isozymes that have been shown to produce partially or fully transformed phenotypes in mammalian cells. We show that GSH, NAC, and oxidized GSH analogs exert potent inhibition of the isozyme cPKC-ç and are somewhat less effective against cPKC- 1. In contrast, the oncogenic isozyme nPKC-â was unaffected by NAC, and it was inhibited by GSH and oxidized GSH analogs very modestly. Our results suggest that the potential impact of non-redox GSH/NAC-mediated PKC inhibition on cellular responses to tumor promoters and indeed, on cell growth regulation in general, may depend upon the pattern of PKC isozyme expression in the cells.

Evidence for a regulatory binding site for arginine-rich peptides on protein kinase C.

The peptides N-biotinyl-RRRCLRRL and N-biotinyl-RKRCLRRL covalently modify protein kinase C (PKC) through reaction of the Cys sulfhydryl group with the active site of the enzyme. The labeling of PKC occurs only in the presence of the cofactors phosphatidylserine, diacylglycerol, and Ca2+ but not in their absence. Low concentrations of the Arg-rich substrate, R4YGSR6Y greatly increase the extent of the reaction of these biotinylated peptides with PKC in the presence of lipid cofactors but in the absence of calcium. This effect can be observed at 50 nM R4YGSR6Y and suggests the presence of a high-affinity binding site for Arg-rich peptides which is separate from the active site but which enhances accessibility of the active site. The study also demonstrates the utility of the biotinylated peptides as active site labels which can detect the conformational change accompanying the activation of PKC.

Irreversible inactivation of protein kinase C by glutathione.

The tripeptide glutathione (GSH) is the predominant low molecular weight thiol reductant in mammalian cells. In this report, we show that at concentrations at which GSH is typically present in the intracellular milieu, GSH and the oxidized GSH derivatives GSH disulfide (GSSG) and glutathione sulfonate each irreversibly inactivate up to 100% of the activity of purified Ca2+- and phosphatidylserine (PS)-dependent protein kinase C (PKC) isozymes in a concentration-dependent manner by a novel nonredox mechanism that requires neither glutathiolation of PKC nor the reduction, formation, or isomerization of disulfide bridges within PKC. Our evidence for a nonredox mechanism of PKC inactivation can be summarized as follows. GSSG antagonized the Ca2+- and PS-dependent activity of purified rat brain PKC with the same efficacy (IC50 = 3 mM) whether or not the reductant dithiothreitol was present. Glutathione sulfonate, which is distinguished from GSSG and GSH by its inability to undergo disulfide/thiol exchange reactions, was as effective as GSSG in antagonizing Ca2+- and PS-dependent PKC catalysis. The irreversibility of the inactivation mechanism was indicated by the stability of the inactivated form of PKC to dilution and extensive dialysis. The inactivation mechanism did not involve the nonspecific phenomena of denaturation and aggregation of PKC because it obeyed pseudo-first order kinetics and because the hinge region of PKC-alpha remained a preferential target of tryptic attack following GSH inactivation. The selectivity of GSH in the inactivation of PKC was also indicated by the lack of effect of the tripeptides Tyr-Gly-Gly and Gly-Ala-Gly on the activity of PKC. Furthermore, GSH antagonism of the Ser/Thr kinase casein kinase 2 was by comparison weak (<25%). Inactivation of PKC-alpha was not accompanied by covalent modification of the isozyme by GSH or other irreversible binding interactions between PKC-alpha and the tripeptide, but it was associated with an increase in the susceptibility of PKC-alpha to trypsinolysis. Treatment of cultured rat fibroblast and human breast cancer cell lines with N-acetylcysteine resulted in a substantial loss of Ca2+- and PS- dependent PKC activity in the cells within 30 min. These results suggest that GSH exerts negative regulation over cellular PKC isozymes that may be lost when oxidative stress depletes the cellular GSH pool.

Protein kinase C-alpha: a novel target for the therapy of androgen-independent prostate cancer? (Review-hypothesis).

Prostate cancer is a leading cause of cancer death among men in Western countries. A major reason for this is that the malignancy often progresses to an androgen-independent phenotype that is highly aggressive and unresponsive to available therapies. Protein kinase C (PKC) is an isozyme family with at least eleven mammalian members that play important roles in cell growth regulation and differentiation. Based on the emerging understanding of the role played by PKC isozymes in the regulation of prostate cancer cell growth and programmed death, in this report we develop the hypothesis that a defective PKC-a-mediated apoptotic pathway in androgen-independent human prostate cancer cells has allowed the cells to acquire a selective growth advantage by overexpression of PKC-a and that this adaptive response renders the cells dependent on constitutively active PKC-a for their survival. Studies reviewed in this report provide strong evidence that expression of constitutive PKC-a activity is required for the survival and growth of androgen-independent human prostate cancer cells, but direct evidence for this is still lacking. We outline experimental approaches that will be required to definitively test the importance of PKC-a to androgen-independent human prostate cancer cell growth and survival. If constitutive PKC-a activity is in fact found to be required for the growth and survival of androgen-independent human prostate cancer, then the development of PKC-a-targeted therapeutics for use in the clinical treatment of prostate cancer will be justified.

Chemosensitization of cancer cells by the staurosporine derivative CGP 41251 in association with decreased P-glycoprotein phosphorylation.

The multidrug resistance (MDR) phenotype of cancer cells often correlates with the level and activity of protein kinase C (PKC). We studied the ability of the staurosporine derivative PKC inhibitor CGP 41251 to reverse the MDR phenotype in MCF-7 human breast carcinoma and CT-26 murine colon adenocarcinoma cells and their doxorubicin (DXR)-selected MDR variants. Nontoxic concentrations of CGP 41251 significantly enhanced the cytotoxic properties of DXR, actinomycin D, vinblastine, and vincristine but not those of 5-fluorouracil. CGP 41251 increased intracellular concentrations of [14C]DXR but did not cause significant differences in P-glycoprotein (P-gp) expression. Pretreatment of MCF-7adr cells with phorbol 12-myristate 13-acetate reduced the CGP 41251 mediated intracellular accumulation of [14C]DXR. At concentrations that induced drug uptake, CGP 41251 significantly decreased the level of P-gp phosphorylation in the cells but did not compete with [3H]azidopine for photoaffinity labeling of P-gp. These data provide evidence that CGP 41251 reverses the MDR phenotype by modulating the phosphorylation of P-gp and/or other PKC substrates critical to the maintenance of the MDR phenotype.

Identification of naturally processed human ovarian peptides recognized by tumor-associated CD8+ cytotoxic T lymphocytes.

Identification of naturally processed peptides recognized by tumorspecific CTLs may lead to epitope-specific tumor vaccines. Because these epitopes may be expressed differently on epithelial tumors and may differ in their ability to induce CTL in vivo, we have isolated the HLA-A2-peptide complexes by immunoaffinity from an established ovarian tumor line transfected with and expressing HLA-A2 gene. High-performance liquid chromatography-fractionated peptides were used to reconstitute epitopes recognized on HLA-A2 by three HLA-A2+ CD8+ CTL lines. These lines recognized at least three of the same groups of fractions (designated SKOV3.A, -B, and -C) but showed differences in the pattern of recognition of other fractions. To gain insight in the epitope distribution by freshly isolated ovarian tumors, we compared the recognition of peaks SKOV3.B and -C with the corresponding peaks from an ovarian tumor (OVA-6) that expressed similar levels of HLA-A2, using one of these lines (CTL-OVA-5) as indicator. CTL-OVA-5 recognized a large number of epitopes from peaks B and C rechromatographed on more resolving high-performance liquid chromatography gradient. Although a number of peaks appeared to be coincident on both SKOV3 and OVA-6, an even higher number appeared either not to overlap or to overlap only partially. These findings, which represent the first analysis of the epitopes presented by a patient tumor, suggest that the use of tumor line-derived peptides for vaccination may require selection of the epitopes corresponding to the ones presented by freshly isolated human tumors.

An N-myristoylated protein kinase C-alpha pseudosubstrate peptide that functions as a multidrug resistance reversal agent in human breast cancer cells is not a P-glycoprotein substrate.

Protein kinase C-alpha (PKC-alpha) activation is an important contributing factor in human breast cancer MCF-7 MDR cell drug resistance. We recently reported the use of N-myristoylated PKC-alpha pseudosubstrate peptides with potent PKC-alpha inhibitory activity as reversal agents of drug resistance in MCF-7 MDR cells. The peptides potently inhibit phosphorylation of the PKC-alpha substrates P-glycoprotein (P-gp), raf kinase and PKC-alpha itself in MCF-7 MDR cells in association with a severalfold induction of intracellular uptake of P-gp substrate chemotherapeutics and a statistically significant twofold increase in cellular chemosensitivity. We now report that the N-myristoylated PKC-alpha pseudosubstrate peptide N-myristoyl-RFARKGALRQKNV (P3) is not a P-gp substrate in MCF-7 MDR cells based on a comparison of the cellular uptake of [125I]-radiolabeled P3 in MCF-7 MDR vs MCF-7 WT cells. The extent of cellular uptake of the radiolabeled peptide in the drug-resistant cell line MCF-7 MDR was either greater than or equivalent to the uptake in the parental drug-sensitive MCF-7 WT cell line over a time course of 30 min to 6 h, and across a peptide concentration range of 25-100 microM. Additionally, treatment of the MCF-7 MDR cells with verapamil (VPL), a known P-gp efflux inhibitor, had no effect on the cellular accumulation of radiolabeled P3. Our results provide direct evidence that the N-myristoylated pseudosubstrate peptide is taken up equivalently by drug-sensitive and MDR cancer cells and therefore has potential value as an MDR reversal agent that operates by a novel mechanism.

Potent induction of human colon cancer cell uptake of chemotherapeutic drugs by N-myristoylated protein kinase C-alpha (PKC-alpha) pseudosubstrate peptides through a P-glycoprotein-independent mechanism.

Phorbol ester protein kinase C (PKC) activators and PKC isozyme over-expression have been shown to significantly reduce intracellular accumulation of chemotherapeutic drugs, in association with the induction of multidrug resistance (MDR) in drug-sensitive cancer cells and enhancement of drug resistance in MDR cancer cells. These observations constitute solid evidence that PKC plays a significant role in the MDR phenotype of cancer cells. PKC-catalyzed phosphorylation of the drug-efflux pump P-glycoprotein was recently ruled out as a contributing factor in MDR. At present, the sole drug transport-related event that has been identified as a component of the role of PKC in MDR is PKC-induced expression of the P-glycoprotein-encoding gene mdr1. The objective of this study was to test the hypothesis that PKC can modulate the uptake of chemotherapeutic drugs in cancer cells independently of P-glycoprotein. We analyzed the effects of selective PKC activators/inhibitors on the uptake of radiolabelled cytotoxic drugs by cultured human colon cancer cells that lacked P-glycoprotein activity and did not express the drug efflux pump at the level of message (mdr1) or protein. We found that the selective PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) significantly reduced uptake of [14C] Adriamycin and [3H] vincristine in human colon cancer cells devoid of P-glycoprotein activity, and that PKC-inhibitory N-myristoylated PKC-alpha pseudosubstrate synthetic peptides potently and selectively induced uptake of the cytotoxic drugs in the phorbol ester-treated and non-treated colon cancer cells. TPA treatment of the cells did not induce expression of either P-glycoprotein or its message mdr1. In contrast with [14C]Adriamycin and [3H] vincristine uptake, [3H] 5-fluorouracil uptake by the cells was unaffected by TPA and reduced by the PKC-inhibitory peptides. These results indicate that PKC activation can significantly reduce the uptake of multiple cytotoxic drugs by cancer cells independently of P-glycoprotein, and that N-myristoylated PKC-alpha pseudosubstrate peptides potently and selectively induce uptake of multiple cytotoxic drugs in cultured human colon cancer cells by a novel mechanism that does not involve P-glycoprotein and may involve PKC isozyme inhibition. Thus, N-myristoylated PKC-alpha pseudosubstrate peptides may offer a basis for the development of agents that reverse intrinsic drug resistance in human colon cancer.

Covalent modification of protein kinase C isozymes by the inactivating peptide substrate analog N-biotinyl-Arg-Arg-Arg-Cys-Leu-Arg-Arg-Leu. Evidence that the biotinylated peptide is an active-site affinity label.

We recently reported that the peptide substrate analog Arg-Lys-Arg-Cys-Leu-Arg-Arg-Leu (RKRCLRRL) irreversibly inactivates the protein kinase C (PKC) isozymes alpha, beta, and gamma in a dithiothreitol-sensitive manner by an active site-directed mechanism. We hypothesized that the inactivation mechanism entailed covalent complex formation between the PKC isozyme and the inactivator peptide. In this report, N-biotinylated analogs of RKRCLRRL that inactivate Ca2+-dependent PKC activity were designed and tested for their ability to covalently label PKC isozymes. A purified PKC isozyme mixture (alpha, beta, gamma, epsilon, zeta) was incubated with the N-biotinylated peptides and then subjected to denaturing gel electrophoresis, transferred to nitrocellulose, and probed for avidin-reactive species. The Ca2+-dependent PKC subfamily members PKC-alpha, -beta, and -gamma comigrated at 82 kDa and were distinguished by isozyme-specific immunoprecipitation. N-Biotinyl-RRRCLRRL covalently labeled all of the isozymes examined. When the isozymes were denatured prior to incubation with the N-biotinylated peptides, no labeling was observed. Inactivation of the Ca2+-dependent PKC subfamily by the N-biotinylated peptides was associated with covalent labeling of the 82-kDa PKC subspecies. The concentration dependence curves observed with N-biotinyl-RRRCLRRL were similar for inactivation and covalent labeling. The rank order of potency of three N-biotinylated peptides was the same for the inactivation and covalent labeling. Both the inactivation and covalent labeling were dithiothreitol-sensitive, and they were each subject to protection by MgATP and a peptide substrate analog. The covalent label was mapped to the catalytic domain of PKC by limited proteolysis of the modified enzyme. These results provide evidence that the N-biotinylated inactivator peptides are active-site affinity labels of PKC. The inactivator peptides most likely function by S-thiolating the active-site Cys residue conserved in PKC. This is the first report to demonstrate covalent labeling of PKC by a peptide substrate analog.

Partial reversal of multidrug resistance in human breast cancer cells by an N-myristoylated protein kinase C-alpha pseudosubstrate peptide.

The predominant characteristics of multidrug resistant (MDR) cancer cells are broad spectrum resistance to chemotherapeutic agents and a pronounced defect in intracellular accumulation of the drugs, in association with overexpression of the drug efflux pump P-glycoprotein. Protein kinase C (PKC) phosphorylates the linker region of P-glycoprotein. Evidence has been presented that the isozyme PKC-alpha may contribute to the drug resistance phenotype of human breast cancer MCF7-MDR cells, PKC-alpha is markedly overexpressed in MCF7-MDR cells, and artificial overexpression of PKC-alpha in MCF7 constructs that overexpress P-glycoprotein significantly enhances the MDR phenotype of the cells in association with increased P-glycoprotein phosphorylation. Verapamil, cyclosporin A, and a number of other agents that compete with cytotoxic drugs for binding sites on P-glycoprotein can potently reverse MDR, but this is accompanied by severe toxicity in vivo. In this report, we demonstrate that an N-myristoylated peptide that contains a sequence corresponding to the pseudosubstrate region of PKC-alpha (P1) partially reverses multidrug resistance in MCF7-MDR cells by a novel mechanism that involves inhibition of PKC-alpha. P1 and two related PKC inhibitory N-myristoylated peptides restored intracellular accumulation of chemotherapeutic drugs in association with inhibition of the phosphorylation of three PKC-alpha substrates in MCF7-MDR cells: PKC-alpha, Raf-1 kinase, and P-glycoprotein. A fourth N-myristoylated peptide substrate analog of PKC, P7, did not affect drug accumulation in the MCF7-MDR cells and failed to inhibit the phosphorylation of the PKC-alpha substrates. The effects of P1 and verapamil on drug accumulation in MCF7-MDR cells were additive. P1 did not affect P-glycoprotein expression. MCF7-MDR cells were not cross-resistant to P1, which suggest that the peptide was not transported by P-glycoprotein. Furthermore, P1 was distinguished from MDR reversal agents such as verapamil and cyclosporin A by its inability to inhibit [3H]azidopine photoaffinity labeling of P-glycoprotein. P1 actually increased [3H] azidopine photoaffinity labeling of P-glycoprotein in MCF7-MDR cells, providing evidence that the effects of P1 on P-glycoprotein in MCF7-MDR cells are not restricted to inhibition of the phosphorylation of the pump. P1 may provide a basis for developing a new generation of MDR reversal agents that function by a novel mechanism that involves inhibition of PKC-alpha-catalyzed P-glycoprotein phosphorylation.

Changes in an HER-2 peptide upregulating HLA-A2 expression affect both conformational epitopes and CTL recognition: implications for optimization of antigen presentation and tumor-specific CTL induction.

The HER-2/neu protooncogene (HER-2) is overexpressed in a significant number of breast and ovarian tumors. Peptides of HER-2 sequence were recently found to reconstitute recognition of cytotoxic T lymphocytes (CTLs) from tumor-associated (TALs) and tumor-infiltrating (TILs) lymphocytes, indicating that they reconstitute natural epitopes recognized by CTLs on HLA-A2+ tumors. Because HER-2 is an important antigen (Ag) for tumor-specific CTL induction and the immunogenicity of peptides for CTL induction is dependent on their presentation as stable complexes with HLA-A2, we identified peptides of high and low stabilizing activity from the sequence of HER-2 and the folate-binding protein (FBP). Distinct sequence patterns in the region positions (P)3-P5 and P1 were found for peptides with high (HSA) and low (LSA) stabilizing ability. A low-HLA-A2-affinity HER-2 peptide, P1 of the CTL epitope, was found to be permissive to substitutions that enhanced HLA-A2-stabilizing ability and conserved CTL recognition. In contrast, the region P3-P5 was not permissive to sequence changes. We conclude that the selective permissivity of P1 and P9 in the tumor epitope sequence may have important implications for optimization of tumor Ag presentation, and "neoantigenicity" of self-antigens, aiming toward induction of tumor-reactive CTLs of defined affinity and specificity for target Ags.