Co-treatment with a C1B5 peptide of protein kinase Cγ and a low dose of gemcitabine strongly attenuated pancreatic cancer growth in mice through T cell activation.

Although gemcitabine is an effective chemotherapeutic for pancreatic cancer, severe side effects often accompany its use. Since we have discovered that locally administered C1B domain peptides effectively control tumor growth without any side effects, the efficacy of co-treatment with this peptide and a low dose of gemcitabine on the growth of pancreatic cancer was examined. Two- and three-dimensional cell culture studies clarified that a co-treatment with C1B5 peptide and gemcitabine significantly attenuated growth of PAN02 mouse and PANC-1 human pancreatic cancer cells in 2D and 3D cultures. Although treatment with the low dose of gemcitabine alone (76%) or the C1B5 peptide alone (39%) inhibited tumor growth moderately, a co-treatment with C1B5 peptide and a low dose of gemcitabine markedly inhibited the growth of PAN02 autografts in the mouse peritoneal cavity (94% inhibition) without any noticeable adverse effect. The number of peritoneal cavity-infiltrating neutrophils and granzyme B+ lymphocytes was significantly higher in the co-treatment group than in the control group. A significant increase of granzyme B mRNA expression was also detected in human T cells by the co-treatment. Taken together, the current study suggests that C1B5 peptide offers a remarkably effective combination treatment strategy to reduce side effects associated with gemcitabine, without losing its tumoricidal effect.

Protein kinase D activity controls endothelial nitric oxide synthesis.

Vascular endothelial growth factor (VEGF) regulates key functions of the endothelium, such as angiogenesis or vessel repair in processes involving endothelial nitric oxide synthase (eNOS) activation. One of the effector kinases that become activated in endothelial cells upon VEGF treatment is protein kinase D (PKD). Here, we show that PKD phosphorylates eNOS, leading to its activation and a concomitant increase in NO synthesis. Using mass spectrometry, we show that the purified active kinase specifically phosphorylates recombinant eNOS on Ser1179. Treatment of endothelial cells with VEGF or phorbol 12,13-dibutyrate (PDBu) activates PKD and increases eNOS Ser1179 phosphorylation. In addition, pharmacological inhibition of PKD and gene silencing of both PKD1 and PKD2 abrogate VEGF signaling, resulting in a clear diminished migration of endothelial cells in a wound healing assay. Finally, inhibition of PKD in mice results in an almost complete disappearance of the VEGF-induced vasodilatation, as monitored through determination of the diameter of the carotid artery. Hence, our data indicate that PKD is a new regulatory kinase of eNOS in endothelial cells whose activity orchestrates mammalian vascular tone.

C1B domain peptide of protein kinase Cγ significantly suppresses growth of human colon cancer cells in vitro and in an in vivo mouse xenograft model through induction of cell cycle arrest and apoptosis.

Two peptides derived from the C1B domain of protein kinase Cγ (PKCγ) were shown to associate with classical PKC isozymes and modulate their activities. These C1B peptides are designated C1B1 (amino acid residues 101-112) and C1B5 (residues 141-151). Since PKC enzyme activity is shown to be involved in colon cancer development, the effect of C1B peptides on the growth of various human colon cancer cell lines was examined in vitro and in vivo. Sub-micromolar to micromolar levels of both C1B peptides induced approximately 60-70% growth attenuation in multiple colon cancer cell lines in a soft agar tumor colony assay; however, C1B5 peptide was not cytotoxic to normal colon epithelial cells in two dimensional culture. The effect of C1B5 peptide on colony growth of COLO205 cells was reversed by treatment with the PKCα/ß inhibitor, Ro-32-0432. C1B peptide treatment attenuated COLO205 cells via two mechanisms: 1) cell cycle arrest and 2) stimulation of apoptosis. This is evident in G 2 arrest and increases in levels of cleaved caspase 3 and p53 phosphorylated at serine 20. Intratumoral injection of C1B5 peptide (20 mg/kg/day, every three days) markedly attenuated the growth of subcutaneous xenografts of COLO205 cells in SCID mice by 76% compared with the control. Taken together, these results strongly suggest that C1B peptides have negligible effects on normal tissues but are potentially effective chemotherapeutic agents for colon cancer.

A phase II study of enzastaurin, a protein kinase C beta inhibitor, in patients with relapsed or refractory mantle cell lymphoma.

BACKGROUND: Protein kinase C beta (PKCbeta), a pivotal enzyme in B-cell signaling and survival, is overexpressed in most cases of mantle cell lymphoma (MCL). Activation of PI3K/AKT pathway is involved in pathogenesis of MCL. Enzastaurin, an oral serine/threonine kinase inhibitor, suppresses signaling through PKCbeta/PI3K/AKT pathways, induces apoptosis, reduces proliferation, and suppresses tumor-induced angiogenesis. PATIENTS AND METHODS: Patients with relapsed/refractory MCL, and no more than four regimens of prior therapy, received 500 mg enzastaurin, orally, once daily. RESULTS: Sixty patients, median age 66 years (range 45-85), Eastern Cooperative Oncology Group performance status of zero to two (48% had baseline International Prognostic Index of 3-5), were enrolled. Most patients had prior CHOP-like chemotherapy and/or rituximab (median = 2 regimens). No drug-related deaths occurred. There was one case each of grade 3 anemia, diarrhea, dyspnea, vomiting, hypotension, and syncope. Fatigue was the most common toxicity. Although no objective tumor responses occurred, 22 patients (37%, 95% CI 25% to 49%) were free from progression (FFP) for > or =3 cycles (one cycle = 28 days); 6 of 22 were FFP for >6 months. Two patients remain on treatment and FFP at >23 months. CONCLUSION: Freedom from progression for >6 months in six patients and a favorable toxicity profile with minimal hematological toxicity indicate that enzastaurin warrants evaluation as maintenance therapy and combination chemotherapy in MCL.

Spatial learning in rats is impaired by microinfusions of protein kinase C-gamma antisense oligodeoxynucleotide within the nucleus accumbens.

The nucleus accumbens (NAcc) has been shown to play a role in motor and spatial learning. Protein kinase C (PKC) has been implicated in the mechanisms of initiation and maintenance of long-term potentiation that is thought to be involved in the storage of long-term memory. In the present study, the importance of de novo synthesis of PKC-gamma within the NAcc in the acquisition and retention of spatial discrimination learning was assessed using an antisense knockdown approach. Separate groups of Long-Evans rats were exposed to acute microinfusions (6microg/microl) of PKC-gamma antisense oligodeoxynucleotide (AS-ODN), control oligodeoxynucleotide (C-ODN) or vehicle into the NAcc at 24 and 3h before each training session. Behavioral findings showed that the blockade of NAcc-PKC-gamma translation caused impairments in the early phase of learning and retention of spatial information. Biochemical experiments showed that PKC-gamma expression was reduced and Ca(2+)/phospholipid-dependent protein kinase C (PKC) activity was blocked significantly in the AS-ODN-treated rats in comparison with control rats. The present findings suggest that NAcc-PKC-gamma plays a role during the early acquisition of spatial learning. Also, retention test results suggest that NAcc-PKC-gamma may be working as an intermediate factor involved in the onset of molecular mechanisms necessary for spatial memory consolidation within the NAcc.

Pooled human albumin primes neutrophils.

Human pooled albumin has traditionally been used both to pacify the artificial surfaces in a cardiopulmonary bypass circuit and also for volume repletion following surgery. In evaluating the routine use of albumin in multiple phases of cardiac surgery, conscientious surgical teams must assess both the physiological and financial price of albumin. Albumin indiscriminately binds many plasma proteins and lipids. In this series of experiments, we explored the influence of highly purified albumin devoid of bound lipids and globulins on both receptor-dependent (FMLP) and receptor-independent (PMA) priming/activation of human neutrophils. We believe that it is important to distinguish the direct influence of albumin from the albumin-bound proteins and lipids. We, therefore, also examined the effect of clinically accessible human pooled albumin on human neutrophils. We observed a dose-dependent priming/activation (elastase release) of human neutrophils by both pooled and purified albumin. We conclude that it is increasingly difficult to justify the routine use of albumin in cardiac surgical patients.

Recombinant human activated protein C for use in severe sepsis.

OBJECTIVE: To review the efficacy and safety of drotrecogin alfa (recombinant human activated protein C) in the treatment of sepsis. DATA SOURCES: Literature was identified through a MEDLINE search (1966-January 2002), the product manufacturer, and the Food and Drug Administration. STUDY SELECTION/DATA EXTRACTION: All relevant information identified from the data sources was evaluated. DATA SYNTHESIS: Drotrecogin alfa reduces coagulation and inflammation in septic patients. A large placebo-controlled clinical trial (n = 1690) of drotrecogin alfa in severely septic patients demonstrated a reduction in mortality (24.7% vs. 30.8%; p = 0.005), with increased bleeding risks (24.9% vs. 17.7%; p <0.001). Patients with more severe sepsis appeared to gain the most benefit. The complete clinical and economic impact of this agent requires further analysis. CONCLUSIONS: Drotrecogin alfa offers a significant advance in the treatment of severe sepsis. Judicious use in appropriate patients is necessary to control cost and maximize clinical benefits.

Regulation of caveolin-1 expression and secretion by a protein kinase cepsilon signaling pathway in human prostate cancer cells.

Caveolin-1, androgen receptor, c-Myc, and protein kinase Cepsilon (PKCepsilon) proteins are overrepresented in most advanced prostate cancer tumors. Previously, we demonstrated that PKCepsilon has the capacity to enhance the expression of both caveolin-1 and c-Myc in cultured prostate cancer cells and is sufficient to induce the growth of androgen-independent tumors. In this study, we have uncovered further evidence of a functional interplay among these proteins in the CWR22 model of human prostate cancer. The results demonstrated that PKCepsilon expression was naturally up-regulated in recurrent CWR22 tumors and that this oncoprotein was required to sustain the androgen-independent proliferation of CWR-R1 cells in culture. Gene transfer experiments demonstrated that PKCepsilon had the potential to augment the expression and secretion of a biologically active caveolin-1 protein that supports the growth of the CWR-R1 cell line. Antisense and pharmacological experiments provided additional evidence that the sequential activation of PKCepsilon, mitogen-activated protein kinases, c-Myc, and androgen receptor signaling drove the downstream expression of caveolin-1 in CWR-R1 cells. Finally, we demonstrate that mitogen-activated protein kinases were required downstream of PKCepsilon to derepress the transcriptional elongation of the c-myc gene. Our findings support the hypothesis that PKCepsilon may advance the recurrence of human prostate cancer by promoting the expression of several important downstream effectors of disease progression.

Differential activation of mitogen-activated protein kinase cascades and apoptosis by protein kinase C epsilon and delta in neonatal rat ventricular myocytes.

Protein kinase C (PKC) epsilon and PKCdelta translocation in neonatal rat ventricular myocytes (NRVMs) is accompanied by subsequent activation of the ERK, JNK, and p38(MAPK) cascades; however, it is not known if either or both novel PKCs are necessary for their downstream activation. Use of PKC inhibitors to answer this question is complicated by a lack of isoenzyme specificity, and the fact that many PKC inhibitors stimulate JNK and p38(MAPK) activity. Therefore, replication-defective adenoviruses (Advs) encoding constitutively active (ca) mutants of PKCepsilon and PKCdelta were used to test if either or both of these PKCs are sufficient to activate ERKs, JNKs, and/or p38(MAPK) in NRVMs. Adv-caPKCepsilon infection (1 to 25 multiplicities of viral infection (MOI); 4 to 48 hours) increased total PKCepsilon levels in a time- and dose-dependent manner, with maximal expression observed 8 hours after Adv infection. Adv-caPKCepsilon induced a time- and dose-dependent increase in phosphorylated p42 and p44 ERKs, as compared with a control Adv encoding beta-galactosidase (Adv-nebetagal). Maximal ERK phosphorylation occurred 8 hours after Adv infection. In contrast, JNK was only minimally activated, and p38(MAPK) was relatively unaffected. Adv-caPKCdelta infection (1 to 25 MOI, 4 to 48 hours) increased total PKCdelta levels in a similar fashion. Adv-caPKCdelta (5 MOI) induced a 29-fold increase in phosphorylated p54 JNK, and a 15-fold increase in phosphorylated p38(MAPK) 24 hours after Adv infection. In contrast, p42 and p44 ERK were only minimally activated. Whereas neither Adv induced NRVM hypertrophy, Adv-caPKCdelta, but not Adv-caPKCepsilon, induced NRVM apoptosis. We conclude that the novel PKCs differentially regulate MAPK cascades and apoptosis in an isoenzyme-specific and time-dependent manner.

Ca2+-independent protein kinase C Apl II mediates the serotonin-induced facilitation at depressed aplysia sensorimotor synapses.

At nondepressed Aplysia sensory to motor synapses, serotonin (5-HT) facilitates transmitter release primarily through a protein kinase A pathway. In contrast, at depressed Aplysia sensory to motor synapses, 5-HT facilitates transmitter release primarily through a protein kinase C (PKC)-dependent pathway. It is known that only two phorbol ester-activated PKC isoforms, the Ca(2+)-dependent PKC Apl I and the Ca(2+)-independent PKC Apl II, exist in the Aplysia nervous system. For the first time, we have now been able to functionally determine which isoform of PKC is involved in a particular form of plasticity. We microinjected cultured sensorimotor pairs of neurons with various PKC constructs tagged with the enhanced green fluorescent protein as a reporter for successful plasmid expression. Our results demonstrate that short-term facilitation of depressed synapses is mediated by PKC Apl II. Dominant-negative PKC Apl II, but not dominant-negative PKC Apl I, disrupted the normal kinetics of 5-HT-induced facilitation by completely blocking its rapid onset. This effect was specific to depressed synapses, because dominant-negative PKC Apl II did not inhibit 5-HT-mediated facilitation of nondepressed synapses. Our results suggest that not only different signal transduction pathways but also different isoforms of a specific cascade may mediate physiological responses according to the state of a synapse.

Hyperphosphorylation of Tau and filopodial retraction following microinjection of protein kinase C catalytic subunits.

Limited proteolysis of protein kinase C (PKC) by calcium-activated proteolysis cleaves the regulatory and catalytic subunits of PKC, generating a free, constitutively activated kinase ("PKM") that, unlike the intact parent enzyme, is not calcium-dependent, and is not restricted to the plasma membrane. These latter properties leave open the possibility that PKM may have access to, and may therefore phosphorylate, substrates normally unavailable to intact PKC. We examined the potential involvement of such aberrant phosphorylation in certain aspects of the neurodegeneration accompanying Alzheimer's disease by microinjecting PKC and PKM, along with a rhodamine-conjugated dextran tracer, into undifferentiated NB2a/d1 mouse neuroblastoma cells. After 4 hr, cultures were fixed and processed for immunofluorescence with monoclonal antibodies (PHF-1, ALZ-50, Tau-1, AT8) directed against tau in various phosphorylation states followed by fluorescein-conjugated secondary antibodies. Microinjected cells were localized via co-injected rhodamine-conjugated dextran tracer under rhodamine illumination, after which antibody immunoreactivity was examined under fluorescein illumination. Microdensitometric analyses indicated that microinjection of PKC did not increase basal immunofluorescent intensities of the antibodies; by contrast, microinjection of PKM induced three- and twofold increases in PHF-1 and ALZ-50 levels, respectively. By contrast, no significant alteration was observed in AT8 and Tau-1 immunofluorescence following either PKC or PKM microinjection. Whereas undifferentiated NB2a/d1 cells typically elaborate short, filopodia-like neurites, phase-contrast microscopy revealed the absence of filopodia or neurites on PKM-injected cells, while a similar percentage of PKC-injected cells. Cell-free analyses confirmed the ability of PKC, in the presence of necessary co-factors, and PKM to increase PHF-1 and ALZ-50 immunoreactivity; no change was observed in AT8 or Tau-1 immunoreactivity. These findings underscore the possibility that an abnormal amplification in limited PKC proteolysis to generate PKM could, under certain pathological conditions, contribute to neuronal degeneration.

Inhibitory effect of staurosporine on protein kinase C stimulation of airway smooth muscle cells.

Phospholipid/calcium-dependent protein kinase (protein kinase C [PKC]) is a critical system in signal transduction of many different cells including airway smooth muscle (ASM) cells. We have previously shown that after administration of different phorbol esters, specific activators of PKC, characteristic electrical and contractile changes of ASM cells can be demonstrated. Similarly, our data showed that stimulation of PKC is implicated in the process of sensitization and the specific antigen challenge response of ASM cells. In this study we examined the effect of staurosporine, a microbial alkaloid, which has been reported to be a specific inhibitor of PKC, on sensitization- and specific antigen challenge-induced electrical and contractile changes of ASM cells. The effect of staurosporine was compared with those of amiloride, furosemide, and compounds NA-0345 and H-7, both synthetic PKC inhibitors. We used ASM preparations isolated from adult male guinea pigs (Camm-Hartley strain). Changes in both membrane potential (Em), measured by a glass microelectrode technique, and isometric force, measured by copper-beryllium strain gauge, were continuously monitored. Experiments were conducted with optimal length (Lmax) of ASM preparations and at 37 degrees C. We found that the exposure of ASM preparations to staurosporine, NA-0345, H-7, amiloride, and furosemide (all in 10(-12) to 10(-4) M) had no measurable effect on the resting membrane potential or isometric force of ASM preparations. In contrast, pretreatment of ASM preparations with staurosporine, NA-0345, H-7, amiloride, or furosemide significantly attenuated (p < 0.001) phorbol myristate acetate-induced changes of ASM preparations.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of the mitogenic activity of eukaryotic translation initiation factor-4E by protein kinase C.

Eukaryotic initiation factor-4E (eIF-4E) binds to the cap structure of eukaryotic mRNAs and is a component of the cap-binding protein complex eIF-4F. eIF-4E is present in cells in limiting concentrations and is phosphorylated both in vivo and in vitro by protein kinase C (PKC). Recently, eIF-4E has been implicated as an intracellular transducer of extracellular growth signals; microinjection of recombinant eIF-4E into quiescent NIH 3T3 cells induced DNA synthesis. In the present report, the mitogenic activity of eIF-4E was examined after coinjection with PKC. Recombinant eIF-4E was phosphorylated by PKC at the same amino acid that is phosphorylated in cultured cells and reticulocytes in response to phorbol ester. At limiting concentrations of eIF-4E, coinjection with PKC induced a fivefold increase in the mitogenic activity of eIF-4E. Injection of PKC alone or coinjection of eIF-4E with cAMP-dependent protein kinase (PKA) or the Raf protein had no effect. These results suggest that the mitogenic activity of eIF-4E is enhanced by PKC-specific phosphorylation and that phosphate addition is a rate-limiting step in eIF-4E activity.

Modulation of maturation and ribosomal protein S6 phosphorylation in Xenopus oocytes by microinjection of oncogenic ras protein and protein kinase C.

Using Xenopus oocytes as a model system, we investigated the possible involvement of ras proteins in the pathway leading to phosphorylation of ribosomal protein S6. Our results indicate that microinjection of oncogenic T24 H-ras protein (which contains valine at position 12) markedly stimulated S6 phosphorylation on serine residues in oocytes, whereas normal ras protein (which contains glycine at position 12) was without effect. The S6 phosphorylation activity in the cell extract from T24 ras protein-injected oocytes was increased significantly. In addition, injection of protein kinase C potentiated the induction of maturation and S6 phosphorylation by the oncogenic ras protein. A similar potentiation was detected when T24 ras protein-injected oocytes were incubated with active phorbol ester. These findings suggest that ras proteins activate the pathway linked to S6 phosphorylation and that protein kinase C has a synergistic effect on the ras-mediated pathway.

Effect of microinjected catalytic fragment of protein kinase C on morphological change in Swiss 3T3 cells.

Although phorbol esters can enhance formation of an active, catalytic domain of protein kinase C (PKC) in intact cells, little is known about the actual importance of the proteolytic pathway in mediating cellular responses to the phorbol esters or other PKC activators. To explore this issue, we examined the effect of microinjected catalytic fragment of PKC on Swiss 3T3 cell morphology. In contrast to the dramatic, rapid response upon phorbol ester treatment, catalytic fragment microinjected in the presence of bovine serum albumin or normal goat immunoglobulin G as carrier protein had no effect. A morphological response similar but not identical to the effect of phorbol ester treatment was obtained, however, if catalytic fragment was microinjected in the presence of normal rabbit immunoglobulin G rather than the usual carrier proteins. The normal rabbit immunoglobulin by itself was inactive. Although the mechanism remains undefined, normal rabbit immunoglobulin but not other carrier proteins modulated PKC activity in vitro. We conclude that the generation of free catalytic fragment of PKC cannot account for the morphological response of Swiss 3T3 cells to the phorbol esters; secondary factors may, however, potentiate its action.

A monoclonal antibody recognising the site of limited proteolysis of protein kinase C. Inhibition of down-regulation in vivo.

A monoclonal antibody to protein kinase C is described that recognises the site of limited proteolysis on the native enzyme. Binding of the antibody to the purified kinase in vitro blocks partial proteolysis by trypsin, and introduction of the Fab fragment into a rodent glioma cell line inhibits phorbol-ester-induced down-regulation of the kinase. These observations are discussed in the context of the domain structure of protein kinase C and the agonist-induced proteolysis of the kinase in vivo.