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1.
Cell Death Dis ; 6: e1815, 2015 Jul 09.
Article in English | MEDLINE | ID: mdl-26158521

ABSTRACT

Approved proteasome inhibitors have advanced the treatment of multiple myeloma but are associated with serious toxicities, poor pharmacokinetics, and most with the inconvenience of intravenous administration. We therefore sought to identify novel orally bioavailable proteasome inhibitors with a continuous daily dosing schedule and improved therapeutic window using a unique drug discovery platform. We employed a fluorine-based medicinal chemistry technology to synthesize 14 novel analogs of epoxyketone-based proteasome inhibitors and screened them for their stability, ability to inhibit the chymotrypsin-like proteasome, and antimyeloma activity in vitro. The tolerability, pharmacokinetics, pharmacodynamic activity, and antimyeloma efficacy of our lead candidate were examined in NOD/SCID mice. We identified a tripeptide epoxyketone, FV-162, as a metabolically stable, potent proteasome inhibitor cytotoxic to human myeloma cell lines and primary myeloma cells. FV-162 had limited toxicity and was well tolerated on a continuous daily dosing schedule. Compared with the benchmark oral irreversible proteasome inhibitor, ONX-0192, FV-162 had a lower peak plasma concentration and longer half-life, resulting in a larger area under the curve (AUC). Oral FV-162 treatment induced rapid, irreversible inhibition of chymotrypsin-like proteasome activity in murine red blood cells and inhibited tumor growth in a myeloma xenograft model. Our data suggest that oral FV-162 with continuous daily dosing schedule displays a favorable safety, efficacy, and pharmacokinetic profile in vivo, identifying it as a promising lead for clinical evaluation in myeloma therapy.


Subject(s)
Antineoplastic Agents/administration & dosage , Fluorine/administration & dosage , Multiple Myeloma/drug therapy , Oligopeptides/administration & dosage , Proteasome Inhibitors/administration & dosage , Animals , Antineoplastic Agents/pharmacokinetics , Cell Line, Tumor , Cell Proliferation/drug effects , Fluorine/pharmacokinetics , Humans , Mice , Multiple Myeloma/pathology , Proteasome Inhibitors/pharmacokinetics , Xenograft Model Antitumor Assays
2.
Bone Marrow Transplant ; 50(3): 375-9, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25599165

ABSTRACT

Cyclophosphamide, bortezomib and dexamethasone (CyBorD) is a highly active three-drug induction regimen for untreated transplant-eligible multiple myeloma patients. Although CyBorD has been evaluated only in the phase 2 setting in a limited number of patients, its high efficacy and ease of administration have led to its widespread use. Given that clinical trial efficacy can overestimate real-life effectiveness, we reviewed our institutional experience with 109 newly diagnosed patients who were treated with CyBorD in a non-clinical trial setting. After a median of four cycles, overall response rate (ORR) and very good partial response rate or better (⩾VGPR) were 95 and 66%, respectively, comparable to phase 2 studies of CyBorD and other three/four-drug induction regimens. All patients subsequently underwent successful stem cell collection and upgraded responses to ORR 98% and ⩾VGPR 79% post transplant. At a median follow-up of 19.8 months after diagnosis, the 2-year OS probability was 95.3% (95%CI: 89-98). The presence of concurrent plasmacytoma at diagnosis was the only prognostic factor predicting poorer survival (HR=5.56; 95%CI: 0.92-33.74; P=0.03). CyBorD was well-tolerated, with no severe peripheral neuropathy and minimal hematologic toxicity. Therefore, CyBorD is a convenient, well-tolerated, highly effective induction regimen in preparation for autologous SCT in real-life clinical practice.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Multiple Myeloma/drug therapy , Boronic Acids/administration & dosage , Bortezomib , Cyclophosphamide/administration & dosage , Dexamethasone/administration & dosage , Female , Humans , Male , Pyrazines/administration & dosage , Remission Induction
3.
Bone Marrow Transplant ; 50(1): 95-9, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25285800

ABSTRACT

Numerous studies have reported the feasibility and safety of autologous SCT (ASCT) in patients with multiple myeloma (MM) and mild to moderate renal impairment, but there are limited data in dialysis-dependent patients. In this retrospective study, we reviewed the toxicities and efficacy outcomes of 33 MM patients with dialysis-dependent renal failure who underwent ASCT at our institution from 1998 to 2012. The most common grade 3 non-hematologic toxicities were mucositis (49%), infection (15%) and bleeding (6%). Atrial dysrhythmias (24%) and delirium (30%) of all grades were also common. Hematologic toxicities included febrile neutropenia (88%); and RBC and platelet transfusions were required by 71 and 100% of patients, respectively. Transplant-related mortality (TRM) was high at 15%, predominantly caused by septic shock. Response to ASCT was at least VGPR (very good PR) in 50%, PR in 46.2% and stable disease (SD) in 3.8%. Median OS was 5.6 years, comparable to our overall institutional data. Overall, seven patients became dialysis independent. We conclude that ASCT can be an effective treatment for dialysis-dependent MM patients, with high response rates and survival. However, toxicities and a high TRM are observed indicating that further studies are needed to enhance the safety of this approach.


Subject(s)
Multiple Myeloma , Renal Dialysis , Renal Insufficiency , Stem Cell Transplantation , Adult , Aged , Autografts , Databases, Factual , Humans , Male , Middle Aged , Multiple Myeloma/complications , Multiple Myeloma/mortality , Multiple Myeloma/therapy , Renal Insufficiency/complications , Renal Insufficiency/mortality , Renal Insufficiency/therapy , Retrospective Studies , Survival Rate
4.
Bone Marrow Transplant ; 50(2): 204-8, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25347006

ABSTRACT

The role of auto-SCT in the treatment of multiple myeloma (MM) in the era of novel agents continues to evolve. It is now clear that the depth of response and clinical outcomes have significantly improved as a result of the combination of these strategies. However, not all patients with MM who undergo auto-SCT are able to sustain a meaningful response and 20% of patients relapse shortly after auto-SCT. In this study, we aimed to assess the impact of early relapse (ER) after auto-SCT on OS for MM patients undergoing single auto-SCT who had received novel agent-based induction regimens. All consecutive patients with MM undergoing single auto-SCT from January 2002 to September 2012 who had novel induction therapy were evaluated. A total of 184 patients were identified. The median OS and PFS for the group of transplanted patients were 93 and 25.4 months, respectively. Median time to relapse was 17.2 months with 40% having relapsed at the time of analysis. ER (<12 months post auto-SCT) was seen in 27 (36%) out of 75 patients who had relapsed, and median OS was significantly shorter than in those with non-ER. Multivariate analysis showed ER as the major independent prognostic factor for OS. On the basis of these findings, we conclude that not only attainment of a good response, but sustainability of it, appears to be a major prognostic variable in MM in the era of novel therapy. Patients with ER post auto-SCT should biologically be characterized in prospective studies to better understand the mechanisms of resistance associated with this particular entity.


Subject(s)
Induction Chemotherapy/methods , Multiple Myeloma/mortality , Multiple Myeloma/therapy , Stem Cell Transplantation , Autografts , Disease-Free Survival , Female , Humans , Male , Middle Aged , Recurrence , Survival Rate , Time Factors
5.
Leukemia ; 27(12): 2351-6, 2013 Dec.
Article in English | MEDLINE | ID: mdl-23670297

ABSTRACT

Several cytogenetic abnormalities are associated with poor outcomes in multiple myeloma (MM). We prospectively analyzed the impact of cytogenetic abnormalities on outcomes during the phase 2 PX-171-003-A1 study of single-agent carfilzomib for relapsed and refractory MM. In the response-evaluable population (257/266), fluorescence in situ hybridization (FISH)/conventional cytogenetic profiles were available for 229 patients; 62 (27.1%) had high-risk cytogenetics--del 17p13, t(4;14) or t(14;16) by interphase FISH or deletion 13 or hypodiploidy by metaphase cytogenetics--and 167 (72.9%) had standard-risk profiles. Generally, baseline characteristics were similar between the subgroups, but International Staging System stage III disease was more common in high- vs standard-risk patients (41.9% vs 27.5%) as was Eastern Cooperative Oncology Group performance status 1/2 (85.5% vs 68.3%). Overall response was comparable between the subgroups (25.8% vs 24.6%, respectively; P=0.85), while time-to-event end points showed a trend of shorter duration in high-risk patients, including median duration of response (5.6 months (95% confidence interval (CI) 3.7-7.8) vs 8.3 months (95% CI 5.6-12.3)) and overall survival (9.3 (95% CI 6.5-13.0) vs 19.0 months (95% CI 15.4-NE); P=0.0003). Taken together, these findings demonstrate that single-agent carfilzomib is efficacious and has the potential to at least partially overcome the impact of high-risk cytogenetics in heavily pre-treated patients with MM.


Subject(s)
Antineoplastic Agents/therapeutic use , Chromosome Aberrations , Multiple Myeloma/drug therapy , Oligopeptides/therapeutic use , Adult , Aged , Aged, 80 and over , Female , Humans , In Situ Hybridization, Fluorescence , Male , Middle Aged , Multiple Myeloma/genetics , Survival Analysis , Treatment Outcome
6.
Phys Rev Lett ; 104(19): 197203, 2010 May 14.
Article in English | MEDLINE | ID: mdl-20866995

ABSTRACT

We predict and experimentally demonstrate that in a medium with externally induced anisotropy, a wave source of a sufficiently small size can excite practically nondiffractive wave beams with stable subwavelength transverse aperture. The direction of beam propagation is controlled by rotating the induced anisotropy axis. Nondiffractive wave beam propagation, reflection, and scattering, as well as beam steering have been directly observed by optically probing dipolar spin waves in yttrium iron garnet films, where the uniaxial anisotropy was created by an in-plane bias magnetic field.

7.
Leukemia ; 23(7): 1337-41, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19225538

ABSTRACT

We have studied a three-drug combination with cyclophosphamide, bortezomib and dexamethasone (CyBorD) on a 28-day cycle in the treatment of newly diagnosed multiple myeloma (MM) patients to assess response and toxicity. The primary endpoint of response was evaluated after four cycles. Thirty-three newly diagnosed, symptomatic patients with MM received bortezomib 1.3 mg/m(2) intravenously on days 1, 4, 8 and 11, cyclophosphamide 300 mg/m(2) orally on days 1, 8, 15 and 22 and dexamethasone 40 mg orally on days 1-4, 9-12 and 17-20 on a 28-day cycle for four cycles. Responses were rapid with a mean 80% decline in the sentinel monoclonal protein at the end of two cycles. The overall intent to treat response rate (>or= partial response) was 88%, with 61% of very good partial response or better (>or=VGPR) and 39% of complete/near complete response (CR/nCR). For the 28 patients who completed all four cycles of therapy, the CR/nCR rate was 46% and VGPR rate was 71%. All patients undergoing stem cell harvest had a successful collection. Twenty-three patients underwent stem cell transplantation (SCT) and are evaluable through day 100 with CR/nCR documented in 70% and >or=VGPR in 74%. In conclusion, CyBorD produces a rapid and profound response in patients with newly diagnosed MM with manageable toxicity.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Multiple Myeloma/drug therapy , Adult , Aged , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Boronic Acids/administration & dosage , Bortezomib , Cyclophosphamide/administration & dosage , Dexamethasone/administration & dosage , Female , Humans , Male , Middle Aged , Multiple Myeloma/surgery , Pyrazines/administration & dosage , Stem Cell Transplantation , Treatment Outcome
8.
Leukemia ; 23(3): 585-90, 2009 Mar.
Article in English | MEDLINE | ID: mdl-18754030
12.
Cell Death Differ ; 14(3): 511-23, 2007 Mar.
Article in English | MEDLINE | ID: mdl-16888648

ABSTRACT

By comparing mRNA profiles in cultured fibroblasts from patients affected with lysosomal storage diseases, we identified differentially expressed genes common to these conditions. These studies, confirmed by biochemical experiments, demonstrated that lysosomal storage is associated with downregulation of ubiquitin C-terminal hydrolase, UCH-L1 in the cells of eight different lysosomal disorders, as well as in the brain of a mouse model of Sandhoff disease. Induction of lysosomal storage by the cysteine protease inhibitor E-64 also reduced UCH-L1 mRNA, protein level and activity. All cells exhibiting lysosomal storage contained ubiquitinated protein aggregates and showed reduced levels of free ubiquitin and decreased proteasome activity. The caspase-mediated apoptosis in E-64-treated fibroblasts was reversed by transfection with a UCH-L1 plasmid, and increased after downregulation of UCH-L1 by siRNA, suggesting that UCH-L1 deficiency and impairment of the ubiquitin-dependent protein degradation pathway can contribute to the increased cell death observed in many lysosomal storage disorders.


Subject(s)
Gene Expression Regulation, Enzymologic , Lysosomal Storage Diseases/metabolism , RNA/metabolism , Signal Transduction , Ubiquitin Thiolesterase/metabolism , Ubiquitin/metabolism , Animals , Apoptosis , Cysteine Proteinase Inhibitors/pharmacology , Fibroblasts/enzymology , Fibroblasts/metabolism , Humans , Leucine/analogs & derivatives , Leucine/pharmacology , Lysosomal Storage Diseases/enzymology , Lysosomal Storage Diseases/genetics , Mice , Oligonucleotide Array Sequence Analysis , Proteasome Endopeptidase Complex/metabolism , Proteasome Endopeptidase Complex/physiology , RNA, Small Interfering , Skin/cytology , Skin/enzymology , Skin/metabolism , Ubiquitin Thiolesterase/genetics
13.
Bone Marrow Transplant ; 36(9): 793-6, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16113669

ABSTRACT

Genetic aberrations have emerged as major prognostic factors for patients with multiple myeloma (MM). We evaluated 126 MM patients for t(4;14) or t(11;14), 13q or p53 deletions and correlated the number of genetic aberrations with patient's clinical outcome following undergoing autologous stem cell transplantation. We demonstrate the significance of genetic-based risk classification that clearly segregate patients into low (no genetic abnormalities or only t(11;14)), intermediate (any one of the genetic abnormalities other than t(11;14)) and high-risk groups (any two or more of the genetic abnormalities other than t(11;14)). High-risk patients do not benefit from stem cell transplant and should be offered alternative therapies.


Subject(s)
Chromosome Deletion , Chromosomes, Human/genetics , Gene Deletion , Multiple Myeloma/genetics , Stem Cell Transplantation , Translocation, Genetic , Tumor Suppressor Protein p53/genetics , Adult , Aged , Complementary Therapies , Female , Humans , Male , Middle Aged , Multiple Myeloma/mortality , Multiple Myeloma/therapy , Risk Factors , Stem Cell Transplantation/methods , Transplantation, Autologous
14.
J Biol Chem ; 276(49): 46172-81, 2001 Dec 07.
Article in English | MEDLINE | ID: mdl-11571282

ABSTRACT

Sialidase (neuraminidase), encoded by the neu-1 gene in the major histocompatibility complex locus catalyzes the intralysosomal degradation of sialylated glycoconjugates. Inherited deficiency of sialidase results in sialidosis or galactosialidosis, both severe metabolic disorders associated with lysosomal storage of oligosaccharides and glycopeptides. Sialidase also plays an important role in cellular signaling and is specifically required for the production of cytokine interleukin-4 by activated T lymphocytes. In these cells, neu-1-encoded sialidase activity is increased on the cell surface, suggesting that a specific mechanism regulates sorting of this enzyme to the plasma membrane. We investigated that mechanism by first showing that sialidase contains the internalization signal found in lysosomal membrane proteins targeted to endosomes via clathrin-coated pits. The signal consists of a C-terminal tetrapeptide (412)YGTL(415), with Tyr(412) and Leu(415) essential for endocytosis of the enzyme. We further demonstrated that redistribution of sialidase from lysosomes to the cell surface of activated lymphocytes is accompanied by increased reactivity of the enzyme with anti-phosphotyrosine antibodies. We speculate that phosphorylation of Tyr(412) results in inhibition of sialidase internalization in activated lymphocytes.


Subject(s)
Cytoplasm/enzymology , Endocytosis , Immunoconjugates , Neuraminidase/metabolism , Abatacept , Animals , Antigens, CD , Antigens, Differentiation/metabolism , Base Sequence , COS Cells , CTLA-4 Antigen , DNA Primers , Membrane Proteins/metabolism , Microscopy, Confocal , Microscopy, Fluorescence , Mutagenesis, Site-Directed , Neuraminidase/chemistry , Neuraminidase/genetics , Phosphorylation , Tyrosine/metabolism
15.
Leukemia ; 15(5): 846-54, 2001 May.
Article in English | MEDLINE | ID: mdl-11368448

ABSTRACT

Eight multiple myeloma patients participated in a phase I trial evaluating the feasibility and safety of subcutaneous vaccination with adenovirus engineered, autologous plasma cells after high-dose therapy. Plasma cells were concentrated from bone marrow harvests by negative selection and high gradient magnetic separation. The mean plasma cell yield was 2.61 x 10(8). Transgene expression measured 48 h after plasma cell infection with an IL-2 expressing adenovirus averaged 2.95 ng/ml/10(6) cells. Vaccine production was successful for 88% of patients. Two months after high-dose therapy, six patients received from one to five injections of 3.5-9.0 x 10(7) cells/vaccine. Vaccines were well tolerated with only minor systemic symptoms reported. Injection with tumor cells induced a local inflammatory response consisting predominantly of CD8+ and/or TIA-1+ T-lymphocytes. Myeloma specific anti-tumor responses, assessed by interferon-gamma (IFN-gamma) release and cytotoxic T cell killing of autologous tumor cells, were not enhanced after vaccination in one evaluable patient. Clinical response, manifested as a decrease in serum paraprotein, was not observed in the one patient who had measurable disease at the time of vaccination. These results demonstrate that the generation of adenovector modified plasma cell vaccines is technically feasible and can be safely administered post-transplant. Further studies of immunlogic and clinical efficacy are required.


Subject(s)
Genetic Therapy , Interleukin-2/genetics , Multiple Myeloma/therapy , Plasma Cells/immunology , Vaccination , Adenoviridae/genetics , Adult , Female , Humans , Male , Middle Aged
17.
J Cardiovasc Surg (Torino) ; 40(6): 811-6, 1999 Dec.
Article in English | MEDLINE | ID: mdl-10776710

ABSTRACT

BACKGROUND: Optimal preservation of donor hearts remains a significant concern during transplantation. Organ shortage led to an increase in the use of damaged hearts. METHODS: To study the effect of preservation solutions on recovery of myocardial metabolism and function after warm ischemia, 10 dogs underwent 30 minutes of warm global ischemia under cardiopulmonary bypass. The animals were then administered 1 liter of Celsior (5 dogs), an extracellular crystalloid solution or 1 liter of University of Wisconsin solution (5 dogs), cooled at 4 degrees C, followed by 60 minutes of cold preservation and 30 minutes of warm blood reperfusion. Interstitial myocardial pH and pO2 changes were measured. The left ventricle dP/dt was measured before and after the ischemic episode, as where creatine kinase, troponine T and lactate serum levels. RESULTS: Tissue pH averaged 6.9+/-0.1, 6.2+/-0.1, 6.7+/-0.1 and 6.8+/-0.1 before and after warm ischemia, following the 60 minutes of cold preservation and the reperfusion period in animals treated with the Celsior solution, compared to 6.8+/-0.1, 6.4+/-0.1, 7+/-0.1 and 6.8+/-0.2 respectively in dogs treated with the University of Wisconsin solution (p<0.05). Oxygen tension in the myocardium averaged 36+/-8 mmHg before warm ischemia and 59+/-31 mmHg after in animals that received Celsior compared to 30+/-10 mmHg and 49+/-7 mmHg in dogs treated with University of Wisconsin (p>0.05). Global myocardial function decreased significantly following reperfusion compared to baseline in both groups of animals. The serum levels of creatine kinase, troponine T and lactate increased significantly during the experiment although there was no significant difference between the 2 groups. CONCLUSION: Both preservation solutions (Celsior and University of Wisconsin) resulted in suboptimal recovery of myocardial function and metabolism when administered after a period of warm ischemia. Strategies to improve recovery of damaged donor hearts remain to be appropriately defined.


Subject(s)
Energy Metabolism/drug effects , Myocardial Ischemia/physiopathology , Organ Preservation Solutions/pharmacology , Organ Preservation , Potassium Compounds/pharmacology , Acid-Base Equilibrium/drug effects , Adenosine/pharmacology , Allopurinol/pharmacology , Animals , Creatine Kinase/blood , Dogs , Energy Metabolism/physiology , Glutathione/pharmacology , Insulin/pharmacology , Lactic Acid/blood , Raffinose/pharmacology , Temperature , Troponin T/blood , Ventricular Function, Left/drug effects , Ventricular Function, Left/physiology
18.
Biochem J ; 276 ( Pt 3): 611-9, 1991 Jun 15.
Article in English | MEDLINE | ID: mdl-1712198

ABSTRACT

Vanadate induces phosphotyrosine accumulation and activates O2 consumption in permeabilized differentiated HL60 cells. NADPH, the substrate of the respiratory burst oxidase, was found to be necessary not only for the increased O2 consumption, but also for tyrosine phosphorylation. The effect of NADPH was not due to reduction of vanadate to vanadyl. Instead, NADPH was required for the synthesis of superoxide, which triggered the formation of peroxovanadyl [V(4+)-OO] and vanadyl hydroperoxide [V(4+)-OOH]. One or both of these species, rather than vanadate itself, appears to be responsible for phosphotyrosine accumulation and activation of the respiratory burst. Accordingly, the stimulatory effects of vanadate and NADPH were abrogated by superoxide dismutase. Moreover, phosphorylation was activated in the absence of NADPH by treatment with V(4+)-OO and/or V(4+)-OOH, generated by treatment of orthovanadate with KO2 or H2O2 respectively. The main source of the superoxide involved in the formation of V(4+)-OO and V(4+)-OOH is the NADPH oxidase. This was shown by the inhibitory effects of diphenylene iodonium and by the failure of undifferentiated cells, which lack oxidase activity, to undergo tyrosine phosphorylation when treated with vanadate and NADPH. By contrast, exogenously generated V(4+)-OO induced marked phosphorylation in the undifferentiated cells, demonstrating the presence of the appropriate tyrosine kinases and phosphatases. A good correlation was found to exist between induction of tyrosine phosphorylation and activation of the respiratory burst, suggesting a causal relationship. Therefore an amplification cycle appears to exist in cells treated with vanadate, whereby trace amounts of superoxide initiate the formation of V(4+)-OO and/or V(4+)-OOH. These peroxides promote phosphotyrosine formation, most likely by inhibition of tyrosine phosphatases. Accumulation of critical tyrosine-phosphorylated proteins then initiates a respiratory burst, with abundant production of superoxide. The newly formed superoxide catalyses the formation of additional V(4+)-OO and/or V(4+)-OOH, thereby magnifying the response. Since vanadium derivatives are ubiquitous in animal tissues, V(4+)-OO and/or V(4+)-OOH could be formed in vivo by reduced O2 metabolites, becoming potential endogenous tyrosine phosphatase inhibitors. Because of their potency, peroxides of vanadate may be useful as probes for the study of protein phosphotyrosine turnover.


Subject(s)
Leukemia, Prolymphocytic/metabolism , Oxygen Consumption , Tyrosine/analogs & derivatives , Vanadates/pharmacology , Granulocytes/metabolism , Humans , Leukemia, Prolymphocytic/drug therapy , NADP/pharmacology , Phosphorylation , Phosphotyrosine , Superoxides/metabolism , Tumor Cells, Cultured , Tyrosine/metabolism , Vanadium/pharmacology
19.
Biochem J ; 269(1): 127-31, 1990 Jul 01.
Article in English | MEDLINE | ID: mdl-1695841

ABSTRACT

The possible role of tyrosine phosphorylation in the activation of granulocytic HL60 cells was examined using vanadate, a phosphotyrosine phosphatase inhibitor. Treatment of permeabilized cells with micromolar concentrations of vanadate resulted in a substantial accumulation of tyrosine-phosphorylated proteins, detected by immunoblotting. At comparable concentrations, vanadate was also found to elicit an NADPH-dependent burst of oxygen utilization. Actin assembly, studied using 7-nitrobenz-2-oxa-1,3-diazole (NBD)-phallacidin, was similarly stimulated by vanadate, though considerably higher concentrations were required to observe this effect. In contrast with these responses, the secretion of lysozyme was not stimulated by vanadate, nor did vanadate affect calcium-induced secretion. Therefore, accumulation of tyrosine-phosphorylated proteins is associated with stimulation of some, but not all, of the responses characteristic of granulocytic cell activation. This indicates that the effects of vanadate are selective and suggests divergence of the signalling pathways leading to the individual effectors.


Subject(s)
Leukemia, Promyelocytic, Acute/metabolism , Neutrophils/metabolism , Signal Transduction , Tyrosine/analogs & derivatives , Vanadates/pharmacology , Actins/metabolism , Adenosine Triphosphate/pharmacology , Amanitins , Calcium/pharmacology , Cell Membrane Permeability , Exocytosis , Guanosine 5'-O-(3-Thiotriphosphate) , Guanosine Triphosphate/analogs & derivatives , Guanosine Triphosphate/pharmacology , Humans , Immunoblotting , Muramidase/metabolism , NADP/pharmacology , Neutrophils/drug effects , Oxygen Consumption/drug effects , Phosphotyrosine , Polymers , Thionucleotides/pharmacology , Tumor Cells, Cultured , Tyrosine/metabolism
20.
J Cell Biol ; 110(6): 1975-82, 1990 Jun.
Article in English | MEDLINE | ID: mdl-2112547

ABSTRACT

Assembly of microfilaments involves the conversion of actin from the monomeric (G) to the filamentous (F) form. The exact sequence of events responsible for this conversion is yet to be defined and, in particular, the role of calcium remains unclear. Intact and electropermeabilized human neutrophils were used to assess more directly the role of cytosolic calcium [( Ca2+]i) in actin assembly. Staining with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin and right angle light scattering were used to monitor the formation of F-actin. Though addition of Ca2+ ionophores can be known to induce actin assembly, the following observations suggest that an increased [Ca2+]i is not directly responsible for receptor-induced actin polymerization: (a) intact cells in Ca2(+)-free medium, depleted of internal Ca2+ by addition of ionophore, responded to the formyl peptide fMLP with actin assembly despite the absence of changes in [Ca2+]i, assessed with Indo-1; (b) fMLP induced a significant increase in F-actin content in permeabilized cells equilibrated with medium containing 0.1 microM free Ca2+, buffered with up to 10 mM EGTA; (c) increasing [Ca2+]i beyond the resting level by direct addition of CaCl2 to permeabilized cells resulted in actin disassembly. Conversely, lowering [Ca2+]i resulted in spontaneous actin assembly. To reconcile these findings with the actin-polymerizing effects of Ca2+ ionophores, we investigated whether A23187 and ionomycin induced actin assembly by a mechanism independent of, or secondary to the increase in [Ca2+]i. We found that the ionophore-induced actin assembly was completely inhibited by the leukotriene B4 (LTB4) antagonist LY-223982, implying that the ionophore effect was secondary to LTB4 formation, possibly by stimulation of phospholipase A2. We conclude that actin assembly is not mediated by an increase in [Ca2+]i, but rather that elevated [Ca2+]i facilitates actin disassembly, an effect possibly mediated by Ca2(+)-sensitive actin filament-severing proteins such as gelsolin. Sequential actin assembly and disassembly may be necessary for functions such as chemotaxis.


Subject(s)
Actins/metabolism , Neutrophils/metabolism , Calcimycin/pharmacology , Calcium/physiology , Cell Membrane Permeability/drug effects , Cell Membrane Permeability/physiology , Dose-Response Relationship, Drug , Electric Stimulation , Flow Cytometry , Humans , Ionomycin/pharmacology , Ionophores/pharmacology , Membrane Potentials/drug effects , Membrane Potentials/physiology , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophils/drug effects , Neutrophils/physiology
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