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Biomedical subjects

M A Schwartz

Publications and source records attributed to M A Schwartz.

At least 19 recordsLinked to original sources
PubMed1996

Integrin regulation of c-Abl tyrosine kinase activity and cytoplasmic-nuclear transport.

The product of the c-abl protooncogene is a nonreceptor tyrosine kinase found in both the cytoplasm and the nucleus. We report herein that cell adhesion regulates the kinase activity and subcellular localization of c-Abl. When fibroblastic cells are detached from the extracellular matrix, kinase activity of both cytoplasmic and nuclear c-Abl decreases, but there is no detectable alteration in the subcellular distribution. Upon adhesion to the extracellular matrix protein fibronectin, a transient recruitment of a subset of c-Abl to early focal contacts is observed coincident with the export of c-Abl from the nucleus to the cytoplasm. The cytoplasmic pool of c-Abl is reactivated within 5 min of adhesion, but the nuclear c-Abl is reactivated after 30 min, correlating closely with its return to the nucleus and suggesting that the active nuclear c-Abl originates in the cytoplasm. In quiescent cells where nuclear c-Abl activity is low, the cytoplasmic c-Abl is similarly regulated by adhesion but the nuclear c-Abl is not activated upon cell attachment. These results show that c-Abl activation requires cell adhesion and that this tyrosine kinase can transmit integrin signals to the nucleus where it may function to integrate adhesion and cell cycle signals.

3T3 Cells↗
PubMed1996

Transformation by Rho exchange factor oncogenes is mediated by activation of an integrin-dependent pathway.

Constitutive activation of growth factor receptor signaling pathways leads to uncontrolled growth, but why tumor cells become anchorage independent is less clear. The fact that integrins transmit signals required for cell growth suggests that constitutive activation of steps downstream from integrins mediates anchorage independence. Since the small GTPase Rho may mediate integrin signal transduction, the effects of serum and the Rho nucleotide exchange factor oncogenes dbl and lbc on cell growth and signaling pathways were examined. Our data show that these oncogenes induce anchorage-independent but serum-dependent growth and stimulation of signaling pathways. These results show, therefore, that anchorage-independent growth results from constitutive activation of integrin-dependent signaling events. They also support the view that Rho is a functionally important mediator of integrin signaling.

3T3 Cells↗
PubMed1996

Involvement of the small GTPase rho in integrin-mediated activation of mitogen-activated protein kinase.

Engagement and clustering of integrins triggers a number of intracellular signaling events, including activation of the mitogen-activated protein (MAP) kinases Erk1 and Erk2. To investigate the mechanism by which integrins mediate the activation of MAP kinases upon binding of NIH 3T3 cells to fibronectin, we assessed the effects of both inhibiting and activating the small GTPase Rho. We observed that inhibition of Rho by the Rho-specific inhibitor C3 exoenzyme or by a dominant negative Rho A (RhoN19) inhibited MAP kinase activation. Conversely, activation of Rho by expression of an activated Rho A mutant (RhoQ63L), or the Rho-specific guanine nucleotide exchange factor lbc, enhanced and partially mimicked activation of Erk2 by plating on fibronectin. These results therefore show that Rho is involved in the integrin-dependent activation of MAP kinase.

A Kinase Anchor Proteins↗
PubMed1996

Protein kinase C regulates alpha v beta 5-dependent cytoskeletal associations and focal adhesion kinase phosphorylation.

Integrins alpha v beta 3 and alpha v beta 5 both mediate cell adhesion to vitronectin yet trigger different postligand binding events. Integrin alpha v beta 3 is able to induce cell spreading, migration, angiogenesis, and tumor metastasis without additional stimulators, whereas alpha v beta 5 requires exogenous activation of protein kinase C (PKC) to mediate these processes. To investigate this difference, the ability of beta 3 or beta 5 to induce colocalization of intracellular proteins was assessed by immunofluorescence in hamster CS-1 melanoma cells. We found that alpha v beta 5 induced colocalization of talin, alpha-actinin, tensin, and actin very weakly relative to alpha v beta 3. alpha v beta 5 was able to efficiently induce colocalization of focal adhesion kinase (FAK); however, it was unable to increase phosphorylation of FAK on tyrosine. Activation of PKC by adding phorbol ester to alpha v beta 5-expressing cells induced spreading, increased colocalization of alpha-actinin, tensin, vinculin, p130cas and actin, and triggered tyrosine phosphorylation of FAK. Unexpectedly, talin colocalization remained low even after activation of PKC. Treatment of cells with the PKC inhibitor calphostin C inhibited spreading and the colocalization of talin, alpha-actinin, tensin, and actin for both alpha v beta 3 and alpha v beta 5. We conclude that PKC regulates localization of cytoskeletal proteins and phosphorylation of FAK induced by alpha v beta 5. Our results also show that FAK can be localized independent of its phosphorylation and that cells can spread and induce localization of other focal adhesion proteins in the absence of detectable talin.

Animals↗
PubMed1996

Physical association of the small GTPase Rho with a 68-kDa phosphatidylinositol 4-phosphate 5-kinase in Swiss 3T3 cells.

Our previous work showed that post-translationally modified Rho in its GTP-bound state stimulated phosphatidylinositol 4-phosphate 5-kinase (PIP5K) activity in mouse fibroblast lysates. To investigate whether Rho physically interacts with PIP5K, we incubated immobilized Rho-GST with Swiss 3T3 cell lysates and tested for retained PIP5K activity. Rho-GST, but not Ras-GST or GST alone, bound significant PIP5K activity. The binding of PIP5K was independent of whether Rho was in a GTP- or GDP-bound state. An antibody against a 68-kDa human erythrocyte type I PIP5K recognized a single 68-kDa protein eluted from Rho-GST column. The Rho-associated PIP5K responded to phosphatidic acid differentially from the erythrocyte type I PIP5K, suggesting that it could be a distinct isoform not reported previously. Rho co-immunoprecipitated with the 68-kDa PIP5K from Swiss 3T3 lysates, demonstrating that endogenous Rho also interacts with PIP5K. ADP-ribosylation of Rho with C3 exoenzyme enhanced PIP5K binding by approximately eightfold, consistent with the ADP-ribosylated Rho functioning as a dominant negative inhibitor. These results demonstrate that Rho physically interacts with a 68-kDa PIP5K, although whether the association is direct or indirect is unknown.

3T3 Cells↗
PubMed1996

Venlafaxine in adults with attention-deficit/hyperactivity disorder: an open clinical trial.

BACKGROUND: It is becoming commonly recognized that adults suffer from attention-deficit/hyperactivity disorder (ADHD). Since medications used in the past of treat adults with ADHD may be ineffective or poorly tolerated by some patients, it is important to determine if newly available medications can safely ameliorate symptoms of ADHD in adults. METHOD: An open clinical trial was undertaken to examine whether venlafaxine was safe and effective in the treatment of adults with ADHD. Ten subjects who met DSM-IV criteria for ADHD were enrolled in this 8-week trial. Individuals were started on 37.5 mg of venlafaxine b.i.d. If moderate ADHD symptoms persisted at the end of Week 4, the dose of venlafaxine was increased to 75 mg b.i.d. Assessments of ADHD symptomatology included the ADHD Rating Scale, Self-Report Version (ARS) and the Clinical Global Impressions (CGI) scale. RESULTS: Nine patients completed the study. At the end of the study, 7 patients were receiving 37.5 mg b.i.d. of venlafaxine. Repeated measures ANOVA demonstrated that treatment with venlafaxine was associated with significant reductions in ADHD symptomatology (p < .02 for the ARS; p < .005 for the CGI). Of the 9 subjects who completed the trial, 7 were considered responders. Venlafaxine was well tolerated, and most patients experienced only mild side effects. CONCLUSION: Venlafaxine may be a promising agent for the treatment of ADHD in adults. Controlled clinical trials are needed to further examine this issue.

Adolescent↗
PubMed1995

Inhibition of cell cycle progression by the alternatively spliced integrin beta 1C.

Integrins regulate cell growth, differentiation, and behavior in many systems. Integrin beta 1C (beta 1S) is an alternatively spliced variant of integrin beta 1 with a specific cytoplasmic domain and is expressed in several human tissues. Human beta 1c transiently expressed in mouse 10T1/2 fibroblasts showed a diffuse pattern of cell surface staining, whereas beta1 localized to focal adhesions. Moderate concentrations of beta 1C had no effect on actin stress fibers or focal adhesions, but markedly inhibited DNA synthesis. Inhibition by beta 1C mapped to the late G1 phase of the cell cycle, near the G1-S boundary. Thus, alternative splicing of beta1 results in transmission of distinct signals that may regulate growth in vivo.

Alternative Splicing↗
PubMed1995

A comparison of conservative therapy and early selective ligation in the treatment of lymphatic complications following vascular procedures.

BACKGROUND: Lymphatic leakage is a relatively uncommon but serious complication following vascular procedures. A conservative approach consisting of bed rest, leg elevation, prophylactic antibiotics, compressive dressings, and intermittent aspiration is the most commonly used treatment. Because of the long time it takes to cure this condition and the potential for infections, a more aggressive approach consisting of wound exploration and ligation of the leaking lymphatic has been proposed. We review our experience of the past 3 years treating 17 of these complications by using 1 of these 2 approaches. PATIENTS AND METHODS: Seventeen groin complications were seen over a period of 3 years. Ten (59%) patients were treated by selective ligation assisted with isosulfan blue dye injection, and the remaining 7 (41%) were treated conservatively. RESULTS: Mean hospital stay was 2.4 days (range 1 to 4) for the operative group versus 19 days (range 14 to 42) for the conservative group. One complication was seen in the operative group, whereas 4 (57%) patients developed groin infections following conservative therapy. One (10%) patient developed a recurrence following ligation that was treated successfully by reoperation. CONCLUSION: Our experience with the use of surgical ligation of leaking lymphatic assisted by isosulfan blue, when compared with conservative treatment, has led to a decrease in hospital stay, lower complication rates, and fewer recurrences. In our view, this approach represents the best form of treatment for postoperative groin lymphatic complications.

Adult↗
PubMed1995

Mapping in vivo associations of cytoplasmic proteins with integrin beta 1 cytoplasmic domain mutants.

Integrins promote formation of focal adhesions and trigger intracellular signaling pathways through cytoplasmic proteins such as talin, alpha-actinin, and focal adhesion kinase (FAK). The beta 1 integrin subunit has been shown to bind talin and alpha-actinin in in vitro assays, and these proteins may link integrin to the actin cytoskeleton either directly or through linkages to other proteins such as vinculin. However, it is unknown which of these associations are necessary in vivo for formation of focal contacts, or which regions of beta 1 integrin bind to specific cytoskeletal proteins in vivo. We have developed an in vivo assay to address these questions. Microbeads were coated with anti-chicken beta 1 antibodies to selectively cluster chicken beta 1 integrins expressed in cultured mouse fibroblasts. The ability of cytoplasmic domain mutant beta 1 integrins to induce co-localization of proteins was assessed by immunofluorescence and compared with that of wild-type integrin. As expected, mutant beta 1 lacking the entire cytoplasmic domain had a reduced ability to induce co-localization of talin, alpha-actinin, F-actin, vinculin, and FAK. The ability of beta 1 integrin to co-localize talin and FAK was found to require a sequence near the C-terminus of beta 1. The region of beta 1 required to co-localize alpha-actinin was found to reside in a different sequence, several amino acids further from the C-terminus of beta 1. Deletion of 13 residues from the C-terminus blocked co-localization of talin, FAK, and actin, but not alpha-actinin. Association of alpha-actinin with clustered integrin is therefore not sufficient to induce the co-localization of F-actin.

3T3 Cells↗
PubMed1995

Integrins: emerging paradigms of signal transduction.

Integrins receive signals from other receptors that lead to activation of ligand binding (inside-out signaling) and matrix assembly. Upon binding ligands, they also activate intracellular signaling pathways. These signals converse with pathways that are initiated by soluble ligands to regulate cell functions. In this way, cell adhesion is coordinated with other events to orchestrate complex cellular behavior.

Animals↗
PubMed1995

Integrin signaling: roles for the cytoplasmic tails of alpha IIb beta 3 in the tyrosine phosphorylation of pp125FAK.

pp125FAK (focal adhesion kinase) a protein tyrosine kinase that may mediate cellular responses to adhesion, is activated and tyrosine-phosphorylated when platelets adhere to fibrinogen via the integrin, alpha IIb beta 3. To determine whether either of the cytoplasmic tails of alpha IIb beta 3 regulates FAK phosphorylation, CHO cells were stably transfected with alpha IIb beta 3 or various cytoplasmic tail truncation mutants. Cells expressing wild-type alpha IIb beta 3 or alpha IIb beta 3 that lacked the COOH-terminal 13 or 18 residues of the 20 residue alpha IIb tail adhered to and spread on fibrinogen or on an anti-alpha IIb antibody, and FAK became tyrosine-phosphorylated. FAK also became phosphorylated in adherent cells lacking the COOH-terminal 35 or 39 residues of the 47 residue beta 3 tail, although the extent of phosphorylation was reduced by about 50% in the latter mutant. Little or no FAK phosphorylation was observed if 46 residues were deleted from the beta 3 tail. None of these beta 3 truncation mutants spread on the anti-alpha IIb antibody. When cells with wild-type alpha IIb beta 3 or truncated beta 3 were detached from a surface, FAK became rapidly dephosphorylated. In contrast, FAK remained phosphorylated in the two alpha IIb truncation mutants for up to 90 minutes in suspension. This persistent phosphorylation was not due to occupancy of alpha IIb beta 3 by adhesive ligands because it was also observed with an alpha IIb tail truncation mutant that contained an additional mutation in the extracellular portion of the receptor that prevents ligand binding. These studies demonstrate that: (1) the beta 3 cytoplasmic tail, including the membrane-proximal portion, is involved in initiation of FAK phosphorylation; (2) FAK phosphorylation can be initiated by cell adhesion in the absence of cell spreading; and (3) the membrane-distal portion of the alpha IIb cytoplasmic tail may normally function to dampen FAK phosphorylation in non-anchored cells.

Amino Acid Sequence↗
PubMed1994

The small GTP-binding protein Rho regulates a phosphatidylinositol 4-phosphate 5-kinase in mammalian cells.

Integrin-mediated adhesion is known to stimulate production of phosphatidylinositol 4,5-bisphosphate (4,5-PIP2) and increase 4,5-PIP2 hydrolysis in response to platelet-derived growth factor (PDGF). We now show that treatment of cells with lovastatin, which inhibits modification of small GTP-binding proteins, reduced PIP2 levels and decreased calcium mobilization in response to PDGF and thrombin. In cell lysates, GTP gamma S stimulated PIP 5-kinase activity, and this effect was blocked by botulinum C3 exoenzyme, suggesting that Rho was responsible. GTP-bound recombinant Rho stimulated PIP 5-kinase activity, whereas GDP-Rho was much less potent and GTP-bound Rac was ineffective. Microinjected botulinum C3 exoenzyme caused diminished calcium mobilization in response to PDGF or thrombin. Conversely, microinjection of activated Rho reversed the decrease in calcium mobilization normally seen in nonadherent cells. These data demonstrate that Rho regulates 4,5-PIP2 synthesis and, indirectly, 4,5-PIP2 hydrolysis. They also raise the possibility that PIP2 synthesis could mediate the effects of Rho on the actin cytoskeleton.

ADP Ribose Transferases↗
PubMed1994

Differing structural requirements for GTPase-activating protein responsiveness and NADPH oxidase activation by Rac.

The Rac GTP-binding proteins regulate the actin cytoskeleton and the superoxide-forming NADPH oxidase of phagocytic leukocytes. These functions of Rac are determined by the GTP/GDP state of the protein, which can be modulated by GTPase-activating proteins (GAPs). The interaction of Ras with both downstream signaling targets and GAPs is mediated via an "effector" domain (amino acids 30-40). We demonstrate that the effector domain of Rac2 is required for both NADPH oxidase activation and actin assembly, but that mutations in this region do not decrease the responsiveness of Rac to GAPs. In contrast, mutations of residues 12 (Gly-->Val) or 61 (Gln-->Leu) inhibit both intrinsic- and GAP-stimulated GTP hydrolysis by Rac2. A double mutation in which both the effector domain and Q61L were modified restored NADPH oxidase activation and membrane ruffling, while the equivalent effector domain and G12V double mutation did not. The Rac2 Q61L mutant had an increased "affinity" for NADPH oxidase activation and for GAP binding as compared to the wild type or G12V proteins. These experiments suggest that Rac contains at least two "effector" interaction sites, and that changes in binding interactions at one of these sites may influence the function of the other.

Base Sequence↗
PubMed1994

Alpha v integrins mediate the rise in intracellular calcium in endothelial cells on fibronectin even though they play a minor role in adhesion.

We have investigated which integrins mediate the elevation of intracellular calcium ([Ca2+]i) triggered by spreading of endothelial cells on fibronectin (FN). Specific anti-integrin monoclonal antibodies immobilized on glass surfaces were used as agonists to trigger cell spreading. These experiments demonstrated that an antibody to alpha v could induce the rise in [Ca2+]i, whereas two antibodies to alpha 5 beta 1 were inactive, despite their ability to induce cell spreading and elevation of intracellular pH. Antibodies in solution were then used as agonists to block association of specific integrins with FN. These experiments also showed that alpha v integrin(s) but not alpha 5 beta 1 mediated the rise in [Ca2+]i in cells spreading on FN. Adhesion assays in the presence of function-blocking anti-integrin antibodies and affinity chromatography on FN columns of surface-labeled cell extracts were carried out to characterize the integrins that bind to FN. Both methods showed that alpha v integrin(s) and alpha 5 beta 1 participate in FN binding; however, the contribution from alpha v integrin(s) was much less than that of alpha 5 beta 1. These results demonstrate that two receptors for FN on the same cells can trigger distinct intracellular signaling pathways and, furthermore, that an integrin whose contribution to adhesion is barely detectable can have a major effect on cellular responses. The results also suggest that the specificity for activation of the calcium signaling pathway resides primarily in the integrin alpha subunit.

Antibodies↗
PubMed1994

Murine and humanized constructs of monoclonal antibody M195 (anti-CD33) for the therapy of acute myelogenous leukemia.

Long-term survival rates of patients with acute myelogenous leukemia treated with intensive chemotherapy are 15-20%, despite efforts to develop new treatment strategies. Murine M195 (131I-M195), an anti-CD33, immunoglobulin (Ig) G2a monoclonal antibody has reactivity restricted to early myeloid cells and myeloid leukemic blasts but not hematopoietic progenitors. Previous trials in patients with relapsed or refractory myeloid leukemia showed that 131I-M195 rapidly targeted to the bone marrow and internalized into target cells. This article describes a therapeutic dose escalation study in which 24 patients received from 50 mCi/m2 to 210 mCi/m2 of 131I-M195 in divided doses. Cytoreduction of peripheral cell counts and bone marrow blasts occurred without nonhematopoietic toxicity. Doses of 131I-M195 greater than 135 mCi/m2 were associated with marrow cytoreduction sufficient to necessitate bone marrow transplant. However, 37% of the patients developed human anti-mouse antibody, preventing retreatment. To decrease immunogenicity and improve effector function, chimeric IgG1 and IgG3, and complementarity-determining region-grafted, humanized IgG1 and IgG3 versions of mouse M195 were developed by genetic engineering techniques. The new versions maintained specificity and biologic function, and they were superior to the mouse M195 in their ability to perform antibody-dependent cellular cytotoxicity against leukemia cells. Humanized M195, but not chimeric M195, showed a 4-8.6 times higher avidity than its mouse counterpart. Because effector function of IgG depends to a large extent on Fc clustering, a homodimeric HuG1 also was developed. Homodimeric HuG1 showed an ability to cause additional dramatic improvements in effector functions, as well as an ability to internalize and retain radioisotope in target leukemia cells. Monomeric and dimeric forms of humanized M195 may be advantageous in the therapy of acute myelogenous leukemia.

Adult↗