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Biomedical subjects

B M Mueller

Publications and source records attributed to B M Mueller.

At least 19 recordsLinked to original sources
PubMed2001

Integrin activation controls metastasis in human breast cancer.

Metastasis is the primary cause of death in human breast cancer. Metastasis to bone, lungs, liver, and brain involves dissemination of breast cancer cells via the bloodstream and requires adhesion within the vasculature. Blood cell adhesion within the vasculature depends on integrins, a family of transmembrane adhesion receptors, and is regulated by integrin activation. Here we show that integrin alpha v beta 3 supports breast cancer cell attachment under blood flow conditions in an activation-dependent manner. Integrin alpha v beta 3 was found in two distinct functional states in human breast cancer cells. The activated, but not the nonactivated, state supported tumor cell arrest during blood flow through interaction with platelets. Importantly, activated alpha v beta 3 was expressed by freshly isolated metastatic human breast cancer cells and variants of the MDA-MB 435 human breast cancer cell line, derived from mammary fat pad tumors or distant metastases in severe combined immunodeficient mice. Expression of constitutively activated mutant alpha v beta 3(D723R), but not alpha v beta 3(WT), in MDA-MB 435 cells strongly promoted metastasis in the mouse model. Thus breast cancer cells can exhibit a platelet-interactive and metastatic phenotype that is controlled by the activation of integrin alpha v beta 3. Consequently, alterations within tumors that lead to the aberrant control of integrin activation are expected to adversely affect the course of human breast cancer.

Animals↗
PubMed2000

Reversible regulation of tissue factor-induced coagulation by glycosyl phosphatidylinositol-anchored tissue factor pathway inhibitor.

Endothelial and tumor cells synthesize tissue factor pathway inhibitor (TFPI-1), which regulates tissue factor (TF) function by TF. VIIa. Xa. TFPI-1 quaternary complex formation (where VIIa and Xa are coagulation factors) and by translocation of these complexes into glycosphingolipid-rich microdomains of the cell membrane. Recombinant TFPI-1 added exogenously to cells is targeted to a degradation pathway. This study analyzes whether quaternary complex formation with endogenous TFPI-1 results also in internalization and degradation. We demonstrate that endogenous TFPI-1 and recombinant TFPI-1 differ in their distribution on the cell surface. Recombinant TFPI-1 is found in phospholipid- and glycosphingolipid-rich membrane domains, whereas endogenous TFPI-1 preferentially localizes to glycosphingolipid-rich microdomains. On quaternary complex formation, endogenous TFPI-1 remains protease sensitive and accessible for antibodies on intact cells, demonstrating that it is not appreciably internalized. Rather, regulation of TF by TFPI-1 is restored within 12 hours, consistent with dissociation of quaternary complexes on the cell surface. Endogenous TFPI-1 can be released from the cell surface by phospholipase treatment, indicating that TFPI-1 either is a glycosyl phosphatidylinositol (GPI)-anchored protein or binds to a GPI-linked receptor. We demonstrate that expression of a recombinant GPI-anchored form of TFPI-1 targets TF. VIIa complexes to glycosphingolipid-rich membrane fractions. Thus, GPI anchoring of TFPI-1 is sufficient for regulation of TF. VIIa complex function by a pathway of reversible inhibition rather than internalization and degradation.

Animals↗
PubMed2000

Diverse functions of protease receptor tissue factor in inflammation and metastasis.

Accumulating evidence suggests that protease receptors and their cognate protease ligands play important roles in cell-signaling events that regulate cell adhesion and migration in inflammation as well as tumor invasion and metastasis. Tissue factor (TF), the cell surface receptor for the serine protease VIIa and the initiator of the coagulation pathways, supports metastatic implantation by activating extracellular, protease-dependent signaling pathways and by intracellular links to the actin cytoskeleton. The adhesion of TF-expressing tumor cells can be mediated by interactions of the receptor-protease complex with specific matrix-associated inhibitors, suggesting a novel bridging mechanism by which proteases participate in migratory functions of cells.

Humans↗
PubMed1999

Urokinase-type plasminogen activator binding to its receptor stimulates tumor cell migration by enhancing integrin-mediated signal transduction.

Urokinase-type plasminogen activator (uPA) and its receptor (uPAR) participate in matrix degradation and cell migration by focusing proteolysis and functioning as a signaling ligand/receptor complex. uPAR, anchored by a lipid moiety in the membrane, is thought to require a transmembrane adapter to transduce signals into the cytoplasm. To study uPAR signaling, we transfected the prostate carcinoma cell line LNCaP, which does not express endogenous uPA or uPAR, with a uPAR encoding cDNA, resulting in high-level surface expression. We studied migration of these cells on fibronectin, which is mediated by the integrin alpha5beta1. Ligation of uPAR with uPA or its amino-terminal fragment enhanced haptotactic migration to fibronectin. In cells on fibronectin, but not on poly-l-lysine, ligation of uPAR also resulted in tyrosine phosphorylation of several proteins, including two proteins involved in integrin signaling, focal adhesion kinase and the crk-associated substrate p130(Cas). Furthermore, after uPAR ligation, uPAR was co-immunoprecipitated with beta1 integrins from the detergent-insoluble fraction of cell lysates. Thus, our data suggest that uPAR occupancy results in an interaction between uPAR and integrins and a potentiation of integrin-mediated signaling, which leads to enhanced cell migration.

Cell Adhesion↗
PubMed1999

Tumor cell adhesion and migration supported by interaction of a receptor-protease complex with its inhibitor.

Tissue factor (TF), the cell-surface receptor for coagulation factor VIIa, supports metastasis. Equally important for this process are (a) interactions of the TF cytoplasmic domain, which binds the mobility-enhancing actin-binding protein 280, and (b) the formation of a proteolytically active TF-VIIa complex on the tumor cell surface. In primary bladder carcinoma cells, we find that this complex localizes to the invasive edge, in proximity to tumor-infiltrating vessels that stain intensely for TF pathway inhibitor (TFPI-1), the major inhibitor of the protease activity of the complex. In culture, binding of VIIa to TF-expressing tumor cells is sufficient to allow cell adhesion, migration, and intracellular signaling on immobilized TFPI-1. Immobilized heparin, a mimic for extracellular matrix-associated proteoglycans, binds physiological concentrations of TFPI-1 in a conformation that supports TF-VIIa-dependent cell adhesion. Consistent with a functional role of TFPI-1 in complex extracellular matrices, we show that TF cooperates with integrin-mediated adhesion and migration on composite matrices that contain ligands for both integrins and the TF-VIIa complex. This study thus provides evidence for a novel mechanism of protease-supported migration that is independent of proteolytic matrix degradation but rather involves protease-dependent bridging of TF's extracellular domain to an ECM-associated inhibitor.

Carcinoma↗
PubMed1998

Low-density-lipoprotein-receptor-related protein (LRP) interacts with a GTP-binding protein.

The low-density-lipoprotein-receptor-related protein (LRP) binds and internalizes numerous ligands, including lipoproteins, proteinase-inhibitor complexes and others. We have shown previously that LRP-mediated ligand internalization is dependent on cAMP-dependent protein kinase (PKA) activity. Here, we investigated whether ligation of LRP increases the intracellular cAMP level and PKA activity via a stimulatory GTP-binding protein. Treatment of LRP-expressing cell lines with the LRP ligands lactoferrin or urokinase-type plasminogen activator caused a significant elevation in cAMP and stimulated PKA activity in a dose-dependent manner. Addition of the 39 kDa receptor-associated protein (RAP), an antagonist for ligand interactions with LRP, blocked the lactoferrin-induced increase in PKA activity, demonstrating a requirement for ligand binding to LRP. Incubation of cell membrane fractions with lactoferrin increased GTPase activity in a time- and dose-dependent manner, and treatment with LRP ligands suppressed cholera-toxin-mediated ADP-ribosylation of the Gsalpha subunit of a heterotrimeric G-protein. Affinity precipitation of LRP with RAP resulted in co-precipitation of two isoforms of Gsalpha from detergent extracts. We thus conclude that LRP is a signalling receptor that associates directly with a stimulatory heterotrimeric G-protein and activates a downstream PKA-dependent pathway.

Cholera Toxin↗
PubMed1998

Requirement for binding of catalytically active factor VIIa in tissue factor-dependent experimental metastasis.

Tissue factor (TF), the initiating cell surface receptor of the coagulation cascade, plays important roles in embryogenesis, angiogenesis, and tumor cell metastasis. It is controversial whether proteolytic function of TF complexed with its serine protease ligand VIIa is required for metastatic tumor dissemination. We show here in a model for TF-dependent experimental hematogenous metastasis, that TF supports metastasis by both proteolytic activity of the TF-VIIa complex and currently undefined functions of the cytoplasmic domain. We demonstrate that ligand binding of VIIa to TF is required for metastasis. Antimetastatic properties of covalently inactivated VIIa provide evidence that ligand binding is insufficient per se to support metastasis, emphasizing that proteolytic activity is necessary for the metastatic process. Ala or Asp mutations of cytoplasmic serine residues were introduced to preclude or mimic phosphorylation. In vivo analysis of these mutants suggests that local protease generation on the tumor cell surface does not serve simply to activate the cytoplasmic domain of TF by serine phosphorylation. Thus, extracellular functions of the catalytically active TF-VIIa complex cooperate with specific functions of the TF cytoplasmic domain to support the complex process of hematogenous tumor cell dissemination.

Animals↗
PubMed1998

A role for tissue factor in cell adhesion and migration mediated by interaction with actin-binding protein 280.

Tissue factor (TF), the protease receptor initiating the coagulation system, functions in vascular development, angiogenesis, and tumor cell metastasis by poorly defined molecular mechanisms. We demonstrate that immobilized ligands for TF specifically support cell adhesion, migration, spreading, and intracellular signaling, which are not inhibited by RGD peptides. Two-hybrid screening identified actin-binding protein 280 (ABP-280) as ligand for the TF cytoplasmic domain. Extracellular ligation of TF is necessary for ABP-280 binding. ABP-280 recruitment to TF adhesion contacts is associated with reorganization of actin filaments, but cytoskeletal adaptor molecules typically found in integrin-mediated focal contacts are not associated with TF. Chimeric molecules of the TF cytoplasmic domain and an unrelated extracellular domain support cell spreading and migration, demonstrating that the extracellular domain of TF is not involved in the recruitment of accessory molecules that influence adhesive functions. Replacement of TF's cytoplasmic Ser residues with Asp to mimic phosphorylation enhances the interaction with ABP-280, whereas Ala mutations abolish coprecipitation of ABP-280 with immobilized TF cytoplasmic domain, and severely reduce cell spreading. The specific interaction of the TF cytoplasmic domain with ABP-280 provides a molecular pathway by which TF supports tumor cell metastasis and vascular remodeling.

Binding Sites↗
PubMed1998

Alphav integrins mediate adhesion and migration of breast carcinoma cell lines.

Integrins with the alphav subunit are involved in cell adhesion and cellular signaling. Some alphav integrins have been associated with tumor progression and dissemination. The objective of this study was to assess the contribution of alphav integrins to the adhesive and migratory behavior of cells derived from breast carcinoma (BCA). The expression and function of alphav integrins was characterized in three BCA cell lines which exhibit different metastatic potentials. These include MCF-7 cells which metastasize inefficiently, MDA-MB-231 cells, which have a moderate metastatic potential, and MDA-MB-435 cells, which metastasize extensively. Each cell type displays a different repertoire of alphav integrins on the cell surface. The complement of alphav integrins on each cell type influences their ability to adhere and migrate. The most striking difference among these cell lines was the expression of the alphavbeta3 integrin. The highly metastatic MDA-MB-435 cells express substantial levels of this receptor, whereas MDA-MB-231 and MCF-7 cells do not. The MDA-MB-435 cells showed a greater ability to adhere and to migrate and this functional difference can largely be attributed to the expression of alphavbeta3 integrin. This characterization is a first step toward determining the role of alphav integrins in animal models of BCA metastasis, and lends support to the hypothesis that the alphavbeta3 integrin can be a contributing factor in metastatic disease.

Antigens, CD↗
PubMed1998

Suppression of human prostate carcinoma metastases in severe combined immunodeficient mice by interleukin 2 immunocytokine therapy.

Immunocytokines are antibody-cytokine fusion proteins that combine the unique targeting ability of antibodies with the multifunctional activities of cytokines to activate effector cells in the tumor microenvironment. Here, we demonstrate the therapeutic efficacy of a tumor-specific immunocytokine, huKS1/4-IL2, which effectively inhibited growth and dissemination of lung and bone marrow metastases of human prostate carcinoma in severe combined immunodeficient mice. This antitumor effect was specific and highly effective, irrespective of reconstitution of these mice with human lymphokine-activated killer cells. Survival times of mice treated with huKS1/4-IL2 were increased 4-fold as compared with animals treated with a mixture of the corresponding antibody and recombinant human interleukin-2 (rhIL2). A persistent antitumor response after treatment with the huKS1/4-IL2 immunocytokine in B, T, and natural killer cell-deficient severe combined immuodeficient-BEIGE mice, depleted of granulocytes, implies a major role for macrophages in this treatment effect. Our data demonstrate that immunocytokine-directed interleukin-2 therapy to tumor sites is an immunotherapeutic approach with potent effects against disseminated metastases of human prostate carcinoma and suggest that this treatment could be effective in an adjuvant setting for patients with minimal residual disease.

Animals↗
PubMed1997

Melanoma cell migration on vitronectin: regulation by components of the plasminogen activation system.

Tumor cell migration and invasion require complex interactions between tumor cells and the surrounding extracellular matrix. These interactions are modified by cell adhesion receptors, as well as by proteolytic enzymes and their receptors. Here, we study the influence of the protease urokinasetype plasminogen activator (uPA) and its receptor (uPAR) on melanoma cell adhesion to, and migration on, the extracellular matrix protein vitronectin (VN). Cell adhesion to VN, but not to type I collagen, is significantly enhanced in the presence of either uPA or its amino-terminal fragment (ATF). Soluble uPAR can inhibit this effect, indicating that uPA/uPAR on melanoma cells can function as a VN receptor. In the absence of bivalent cations, uPA/uPAR can promote cell attachment on VN, but not cell spreading, suggesting that the glycosylphosphatidylinositol (GPI)-anchored uPAR alone is unable to organize the cytoskeleton. Chemotactic melanoma cell migration on a uniform VN matrix is inhibited by uPA and ATF, implying that cell motility decreases when uPA/uPAR acts as a VN receptor. In contrast, plasminogen activator inhibitor I (PAI-I) can stimulate melanoma cell migration on VN, presumably by inhibiting uPA/uPAR-mediated cell adhesion to VN and thereby releasing the inhibition of cell migration induced by uPA. Together, our data implicate components of the plasminogen activation system in the direct regulation of cell adhesion and migration, thereby modulating the behavior of malignant tumor cells.

Cell Movement↗
PubMed1997

Proteases and protease inhibitors in tumor progression.

Our understanding of the role of matrix degrading proteases in cancer has dramatically expanded over the last two decades. From correlative observations linking proteases to cancer progression, we have accumulated evidence supporting a causal role for proteases in various steps of tumor progression and have become increasingly aware of the complex interactions that exist among proteases. Specific natural inhibitors of these proteases have also been identified and their role as potent cytostatic agents in cancer has been suggested. In this article some of the concepts on the role of proteases in cancer are discussed and examples of cooperation between matrix metalloproteinases and the plasmin/plasminogen activators system are presented. The role of protease inhibitors such as tissue inhibitor of metalloproteinases-2 (TIMP-2) and plasminogen activator inhibitor-2 (PAI-2) as inhibitors of tumor growth, invasion and metastasis is discussed.

Amino Acid Sequence↗
PubMed1997

Receptor-mediated endocytosis of urokinase-type plasminogen activator is regulated by cAMP-dependent protein kinase.

Internalization of the urokinase-type plasminogen activator (uPA) requires two receptors, the uPA receptor (uPAR) and the low density lipoprotein receptor-related protein (LRP)/alpha2-macroglobulin (alpha2M) receptor. Here, we address whether protein kinases are involved in the internalization of uPA by human melanoma cells. Initially, we found that the internalization of uPA was significantly inhibited by the serine/threonine protein kinase inhibitors staurosporine, K-252a and H-89, but not by the tyrosine kinase inhibitors, genistein and lavendustin A. Internalization of uPA was also inhibited by a pseudosubstrate peptide for cAMP-dependent protein kinase (PKA), but not by a pseudosubstrate peptide for protein kinase C. We confirmed a requirement for PKA-activity and implicated a specific isoform by using an antisense oligonucleotide against the regulatory subunit RI alpha of PKA which suppresses PKA-I activity. Exposure of cells to this oligonucleotide led to a specific, dose-dependent decrease in RI alpha protein and to a significant inhibition in the rate of uPA internalization. We further demonstrate that treatment of melanoma cells with either H-89 or PKA RI alpha antisense oligonucleotides also resulted in a decreased internalization of two other ligands of LRP, activated alpha2M and lactoferrin, indicating that PKA activity is associated with LRP. Finally, we demonstrate that PKA activity is also required for the internalization of transferrin, but not for the internalization of the epidermal growth factor or adenovirus 2, suggesting that in melanoma cells, PKA activity is not generally required for clathrin-mediated endocytosis, but is rather associated with specific internalization receptors.

Adenoviruses, Human↗
PubMed1996

Requirement of receptor-bound urokinase-type plasminogen activator for integrin alphavbeta5-directed cell migration.

The urokinase plasminogen activator (uPA) interacts with its cell surface receptor (uPAR), providing an inducible, localized cell surface proteolytic activity, thereby promoting cellular invasion. Evidence is provided for a novel function of cell surface-associated uPA.uPAR. Specifically, induction of cell surface expression of uPA. uPAR by growth factors or phorbol ester was necessary for vitronectin-dependent carcinoma cell migration, an event mediated by integrin alphavbeta5. Cell migration on vitronectin was blocked with either a soluble form of uPAR, an antibody that disrupts uPA binding to uPAR, or a monoclonal antibody to alphavbeta5. Moreover, plasminogen activator inhibitor type 2 blocked this migration event but did not affect adhesion, suggesting a direct role for uPA enzyme activity in this process and that migration but not adhesion of these cells is regulated by uPA.uPAR. Growth factor-mediated induction of uPA.uPAR on the carcinoma cell surface promotes a specific motility event mediated by integrin alphavbeta5, since cells transfected with the beta3 integrin subunit expressed alphavbeta3 and migrated on vitronectin independently of growth factors or uPA.uPAR expression. This relationship between alphavbeta5 and the uPA.uPAR system has significant implications for regulation of motility events associated with development, angiogenesis, and tumor metastasis.

Cell Movement↗
PubMed1996

Different roles for plasminogen activators and metalloproteinases in melanoma metastasis.

Malignant human melanoma cells produce many matrix-degrading enzymes, including plasminogen activators and matrix metalloproteinases. These enzymes have substrate specificity for different components of ECM and most of them have been demonstrated to contribute to melanoma cell-mediated dissolution of matrices and to melanoma cell invasion. The degradation of complex matrices in vitro requires the cooperation of proteases with specificity for glycoproteins and collagens. The contribution of proteases to spontaneous melanoma metastasis was studied by overexpressing specific protease inhibitors in human melanoma cells. Overexpression of PAI-2 inhibited the spread of distant metastasis indicating a role for uPA/plasmin in melanoma invasion. Overexpression of TIMP-2, in contrast, reduced the growth rate of subcutaneous tumors, but did not inhibit metastasis, indicating that MMP activities promote melanoma growth in the skin and may not be required for metastatic dissemination. Thus, uPA and MMP activities are involved in different processes, but they both contribute to melanoma malignancy.

Animals↗
PubMed1996

Tissue factor in cancer angiogenesis and metastasis.

Tumor cells frequently express tissue factor, a transmembrane glycoprotein that functions as the cellular receptor and catalytic cofactor for the serine protease factor VIIa. In human tumors, tissue factor expression correlates spatially with neovascularization, indicating that tissue factor function may be linked to angiogenic properties of malignant tumors. Tissue factor also supports hematogenous tumor dissemination. The role of tissue factor in metastasis appears to involve both the coagulation pathway triggered by interactions of the tissue factor extracellular domain as well as cellular events dependent on the short cytoplasmic domain that may participate in tissue factor-mediated outside-in signaling.

Humans↗
PubMed1995

Therapeutic efficacy of a doxorubicin immunoconjugate in a preclinical model of spontaneous metastatic human melanoma.

Doxorubicin (DOX) was conjugated to a monoclonal antibody (mAb 425) directed against the human epidermal growth factor receptor. The immunoreactivity of these conjugates, with an average of six to eight molecules of DOX per antibody, was largely conserved, and their in vitro cytotoxicity against metastatic human melanoma cells (M24 met) was improved over that of free DOX. An evaluation of the therapeutic efficacy of mAb 425-DOX indicated that this immunoconjugate suppressed the growth of primary and secondary M24 met tumors in mice with severe combined immunodeficiency and prolonged the life span of these animals, whereas an equivalent dose of free DOX was ineffective. Conjugation of DOX to an irrelevant mAb also increased its antitumor effect over that of equivalent amounts of free drug but to a lesser extent than that achieved by the mAb 425-DOX conjugate. These results demonstrate targeted delivery and striking antitumor activity of DOX immunoconjugates in a preclinical model of spontaneous, metastatic human melanoma that was insensitive to free DOX.

Animals↗