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Biomedical subjects

B M Mueller

Publications and source records attributed to B M Mueller.

At least 37 records · Page 2Linked to original sources
PubMed1995

Tissue factor-initiated thrombin generation activates the signaling thrombin receptor on malignant melanoma cells.

The human melanoma cell line M24met expresses tissue factor, the cellular initiator of the blood coagulation cascade. Blocking of the coagulation pathways at the level of tissue factor, factor Xa, or thrombin inhibits hematogenous M24met metastasis in SCID mice, implicating a role for thrombin generation in this process. Dependent on cell surface tissue factor activity, M24met cells generate thrombin in vitro. Thrombin and the thrombin receptor agonist peptide TRP-14 activate a signaling pathway in M24met cells that involves an increase in intracellular calcium and induces cell proliferation. Immunofluorescence evidences expression of the signaling thrombin receptor on these cells. Thus, M24met melanoma cells express both the initiating cell surface receptor for the coagulation pathways and the central signaling receptor of the coagulation system, suggesting the in situ generation of proliferative signals which can contribute to the malignant phenotype.

Animals↗
PubMed1995

Overexpression of plasminogen activator inhibitor 2 in human melanoma cells inhibits spontaneous metastasis in scid/scid mice.

A metastatic human melanoma cell line that produces urokinase-type plasminogen activator was stably transfected with cDNA encoding human plasminogen activator inhibitor 2 (PAI-2). Transfected clones expressed PAI-2 at levels two to nine times higher than both the parental cell line and mock transfectants, as detected by ELISA of cell lysates and conditioned medium. The clone with the highest PAI-2 expression exhibited complete inhibition of soluble and cell-surface-bound plasminogen activator activity. The level of PAI-2 overexpression in these clonal cell lines correlated positively with the inhibition of their ability to degrade extracellular matrix in vitro. Parental, mock-transfected, and PAI-2-transfected cell lines produced rapidly growing tumors when injected s.c. into the skin of mice with severe combined immunodeficiency. The tumors producing the highest levels of PAI-2 were surrounded by a dense tumor capsule. Both parental cells and mock-transfected cells invariably metastasized from s.c. tumors to lymph nodes and lungs of mice. PAI-2-transfected cell lines produced significantly less or no metastases. Taken together, these data indicate a critical role for plasminogen activator activity in melanoma invasion and metastasis.

Animals↗
PubMed1995

The urokinase-type plasminogen activator receptor, a GPI-linked protein, is localized in caveolae.

The urokinase plasminogen activator receptor (uPAR), a glycosylphosphatidylinositol-linked glycoprotein, plays a central role in the regulation of pericellular proteolysis and participates in events leading to cell activation. Here, we demonstrate that uPAR, on a human melanoma cell line, is localized in caveolae, flask-shaped microinvaginations of the plasma membrane found in a variety of cell types. Indirect immunofluorescence with anti-uPAR antibodies on the melanoma cells showed a punctated staining pattern that accumulated to stretches along sides of cell-cell contact and membrane ruffles. uPAR colocalized with caveolin, a characteristic protein in the coat of caveolae, as demonstrated by double staining with specific antibodies. Further, uPAR could be directly localized in caveolae by in vivo immunoelectron microscopy. Both uPAR and its ligand, uPA, were present in caveolae enriched low density Triton X-100 insoluble complexes, as shown by immunoblotting. From such complexes, caveolin could be coprecipitated with uPAR-specific antibodies suggesting a close spatial association between uPAR and caveolin that might have implications for the signal transduction mediated by uPAR. Further, functional studies indicated that the localization of uPAR and its ligand in caveolae enhances pericellular plasminogen activation, since treatment of the cells with drugs that interfere with the structural integrity of caveolae, such as nystatin, markedly reduced cell surface plasmin generation. Thus, caveolae promote efficient cell surface plasminogen activation by clustering uPAR, uPA, and possibly other protease receptors in one membrane compartment.

Caveolin 1↗
PubMed1994

Effect of tissue inhibitor of the matrix metalloproteinases-2 expression on the growth and spontaneous metastasis of a human melanoma cell line.

In this study, we examined the effect of expression of tissue inhibitor of metalloproteinases-2 (TIMP-2) on the growth and dissemination of a highly metastatic human melanoma cell line (M24net). M24net melanoma cells express a number of matrix metalloproteinases (MMPs), including gelatinase A and B (MMP-2 and MMP-9) and interstitial collagenase (MMP-1) (A. M. P. Montgomery et al., Cancer Res., 53: 693-700, 1993). The activity of these proteases was effectively down-regulated by transfecting M24net cells with complementary DNA-encoding human TIMP-2. Overexpression of TIMP-2 markedly reduced melanoma growth in the skin of immunodeficient mice but did not prevent these highly malignant cells from spontaneously metastasizing to the lungs and lymph nodes of inoculated mice. We provide a mechanism to account for the growth inhibitory property of TIMP-2 based on its ability to regulate M24net cell growth in three-dimensional interstitial collagen. In the presence of this matrix, M24net cells assume a differentiated morphology and have a reduced growth rate. We present evidence that overexpression of TIMP-2 increases the susceptibility of M24net cells to growth inhibition and morphological differentiation by occluding interstitial collagen.

Animals↗
PubMed1994

A recombinant antibody-interleukin 2 fusion protein suppresses growth of hepatic human neuroblastoma metastases in severe combined immunodeficiency mice.

A genetically engineered fusion protein consisting of a human/mouse chimeric anti-ganglioside GD2 antibody (ch14.18) and recombinant human interleukin 2 (rhIL-2) was tested for its ability to target rhIL-2 to tumor sites and stimulate immune effector cells sufficiently to achieve effective tumor cell lysis in vivo. The ch14.18-IL-2 fusion protein proved more effective than equivalent doses of rhIL-2 in suppressing dissemination and growth of human neuroblastoma in an experimental hepatic metastases model of scid (severe combined immunodeficiency) mice reconstituted with human lymphokine-activated killer cells. The ch14.18-IL-2 fusion protein was also more proficient than equivalent doses of rhIL-2 in prolonging the life-span of these animals. This recombinant antibody-cytokine fusion protein may prove useful for future treatment of GD2-expressing human tumors in an adjuvant setting.

Animals↗
PubMed1994

Binding of urokinase to its receptor promotes migration and invasion of human melanoma cells in vitro.

Previously, we reported that urokinase-type plasminogen activator (uPA) plays a pivotal role in extracellular matrix dissolution by malignant melanoma cells. Here, we demonstrate that a highly metastatic melanoma cell line (M24met) that secretes uPA expresses high levels of the uPA receptor (uPAR), 2.4 x 10(6) binding sites/cell with a KD of 5.67 x 10(-10) M. The receptor was identified as a 55,000-60,000 kDa cell surface protein. Although M24met cells secrete uPA, they are unable to efficiently utilize this enzyme for invasion, unless it is bound to its receptor. This contention is based on the finding that an antibody against uPAR (monoclonal antibody 3936) inhibited invasion of M24met cells through a reconstituted basement membrane (Matrigel) up to 33%, while a reduction of uPA catalytic activity by its plasminogen activator inhibitor-2 resulted in 46% inhibition of invasion. Furthermore, uPAR is involved in signal transduction events in M24met cells, since both uPA and its amino-terminal fragment stimulated the migration of melanoma cells toward Matrigel, resulting in maximal increases of 32 and 72%, respectively. Our results indicate that both uPA and uPAR are involved in melanoma metastasis and that uPAR contributes to at least two important steps in this process, matrix dissolution and migration.

Cell Movement↗
PubMed1993

Melanoma-mediated dissolution of extracellular matrix: contribution of urokinase-dependent and metalloproteinase-dependent proteolytic pathways.

Constitutive overexpression of both urokinase and matrix metalloproteinase (MMP) activity is frequently observed in individual malignant tumors. In this study we describe the combined contribution of these distinct enzyme systems to the invasive phenotype of a highly metastatic human melanoma cell line (M24met). M24met cells were found to secrete a spectrum of MMPs, including interstitial collagenase, type IV collagenases (M(r) 92,000 and 72,000 progelatinases), and stromelysin. Urokinase, but not tissue-type plasminogen activator, was detected in M24met-conditioned media and on cell surfaces. The contribution of these enzymes to extracellular matrix dissolution was determined by exploiting specific inhibitors, namely tissue inhibitor of the metalloproteinases-2 and plasminogen activator inhibitor-2. Due to the coexpression of urokinase and MMP-dependent activity, M24met cells were observed to degrade multiple components of the extracellular matrix and to significantly degrade both interstitial and basement membrane matrices. Urokinase-dependent removal of matrix glycoprotein was observed to precede MMP-dependent collagenolysis as a prerequisite rate-limiting step. We present evidence which suggests that this temporal relationship is imposed by the structural architecture of the matrix such that matrix glycoprotein serves to protect associated collagen from MMP-dependent degradation. In addition to mediating significant collagenolysis, MMP activity was further implicated in the dissolution of matrix tropoelastin. Urokinase/plasmin activity was not found to be required for MMP-zymogen activation.

Amino Acid Sequence↗
PubMed1993

Therapeutic potential of chimeric and murine anti-(epidermal growth factor receptor) antibodies in a metastasis model for human melanoma.

On many tumors, high numbers of epidermal growth factor (EGF) receptors provide a target for antibody-mediated tumor therapy. We evaluate here the therapeutic potential of a mouse/human chimeric anti-(EGF receptor) antibody and compare it to the parental murine antibody in a xenograft model for metastatic melanoma. Our model is based on the human cell line M24met, which overexpresses the EGF receptor and metastasizes spontaneously in SCID mice. Both the chimeric anti-(EGF receptor) antibody (ch225) and the mouse monoclonal antibody (m225) exhibited saturable, high-affinity binding to M24met cells and were equivalent in their ability to target M24met tumors in mice. Neither anti-(EGF receptor) antibodies nor EGF modulated the growth of M24met cells in vitro. Further analysis revealed that the EGF receptor on these cells is not phosphorylated upon EGF binding, indicating an anomalous receptor on these cells. In antibody-dependent cellular cytotoxicity experiments, ch225 and m225 were potent mediators of M24met cytolysis by effector cells. Antibody-mediated cytotoxicity revealed a marked species preference, with ch225 activating human peripheral blood mononuclear cells and m225 activating mouse splenocytes and to a lesser degree mouse macrophages. Neither antibody mediated cytolysis in the presence of human complement. In SCID mice, m225 suppressed spontaneous metastasis considerably while ch225 had only a modest effect. Our data indicate that in the M24met melanoma tumor model, anti-(EGF receptor) antibodies suppress spontaneous metastasis solely by activating immune effector cells.

Animals↗
PubMed1993

Mechanisms of cellular cytotoxicity mediated by a recombinant antibody-IL2 fusion protein against human melanoma cells.

Functional characteristics were established for a genetically engineered fusion protein between human IL2 and mouse/human chimeric mAb 225 directed against human epidermal growth factor receptor (EGFR), aberrantly expressed on human melanoma cells. The emphasis of these studies was on the mechanism(s) of action by which the ch225-IL2 fusion protein mediated cytotoxic killing of human melanoma cells by different human immune effector cells. Ch225-IL2 fusion protein bound to human EGFR with the high affinity of the parental antibody, and was as active as the equivalent amount of rhIL2. Ch225-IL2 enhanced cellular cytotoxicity mediated by freshly separated PBMC, isolated natural killer (NK) cells and activated T cells against melanoma cell lines. NK cells, which constitutively express both Fc gamma RIII and IL2R, interacted with ch225-IL2, mainly through Fc gamma RIII, while the involvement of IL2R was secondary. However, the effect of ch225-IL2 on activated T cells was most likely mediated through IL2R. These results suggest that the genetically engineered ch225-IL2 fusion protein may become a potent immunotherapeutic agent capable of stimulating various immune effector populations to effectively kill human melanoma cells.

Animals↗
PubMed1992

Expression of tissue factor by melanoma cells promotes efficient hematogenous metastasis.

Metastasis is a multistep process which requires highly adapted interactions of tumor cells with host target organs. Compared with nonmetastatic cells, metastatic human melanoma cells express 1000-fold higher level of tissue factor (TF), the major cellular initiator of the plasma coagulation protease cascades. To explore whether TF may contribute to metastatic tumor dissemination, we analyzed the effect of specific inhibition of TF function on human melanoma metastasis in severe combined immunodeficient (SCID) mice. Using species-specific antibodies to TF, we demonstrate that initial adherence in insufficient for successful tumor cell implantation in a target organ. Rapid arrest of human tumor cells in the lungs of mice was not diminished by inhibition of TF. However, inhibition of TF receptor function and consequent reduction in local protease generation abolished prolonged adherence of tumor cells, resulting in significantly reduced numbers of tumor cells retained in the vasculature of the lungs. The growth of pulmonary metastases was also significantly inhibited by a blocking anti-TF monoclonal antibody and Fab fragments thereof, whereas a noninhibitory antibody lacked antimetastatic effects. Cell surface expression of functional TF thus contributes to melanoma progression by allowing metastatic cells to provide requisite signals for prolonged adhesive interactions and/or transmigration of tumor cells across the endothelium, resulting in successful metastatic tumor implantation.

Animals↗
PubMed1992

Long-term growth suppression of human glioma xenografts by chemoimmunoconjugates of 4-desacetylvinblastine-3-carboxyhydrazide and monoclonal antibody 9.2.27.

A conjugate of 4-desacetylvinblastine-3-carboxyhydrazide (DAVLBHY) and the glioma-reactive monoclonal antibody (mAb) 9.2.27 induced long-term suppression of tumor growth in athymic nude mice engrafted with U87MG human glioma cells. In vitro, DAVLBHY had the strongest antiproliferative activity (inhibitory concentration at which incorporation of [3H]thymidine is at 50% of untreated control is 2.0 x 10(-9) M) of seven cytotoxic drugs tested and so was chosen for conjugation to mAb 9.2.27, which reacts specifically with the core protein of chondroitin sulfate proteoglycans found in human glioblastomas. After conjugation of DAVLBHY to the carbohydrate residues of mAb 9.2.27 it retained its full binding capacity. For in vivo studies, DAVLBHY and several conjugate derivatives were evaluated by using two dosages of i.v. injections, each starting 2 days after s.c. tumor inoculation. The control tumors reached a volume of nearly 3000 mm3 within 30 days. Tumor growth was delayed by about 20 days with four i.v. injections of 0.5 mg/kg 9.2.27-DAVLBHY, which was slightly superior to the unconjugated drug. Moreover, 9.2.27-DAVLBHY produced a highly significant suppression of growth so that the average tumor volume was only 3% of that observed in untreated controls after 28 days. Four injections of this conjugate at a larger dose, 2.0 mg/kg, prevented recurrence of the tumors for 130 days in all animals tested, thus demonstrating a significant increase in the therapeutic index, since the unconjugated drug provided limited inhibition of tumor growth for only 40 days. The specificity of the antitumor effect was demonstrated in a comparison with the control conjugate, KS1/4-DAVLBHY, which despite partial tumor suppression had only a transient effect. The specific antitumor effect of 9.2.27-DAVLBHY was unexpected, since the target antigen is expressed at a relatively low density (40,000 sites/cell) on U87MG glioma cells.

Animals↗
PubMed1992

Activation of methotrexate-alpha-alanine by carboxypeptidase A-monoclonal antibody conjugate.

Carboxypeptidase A (CP-A) and monoclonal antibody KS1/4 directed against an antigen on human lung adenocarcinoma cells (UCLA-P3) were derivatized by treatment with succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate and N-succinimidyl 3-(2-pyridyldithio)propionate, respectively. The derivatized proteins were reacted to produce thioether-linked enzyme-antibody conjugates. Sequential HPLC size-exclusion and DEAE chromatography separated the conjugate preparation from unreacted enzyme and antibody. On the basis of SDS-PAGE analysis and measurement of catalytic activity, the preparation contained approximately equal amounts of 1:1 and 2:1 (enzyme:antibody) conjugates; binding activity of the conjugate (1.8 x 10(5) molecules/cell) was similar to that of unreacted antibody. In vitro cytotoxicity studies with UCLA-P3 cells demonstrated the ability of cell-bound conjugate to convert the prodrug methotrexate-alpha-alanine (MTX-Ala) to methotrexate (MTX). In the absence of conjugate, ID50 values for MTX-Ala and MTX were 8.9 x 10(-6) and 5.2 x 10(-8) M, respectively. ID50 for the prodrug improved to 1.5 x 10(-6) M with cells containing bound conjugate. This potentiation of MTX-Ala cytotoxicity by conjugate-bound CP-A, which was at least 30-fold greater than that produced by a comparable amount of free enzyme, is attributed to enhanced effectiveness of MTX generated at the cell surface as opposed to the surrounding medium. Examination of the time course of cytotoxicity over a 96-h period showed that the conjugate-prodrug combination (at 2.5 x 10(-6) M) was nearly as effective as MTX in preventing cell replication. These results demonstrate the chemotherapeutic potential of carboxypeptidase-monoclonal antibody conjugates used in conjunction with MTX peptide prodrugs.

Adenocarcinoma↗
PubMed1992

Involvement of integrin alpha V gene expression in human melanoma tumorigenicity.

Human melanoma originates in the skin and can lead to wide-spread metastatic disease. Analysis of melanoma biopsy material has shown that the vitronectin receptor, integrin alpha v beta 3, is a specific marker of the most malignant cells, i.e., vertically invasive primary lesions or distant metastases (Albelda, S. M., S. A. Mette, D. E. Elder, R. Stewart, L. Damjanovich, M. Herlyn, and C. A. Buck. 1990. Cancer Res. 50:6757-6764), suggesting a role for this adhesion receptor in the malignant growth of human melanoma tumors. A cell model was established to analyze the role of alpha v integrins on the tumorigenicity of human melanoma. From M21 human melanoma cells, stable variants were selected that lack alpha v gene expression and thus fail to express integrin alpha v beta 3 (M21-L cells). These cells not only lost the ability to attach to vitronectin but showed a dramatic reduction in tumorigenicity when transplanted into athymic nude mice, compared with M21 cells, even though both cell types showed identical beta 1 integrin expression and growth properties in vitro. M21-L cells were stably transfected with a cDNA-encoding alpha v. This resulted in the functional expression of integrin alpha v beta 3 on these cells and completely restored their tumorigenicity. Thus, integrin alpha v gene expression and the resulting adhesive phenotype are directly involved in the proliferation of human melanoma in vivo.

Cell Division↗
PubMed1991

Suppression of spontaneous melanoma metastasis in scid mice with an antibody to the epidermal growth factor receptor.

The human melanoma cell line M24met metastasizes spontaneously from s.c. tumors to multiple distant sites in mice with severe combined immunodeficiency. Metastasis to lymph nodes and lungs is found in 100% of the animals. M24met has an undifferentiated phenotype and extra copies of the short arm of chromosome 7. This cell line expresses the epidermal growth factor receptor, and 425.3, a monoclonal antibody to the epidermal growth factor receptor, binds to 291,000 receptor molecules per M24met cell with a KD of 2.3 x 10(-10) M. This antibody has no effect on the proliferation of M24met cells under tissue culture conditions and does not mediate effector cell or complement-dependent cytotoxicity of these cells in vitro. However, treatment of established s.c. M24met tumors in mice with severe combined immunodeficiency with monoclonal antibody 425.3 specifically suppresses spontaneous metastasis of these tumors. Total doses of 4, 2, and 1 mg antibody per mouse decrease the number and size of melanoma metastases and prolong the life span of treated animals. Treatment with 4 mg of the F(ab')2 fragment of monoclonal antibody 425.3 does not influence M24met melanoma metastasis, implying a significant contribution of the Fc portion to the antimetastatic effect of this antibody.

Animals↗
PubMed1991

Effect of a chimeric anti-ganglioside GD2 antibody on cell-mediated lysis of human neuroblastoma cells.

An anti-GD2 ganglioside human/mouse chimeric monoclonal antibody, ch14.18, like its murine counterpart, 14.G2a, was shown to bind to human neuroblastoma cells. This chimeric antibody proved to be more effective than 14.G2a in mediating the lysis of neuroblastoma cells with human effector cells, such as granulocytes and natural killer cells within the peripheral blood mononuclear cell population. A comparison of these two effector cell populations isolated from the same donor revealed granulocytes to be more effective than peripheral blood mononuclear cells in lysing neuroblastoma cells, which were coated with monoclonal antibody ch14.18. Addition of recombinant human granulocyte-macrophage colony-stimulatory factor increased ch14.18-mediated lysis of neuroblastoma cells by granulocytes but not by peripheral blood mononuclear cells. In fact, granulocytes were effective in mediating lysis of neuroblastoma cells coated with ch14.18 irrespective of whether they were obtained from normal adults or from neuroblastoma patients.

Animals↗
PubMed1991

Potential of the scid mouse as a host for human tumors.

Animal models of human tumors and their metastases that effectively mimic clinical disease are in considerable demand. While it is certainly true that athymic nude mice provide us with useful models to study a large number of human tumors in vivo, it is also well known that nude mice usually do not develop spontaneous metastases and are not suitable for all types of tumors. Therefore, the scid mouse that allows disseminated growths for a number of human tumors, particularly hematologic disorders and malignant melanoma, can be used preferentially for the investigation of such malignancies. The potential to study the interaction of human immune cells and human tumor in an in vivo model is another unique feature of scid mice that will add to their usefulness in experimental cancer research.

Animals↗