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The tumor cell laminin receptor is a cell-surface protein that binds laminin with high affinity (Kd = 1.0 nM). The putative ligand-binding domain of the laminin receptor has been molecularly cloned and sequenced. In the present study, we used the predicted amino acid sequence of the laminin receptor to generate synthetic peptide antigens and produced immunoglobulin M (IgM) anti-laminin receptor monoclonal antibodies. The disulfide bond group of the IgM molecule was used to couple the antibodies to the surface of liposomes encapsulating doxorubicin. The anti-laminin receptor monoclonal antibodies coupled to the liposomes bound avidly to the surface of MDA-MB-435S (MDA-435) human breast carcinoma cells, which have high numbers of laminin receptors. These antibody-coupled liposomes exhibited a low degree of binding to Hs 578Bst (Hs 578) normal human breast epithelial cells, which express a low number of laminin receptors. Excess liposomes competed for the binding of unbound laminin to the tumor cell surface, and excess laminin competed for binding with the liposomes. Antibody-coupled liposomes encapsulating doxorubicin were specifically more efficient in inhibiting colony formation by MDA-435 cells in vitro than unbound doxorubicin or liposomes without anti-laminin receptor monoclonal antibodies. Unbound doxorubicin inhibited thymidine uptake by 10%-20% in both Hs 578 and MDA-435 cells, whereas the antibody-coupled liposomes encapsulating doxorubicin inhibited thymidine uptake by 90% in MDA-435 cells but only 15% in Hs 578 cells. Thus, use of anti-laminin receptor monoclonal antibodies coupled with liposomes encapsulating doxorubicin represents a new strategy for selective targeting of doxorubicin to carcinoma cells with exposed laminin receptors.

Human melanoma cells resistant to killing by monoclonal antibody R24 plus human complement became susceptible after treatment with doxorubicin (adriamycin). Treatment with doxorubicin prevented the rapid degradation of surface-bound complement component C3b that has been identified as a protective mechanism of complement-resistant melanoma cells. Doxorubicin caused the increased complement susceptibility as free drug and after immobilization onto glass beads to prevent cellular uptake. Immobilized doxorubicin was more effective than free drug, causing enhanced complement susceptibility at concentrations where the free drug was no longer active. In contrast to free doxorubicin, which exhibited a direct cytotoxic effect leading to cell death within 4 days, immobilized doxorubicin did not affect cell viability. These findings suggest that combination therapy of the complement-activating monoclonal antibody R24 with the complement-enhancing drug doxorubicin may be a promising approach for the treatment of melanoma.

The activation of bovine trypsinogen by an extracellular acid proteinase from A. fumigatus is described. The enzyme activates trypsinogen optimally at pH 3.5 and 32 degrees C. The effect of substrate and enzyme concentrations on the activation has been studied and the Km-value has been determined.

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor (EGF) on type-IV-procollagen (basement-membrane collagen) and laminin synthesis, turnover, and secretion was studied in human A431 squamous epidermoid carcinoma cells. Type-IV procollagen and laminin were biochemically and immunologically identified in the medium and cell extracts using immuno-precipitation followed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. EGF or TPA produced a sixfold increase in type-IV-procollagen and laminin secretion within 2 h; this was accompanied by a three- to fourfold increase in the levels of cell-associated type-IV procollagen and laminin, respectively. The level of type-IV-procollagen and laminin synthesis and secretion remained elevated for at least 16 h after the administration of either EGF or TPA. A combination of EGF and TPA was more effective than either agent alone in promoting the secretion of laminin but not of type-IV procollagen. EGF and/or TPA did not, however, produce a selective increase in the synthesis of collagen and laminin, since total protein synthesis was also increased to the same degree by these agents. As determined by labeling and chase studies, neither EGF nor TPA had any appreciable effect upon type-IV-procollagen or laminin degradation. These results indicate that the synthesis of components associated with the basement membrane in A431 cells (i.e., type-IV procollagen and laminin) can be rapidly modulated by EGF and a tumor promoter, i.e., TPA.(ABSTRACT TRUNCATED AT 250 WORDS)

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Human melanoma cells resistant to killing by the R24 mAb and human complement rapidly degrade surface-deposited C3b (M. Panneerselvam, S. Welt, L. J. Old, C.-W. Vogel. 1986. J. Immunol. 136:2534). We report that C-resistant melanoma cells express a membrane proteinase that can cleave C3b, generating a cleavage product with a molecular mass of approximately 30 kDa. The C3-cleaving proteinase was identified on the melanoma cells by its cross-reaction with antiserum to p57, a C3-cleaving proteinase previously isolated from human E membranes (C. Charriaut-Marlangue, M. Barel, R. Frade. 1986. Biochem. Biophys. Res. Commun. 140:1113). Preincubation of the C-resistant melanoma cells with anti-p57 IgG or their F(ab')2 fragments increased their susceptibility to complement killing from 25% to approximately 50% and reduced the rate of C3b cleavage and the amount of the 30-kDa fragment generated on the cells. Anti-p57 IgG stained C-resistant melanoma cells by indirect immunofluorescence and precipitated a protein with an apparent molecular mass of 65 kDa. This membrane protein, termed p65, was not detectable on C-susceptible melanoma cells. Membrane extracts from C-resistant melanoma cells also showed C3-cleaving activity when incubated with purified C3 or C3b, similarly generating a C3 fragment of approximately 35 kDa. This fluid-phase C3 cleaving activity could be partially inhibited by anti-p57 IgG. These data suggest that p65 is a C3-cleaving proteinase, antigenically related to p57, that is expressed on C-resistant melanoma cells and responsible for the C resistance of these cells. We propose that the membrane-bound C3-cleaving proteinase represents another C regulatory protein protecting host cells against killing by C.

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