sepharose and Fibrosarcoma

Heat shock protein-based vaccines have been shown to immunize against cancer and infectious diseases in both prophylactic and therapeutic protocols. So far, four classes of heat shock proteins (HSPs) preparation: gp96, HSP90 (hsp86, hsp84), HSP70 (hsc70, hsp70) and calreticulin have been used successfully. The methods for purifying them individually are now readily available. However, since tumors are not always available in large quantity, a major challenge remains the development of a procedure to simultaneously isolate these HSPs from the same sample. We report here that hsp40, hsp60, hsc70, hsp70, hsp84, hsp86, and gp96 (grp94) but not BiP (grp78) and calreticulin can be separated from a single tumor sample in one step using heparin-agarose chromatography. Interestingly this procedure separates the HSP70 isoforms hsp70 from hsc70, but not the HSP90 isoforms hsp84 and hsp86. The three main immunogenic HSPs, gp96, hsp86/84, and hsc70 can be further isolated to homogeneity using additional purification methods. In addition, we have shown that the interaction of the chaperoned peptides with hsc70 and gp96 is not compromised during heparin chromatography. These observations provide a new method for preparation of multiple HSP-based vaccines, circumventing the sample size limitation, as well as providing the possibility to study how multiple HSPs can synergize in eliciting immunity.

Topics: Animals; Antigens, Neoplasm; Cancer Vaccines; Carrier Proteins; Chromatography, Affinity; Endoplasmic Reticulum Chaperone BiP; Fibrosarcoma; Heat-Shock Proteins; HSC70 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Mice; Mice, Inbred BALB C; Neoplasm Proteins; Sepharose

A type IV collagen-binding protein of 41 kDa was isolated from the mushroom Hypsizigus marmoreus and the protein was designated as HM41. The Western blotting analysis with anti-HM41 antibodies demonstrated that HM41 was unrelated to HM23, which had been shown to have an affinity for type IV collagen. The microsequence analysis of the membrane-blotted peptides generated by fragmentation with cyanogen bromide showed no homologous proteins reported. HM41 had cell adhesion-promoting activity for murine Lewis lung carcinoma LL2 cells and human fibrosarcoma HT1080 cells. These results indicate that HM41 is a hitherto undescribed fungus protein that can interact both with animal extracellular matrix protein type IV collagen and with animal tumor cells.

Topics: Agaricales; Animals; Carcinoma, Lewis Lung; Cell Adhesion; Chromatography, Affinity; Collagen; Fibrosarcoma; Fungal Proteins; Humans; Integrins; Mice; Receptors, Collagen; Sepharose; Tumor Cells, Cultured

We have previously developed a simple and quantitative method for assessment of in vivo tumor cell-induced angiogenesis by means of microencapsulation of tumor cells in agarose hydrogel and mouse hemoglobin ELISA (mHb-ELISA). In this article, we report that the new blood vessels induced with agarose-encapsulated tumor cells have the same sensitivity to tumor necrosis factor-alpha (TNF-alpha) as the original solid-tumor vessels. Agarose beads (average diameter = 200 microns), in which Meth-A fibrosarcoma cells were microencapsulated, were subcutaneously implanted in non-syngeneic ddY mice. Ten days later, extensive angiogenesis was observed on the implanted sites of Meth-A agarose heads, whereas no new blood vessels were induced with cell-free agarose heads. The vascular permeability of the new blood vessels induced with agarose-microencapsulated Meth-A cells was selectively and significantly enhanced by the i.v. injection of TNF-alpha, and it reached the maximum level at 2 h after the injection of TNF-alpha. At 4 h after the injection of TNF-alpha, the vascular permeability was reduced to the basal level. This permeability profile in Meth-A agarose beads in ddY mice is very similar to that in Meth-A solid tumor in syngeneic BALB/c mice. On the other hand, TNF-alpha-treatment did not affect the vascular permeability of other normal tissues or inflammatory tissue in ddY mice. These results strongly suggest that the new blood vessels induced with agarose-microencapsulated tumor cells have the specific characteristics of tumor vessels. Our in vivo angiogenesis assay system should be useful not only to screen anti-angiogenetic agents, but also to elucidate the mechanism of tumor angiogenesis.

Topics: Animals; Capillary Permeability; Female; Fibrosarcoma; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Neovascularization, Pathologic; Polyethylene Glycols; Sepharose; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The laminin alpha1 chain carboxyl-terminal globular domain (G domain) contains multiple biological activities. Recently, we identified five cell binding sequences from the G domain by screening with overlapping 12-mer peptides encompassing the entire domain. The structures of these five sequences in the alpha1 chain are conserved in the corresponding regions of the different laminin alpha chains. Here we characterize the adhesion activities of the corresponding peptide segments from both the mouse laminin alpha2 chain and Drosophila laminin alpha chain using peptide-coated plastic plates and peptide-conjugated Sepharose beads. Using several cell lines, the laminin alpha2 chain peptides showed cell attachment and/or spreading activities with cell type specificities. Cell spreading on MG-10 was inhibited by integrin antibodies. Four of the Drosophila laminin peptides showed cell attachment activities. These results suggest that biologically active regions in the G domain are conserved in the laminin alpha1 and alpha2 chains, and that these regions in laminin play an important role in cell surface receptor interactions.

Topics: Amino Acid Sequence; Animals; Binding Sites; Cell Adhesion; Cells, Cultured; Conserved Sequence; Dose-Response Relationship, Drug; Drosophila; Fibrosarcoma; Humans; Laminin; Melanoma; Mice; Models, Molecular; Muscle, Skeletal; Peptide Fragments; Rats; Sepharose; Tumor Cells, Cultured

When nonadherent splenic cells from normal and tumor-bearing (mouse fibrosarcoma, MFS) Swiss mice were added to wells made in agarose layers in plastic petri dishes, subpopulations of cells from tumor-bearing mice were seen to migrate out of the wells, whereas those from normal mice did not. The proportion of migratory cells among the lower density (less than 1.057 to less than 1.069 g/ml) cells was larger than that of higher density (1.069 to less than 1.087 g/ml) cells. When the plastic surface underneath the agarose layer was covered with a monolayer of MFS cells, the splenic cells from normal mice also migrated out of the wells. About 20% dead MFS cells were observed in the zone of migration when the migratory cells were from normal mice, and about 30% when the migratory cells were from tumor bearing mice. Apart from revealing the differences between the migratory behavior of splenic cells, the present work also suggests a novel application of agarose methodology in the study of interaction of cytotoxic cells with malignant cells.

Topics: Animals; Cell Movement; Cell Survival; Cells, Cultured; Culture Media; Fibrosarcoma; Lymphocytes; Mice; Sarcoma, Experimental; Sepharose; Spleen

Several variant cell lines that are deficient in surface laminin have been isolated from heterogeneous murine tumour populations. The parent populations from which these variant lines were isolated contain cells that express high levels of surface laminin as indicated by immunofluorescence, immunoperoxidase/electron microscopy and immunoprecipitation. The laminin-deficient lines were compared with their respective parent populations for motility by both the Boyden Chamber assay and the agarose assay. In both assays, the laminin-positive populations were much more motile than the laminin-deficient lines. The addition of exogenous laminin to the laminin-deficient lines significantly increased their motility. These observations are of interest since cell motility is thought to contribute to tumour cell metastasis, and the laminin-positive cell populations are highly tumorigenic and metastatic, while the laminin-deficient cells are of low tumorigenicity and are virtually non-metastatic.

Topics: Animals; Cell Line; Cell Movement; Fibrosarcoma; Laminin; Methods; Mice; Microscopy, Phase-Contrast; Sepharose; Stimulation, Chemical

Topics: Animals; Antibodies; Antigens; Antigens, Neoplasm; Antigens, Surface; Fibrosarcoma; Immune Sera; Mice; Mice, Inbred C57BL; Sarcoma, Experimental; Sepharose; Tissue Distribution

A novel method is described for production of heterologous antisera to a specific tumor-associated murine antigen by immunization with agarose-trapped immune complexes.

Topics: Animals; Antigen-Antibody Complex; Fibrosarcoma; Fluorescent Antibody Technique; Mice; Neoplasms, Experimental; Precipitin Tests; Rabbits; Sepharose; Spleen

Topics: Absorption; Animals; Antibodies, Anti-Idiotypic; Chemical Fractionation; Chromatography; Dextrans; Female; Fibrosarcoma; Immunoglobulin Fab Fragments; Isoantibodies; Mice; Mice, Inbred C3H; Neoplasm Transplantation; Neoplasms, Experimental; Papain; Sepharose; Transplantation Immunology; Transplantation, Homologous