sepharose and Neoplasm-Metastasis

ADAM 12 is a member of a family of disintegrin-containing metalloproteases that have been implicated in a variety of diseases including Alzheimer's disease, arthritis, and cancer. We purified ADAM 12 from the urine of breast cancer patients via Q-Sepharose anion exchange and gelatin-Sepharose affinity chromatography followed by protein identification by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Four peptides were identified that spanned the amino acid sequence of ADAM 12. Immunoblot analysis using ADAM 12-specific antibodies detected an approximately 68-kDa band identified as the mature form of ADAM 12. To characterize catalytic properties of ADAM 12, full-length ADAM 12-S was expressed in COS-7 cells and purified. Substrate specificity studies demonstrated that ADAM 12-S degrades gelatin, type IV collagen, and fibronectin but not type I collagen or casein. Gelatinase activity of ADAM 12 was completely abrogated by zinc chelators 1,10-phenanthroline and EDTA and was partially inhibited by the hydroxamate inhibitor Marimastat. Endogenous matrix metalloprotease inhibitor TIMP-3 inhibited activity. To validate our initial identification of this enzyme in human urine, 117 urine samples from breast cancer patients and controls were analyzed by immunoblot. The majority of samples from cancer patients were positive for ADAM 12 (67 of 71, sensitivity 0.94) compared with urine from controls in which ADAM 12 was detected with significantly lower frequency. Densitometric analyses of immunoblots demonstrated that ADAM 12 protein levels were higher in urine from breast cancer patients than in control urine. In addition, median levels of ADAM 12 in urine significantly increased with disease progression. These data demonstrate for the first time that ADAM 12 is a gelatinase, that it can be detected in breast cancer patient urine, and that increased urinary levels of this protein correlate with breast cancer progression. They further support the possibility that detection of urinary ADAM 12 may prove useful in the development of noninvasive diagnostic and prognostic tests for breast and perhaps other cancers.

Topics: ADAM Proteins; ADAM12 Protein; Adult; Aged; Amino Acid Sequence; Animals; Blotting, Western; Breast Neoplasms; Caseins; Catalysis; Chelating Agents; Chromatography, Affinity; Chromatography, Ion Exchange; Collagen Type I; Collagen Type IV; COS Cells; Databases as Topic; Densitometry; Disease Progression; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Extracellular Matrix; Female; Fibronectins; Gelatin; Humans; Hydroxamic Acids; Immunoblotting; Membrane Proteins; Metalloendopeptidases; Middle Aged; Molecular Sequence Data; Neoplasm Metastasis; Peptides; Phenanthrolines; Plasmids; Recombinant Proteins; Sensitivity and Specificity; Sepharose; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Substrate Specificity; Ultracentrifugation; Zinc

Bone metastasis is the major reason for death caused by breast cancer. We used human breast cancer (MCF-7) cells that are poorly metastatic but show highly inducible migration to determine bone-derived factors that induce migration of initially non-disseminating breast cancer cells. We have found that a lipid fraction from human osteoblast-like MG63 cell-conditioned medium (MG63CM) contains a migration-inducing factor for MCF-7 cells. In this fraction, we have identified oxysterol (OS) as a lipid mediator for tumor cell migration. In MCF-7 cells, insulin-like growth factor 1 elevates the expression of OS-binding protein-related protein 7. Binding of OS to OS-binding protein or OS-binding protein-related protein is known to trigger elevation of sphingomyelin, a sphingolipid that organizes lipid microdomains in the cell membrane. In MCF-7 cells, OS increases the intracellular concentration of sphingomyelin and other phospholipids and induces the translocation of the small GTPase p21Ras to GM1- and cholesterol-rich membrane areas. The induction of migration by MG63CM is prevented by incubation of MG63 cells with mevinolin, a statin-type cholesterol biosynthesis inhibitor that depletes the conditioned medium of OS. Osteoblast-derived OS may, thus, be a yet unrecognized lipid mediator for bone metastasis of breast cancer and a new target for anti-metastasis chemotherapy with statins.

Topics: Arachidonic Acid; Aspirin; Breast Neoplasms; Cell Membrane; Cell Movement; Cholesterol; Chromatography, High Pressure Liquid; Coculture Techniques; Culture Media, Conditioned; Dinoprostone; Electrophoresis, Polyacrylamide Gel; HSP70 Heat-Shock Proteins; Humans; Insulin-Like Growth Factor I; Lipid Metabolism; Lovastatin; MAP Kinase Signaling System; Microscopy, Fluorescence; Neoplasm Metastasis; Osteoblasts; Phospholipids; Phosphorylation; Prostaglandins B; Protein Transport; Proto-Oncogene Proteins p21(ras); Reverse Transcriptase Polymerase Chain Reaction; Sepharose; Signal Transduction; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sphingolipids; Sterols; Trypsin; Tumor Cells, Cultured

Matrilysin produced by human colon cancer cells may be involved in the progression and metastasis of cancer. In the present study, we investigated the association of matrilysin with angiogenesis. One microgram of recombinant matrilysin is confirmed to have increased [3H]-thymidine uptake in human umbilical vein endothelial cells. Then we used micro encapsulation and a mouse hemoglobin enzyme-linked immunosorbent assay system for in vivo quantitation of angiogenesis with BALB/c nu/nu athymic mice. Hundred micrograms of recombinant matrilysin induced angiogenesis to the same degree as 10 microg of basic fibroblast growth factor (bFGF). Angiogenesis was observed at the site implanted with human colon cancer WiDr cells in agarose micro beads. This was inhibited by subcutaneous injection of matrilysin-specific antisense oligonucleotide significantly by 53%. In conclusion, matrilysin may be associated with angiogenesis of human colon cancer through the direct proliferative action on endothelial cells.

Topics: Animals; Cell Division; Cells, Cultured; Colonic Neoplasms; Colorectal Neoplasms; Culture Media, Serum-Free; DNA; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Fibroblast Growth Factor 2; Humans; Immunoblotting; Matrix Metalloproteinase 7; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Neoplasm Transplantation; Neovascularization, Pathologic; Oligonucleotides, Antisense; Sepharose; Umbilical Veins

We studied the influence of 10 synthetic glycoamine analogs on colony formation in 0.3 and 0.9% agarose by metastatic human breast carcinoma (MDA-MB-435) and melanoma (TXM-13) cells. Nine synthetic analogs significantly inhibited the colony formation in 0.9% agarose of MDA-MB-435 human breast carcinoma cells; five compounds caused a 73-83% reduction of colony formation. Seven synthetic glycoamines caused a significant inhibition of colony formation in 0.9% agarose by TXM-13 melanoma cells with the inhibitory effect ranging from 71 to 87%. The 50% inhibition (I50) doses and relative activity rank of the compounds were similar for both breast carcinoma and melanoma cell lines. The murine B16 melanoma cell aggregation assay was employed to elucidate the potential mechanism(s) of the inhibitory activity of synthetic glycoamines. The relative activity ranks of the compounds based on the independently determined I50 doses for both cell aggregation and clonogenic growth assays were very similar for the four most active synthetic analogs and clearly indicated the importance of hydrophobic amino acid in mediating the bioactivity of synthetic glycoamines. In both experimental systems (clonogenic growth in agarose and cell aggregation assay) the leading compound was N-(1-deoxy-D-fructos-1-yl)-D-leucine (Fru-D-Leu) and the least active analog was N-(l-deoxy-D-fructos-1-yl)-glycine (Fru-Gly). These results show that synthetic glycoamines may act by competing for specific carbohydrate-lectin interactions, particularly those involving beta-galactoside-specific lectins expressed on metastatic cells.

Topics: Amino Sugars; Animals; Binding, Competitive; Breast Neoplasms; Carbohydrate Sequence; Carcinoma; Colony-Forming Units Assay; Female; Galactosides; Glucosamine; Humans; Lectins; Magnetic Resonance Spectroscopy; Mass Spectrometry; Melanoma; Mice; Molecular Sequence Data; Molecular Structure; Neoplasm Metastasis; Sepharose; Structure-Activity Relationship; Tumor Cells, Cultured; Tumor Stem Cell Assay

Insulin-like growth factor I (IGF-I) stimulates the proliferation of highly metastatic NL-17 cells to a greater extent than poorly metastatic NL-44 cells, both of which are derived from mouse colon carcinoma 26. The NL-17 cells have been compared with NL-44 cells for the signal transduction pathway of IGF-I. IGF-I receptors of both cell types were identified by affinity labeling, and there was no significant difference between the two cell types in the amount or the autophosphorylation activity of the IGF-I receptors. However, when IGF-I-dependent tyrosine phosphorylation of cellular components was examined, remarkable tyrosine phosphorylation of proteins with molecular weights of 150,000 (pp150) and 160,000 (pp160) was found in NL-17 cells. In contrast, this phosphorylation stayed at significantly lower levels in NL-44 cells than in NL-17 cells. The phosphorylation of pp150 and pp160 was induced within 10 s after the addition of IGF-I and reached its maximal level by 30 s. After the removal of IGF-I, the phosphorylation of pp150 and pp160 was reduced to the basal level within 30 min. This phosphorylation was not induced by platelet-derived or epidermal growth factor. The pp150 and pp160 were not absorbed by wheat germ agglutinin-agarose. They were found in the soluble fraction of cytoplasm but not in the membrane or the cytoskeleton. The pp150 and pp160 might be endogenous substrates of IGF-I receptor kinase. These results suggest that tyrosine phosphorylation of pp150 and pp160 mediates the higher proliferative response of NL-17 cells to IGF-I.

Topics: Animals; Antibodies, Monoclonal; Cell Line; Colonic Neoplasms; Epidermal Growth Factor; Insulin; Insulin-Like Growth Factor I; Kinetics; Mice; Molecular Weight; Neoplasm Metastasis; Neoplasm Proteins; Phosphoproteins; Phosphorylation; Phosphotyrosine; Platelet-Derived Growth Factor; Receptors, Cell Surface; Receptors, Somatomedin; Sepharose; Tyrosine

The purpose of this study was to determine whether the degree of anchorage-independent growth of human tumor cells in increasing concentrations of agarose correlated with the capacity of the cells to produce experimental metastases in nude mice. Human melanoma, breast carcinoma, and colon carcinoma cells from parental lines and variants selected in vivo for metastasis and in vitro cloned lines were plated into medium containing 0.3%, 0.6%, 0.9%, or 1.2% of agarose. These cells were also injected into nude mice: intravenously for melanoma, into the mammary fat pad for breast carcinoma, and into the spleen for colon carcinoma. Production of tumor cell colonies in dense agarose (greater than 0.6%) correlated with production of experimental metastases in the lung (melanoma, breast carcinoma) or liver (colon carcinoma). We conclude that the degree of anchorage-independent growth of tumor cells can predict their biological behavior and metastatic potential in vivo. Thus, this technique may be useful for the isolation of metastatic cells from heterogeneous human neoplasms.

Topics: Animals; Breast Neoplasms; Clone Cells; Colonic Neoplasms; Culture Media; Humans; Lung Neoplasms; Melanoma; Mice; Mice, Nude; Neoplasm Metastasis; Neoplasm Transplantation; Sepharose; Tumor Cells, Cultured

Two highly glycosylated membrane sialoglycoproteins designated P2A and P2B were isolated from the lymphoreticular tumor cell line called MDAY-D2 and shown to be structurally similar to the lymphocyte glycoprotein called leukosialin and to lysosome-associated membrane glycoprotein termed LAMP-1, respectively. The loss of sialic acid and polylactosamine sequences in glycosylation mutants of MDAY-D2 has been correlated previously with enhanced cell adhesion on extracellular matrix proteins. Since these two glycoproteins bear the majority of the sialylated oligosaccharides found in membrane fractions of MDAY-D2, they were tested for binding activity on extracellular matrix proteins. Both isolated glycoproteins bound to immobilized collagen type I with affinities that were dependent on their glycosylation. Enzymatic removal of sialic acid, polylactosamine, or complete asparagine-linked chains from purified P2B enhanced its binding to collagen, laminin, and fibronectin. In contrast, P2A bound specifically to collagen type I and the interaction required the presence of sialic acid residues which were sensitive to neuraminidase digestion but not to endoglycosidase F. The results suggest that oncodevelopmental regulation of oligosaccharide expression on P2A and P2B glycoproteins may modulate their binding to extracellular matrix glycoproteins.

Topics: Amino Sugars; Collagen; Extracellular Matrix; Glycosylation; Membrane Glycoproteins; Molecular Weight; N-Acetylneuraminic Acid; Neoplasm Metastasis; Sepharose; Sialic Acids; Sialoglycoproteins; Tumor Cells, Cultured

Spontaneous primary mammary tumors of C3H-Avy mice differ in metastatic colonization potential, some producing many lung deposits (high-colonization potential) and others producing few or none (low-colonization potential) after iv inoculation of cells. The degree of metastasis from undisturbed neoplasms also varies from tumor to tumor. This study examined whether these differences between tumors could be accounted for by differences in clonogenic or stem cell content. Tests for clonogenic cells were: growth in 0.3% agarose and limiting dilution assays. Mammary tumor cells of known colonization potential were inoculated iv at serially reduced doses, and the relationship between number of cells injected and number of lung deposits formed was determined. Parallel in vitro dose-response assays in 0.3% agarose were performed with the use of cells from the same primary tumors. Colony-forming efficiency in 0.3% agarose cultures varied between individual primary mammary tumors and was positively associated with experimental metastatic potential, suggesting that the stem or clonogenic cell content of primary tumors is one of the important determinants of the metastatic phenotype.

Topics: Agar; Animals; Colony-Forming Units Assay; Culture Media; Female; Lung Neoplasms; Mammary Neoplasms, Experimental; Mice; Mice, Inbred C3H; Neoplasm Metastasis; Sepharose; Tumor Stem Cell Assay

Twenty-seven specimens for in vitro agarose clonogenicity testing were obtained from 25 patients with small-cell carcinoma of the lung (SCCL). The specimens were obtained from bone marrows, pleural effusions, lymph nodes, and liver biopsies. Colony formation was seen in 14 of 15 specimens that were histologically involved with SCCL, but no colony growth was seen in the 12 patient specimens without histocytopathological evidence of SCCL, including seven bone marrow specimens. Cytological examination of the agarose colonies confirmed their SCCL origin. Colonies reached sizes of 50 to 1000 cells in 7 to 10 days, indicating an in vitro doubling time of less than 24 hr, remarkably shorter than the population doubling times measured in patients. None of the 100 clones picked from these specimens demonstrated the ability to continuously replicate in vitro. These results show an excellent correlation between agarose colony formation and histological tumor involvement and a more rapid in vitro doubling time than that seen in vivo and demonstrate that standard tissue culture conditions do not allow demonstration of a self-renewing stem cell in fresh tumor specimens of SCCL.

Topics: Carcinoma, Small Cell; Cell Division; Cells, Cultured; Clone Cells; Culture Media; Humans; Lung Neoplasms; Neoplasm Metastasis; Sepharose

Topics: Adenocarcinoma; Animals; Binding Sites, Antibody; Carcinoembryonic Antigen; Chromatography, Affinity; Colonic Neoplasms; Goats; Humans; Immunoelectrophoresis; Immunoglobulin G; Iodine Radioisotopes; Liver Neoplasms; Neoplasm Metastasis; Radioimmunoassay; Sepharose

Topics: Adenocarcinoma; Aluminum Hydroxide; Animals; Animals, Newborn; Antibody Formation; Carcinoembryonic Antigen; Colonic Neoplasms; Goats; Horses; Immune Sera; Immune Tolerance; Immunization, Secondary; Immunodiffusion; Immunoelectrophoresis; Liver Neoplasms; Neoplasm Metastasis; Rabbits; Radioimmunoassay; Sepharose