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Biomedical subjects

C N Rao

Publications and source records attributed to C N Rao.

At least 55 records · Page 3Linked to original sources
PubMed1986

Laminin increases the release of type IV collagenase from malignant cells.

We have studied the effect of laminin on type IV collagenolytic activity elaborated by malignant cells in culture. Laminin (at concentrations of 4-8 micrograms/ml) added to serum-free culture supernatants of subconfluent A2058 human melanoma cells significantly increased the release of the type IV collagenolytic activity (200-300%). The induction of type IV collagenase was more pronounced (580%) using a fragment of laminin which binds to the cell surface laminin receptor. A monoclonal antibody against the human laminin receptor blocked the effect of laminin on type IV collagenase, suggesting that occupation of the laminin receptor may be necessary for the effect. Increase in the type IV collagenolytic activity mediated by laminin was also demonstrated in two other malignant cell lines, HT fibrosarcoma (168%) and mouse melanoma (B16-F10) (271%). The increase in type IV collagenase was found to be specific for laminin because another cell-binding matrix protein, fibronectin, did not have any effect, and epidermal growth factor and transferrin actually decreased the type IV collagenase in human melanoma culture medium (epidermal growth factor, 50% at 20 ng/ml; and transferrin, 20% at 10 micrograms/ml). These studies suggest that tumor cell binding to laminin, which comprises the first step of basement membrane invasion, will induce the second step, namely the collagenolytic dissolution of the basement membrane.

Animals↗
PubMed1986

Altered levels of laminin receptor mRNA in various human carcinoma cells that have different abilities to bind laminin.

The human laminin receptor was purified and molecularly cloned to investigate its biosynthetic regulation. Laminin receptor from normal and neoplastic tissue was preparatively affinity purified to homogeneity based on the high affinity of the receptor for laminin. The apparent molecular weight of the receptor from different carcinoma sources and from normal placental tissue is in the range of 68-72 kDa. Isoelectric focusing and two-dimensional gel electrophoresis indicated that the receptor protein consists of one major polypeptide chain with a pI value of 6.4 +/- 0.2. Laminin receptor cDNA clones were isolated after screening a human endothelial lambda gt11 cDNA library with a monoclonal antibody directed against a domain of the laminin receptor involved in ligand binding. Definitive identification of the cDNA clones was based on comparison of cDNA sequence with the amino acid sequence of a cyanogen bromide-generated octapeptide of purified placental laminin receptor. The laminin receptor mRNA is approximately 1700 bases long. The level of laminin receptor mRNA in a variety of human carcinoma-derived cell lines correlated with the number of laminin receptors on the cell surfaces of those cells. This suggests that the amount of laminin receptor mRNA may be a rate-limiting control step in the biosynthesis of the laminin receptor, and hence in the regulation of cellular attachment to basement membranes via laminin.

Amino Acid Sequence↗
PubMed1986

Butyrate induced reduction of tumor cell laminin receptors.

Laminin, a glycoprotein of basement membranes, binds to the surface of cultured human pancreatic carcinoma cells (PANC-1). The binding is saturable, with a high proportion of specific binding, as determined by competition of labeled ligand with 80-fold excess unlabeled ligand. Pretreatment of the carcinoma cells with butyrate markedly reduces the amount of specific laminin binding. The butyrate effect on laminin binding is dose dependent and is observed at a concentration of butyrate which does not significantly reduce cellular protein synthesis or increase laminin production. Scatchard analysis indicates that butyrate reduces the total number of laminin binding sites without affecting the binding coefficient (Kd = 2 nm). The laminin receptor protein isolated from the PANC-1 cell extract has a molecular weight of approximately 70,000. After butyrate treatment the total amount of extractable laminin receptor protein is significantly reduced. Thus butyrate reduces laminin binding to PANC-1 carcinoma cells by a mechanism which involves decreased expression of laminin receptor proteins in the plasma membrane.

Adenocarcinoma↗
PubMed1985

Binding domain for laminin on type IV collagen.

Binding of type IV collagen to laminin was studied by attaching one member of the ligand pair to a solid phase. When laminin was bound to a solid phase, type IV collagen exhibited saturable binding. Digestion of type IV collagen with high concentrations of pepsin destroyed the laminin binding activity. Type IV collagen was also found to bind to fibronectin but the binding activity was not destroyed by pepsin treatment. Rotary shadowing electron microscopy of the pepsin digested type IV collagen indicated that the carboxy terminal end region of about 100 nm is cleaved. Rotary shadowing electron microscopy studies demonstrate that the carboxy terminal end of type IV collagen has a major laminin binding site.

Animals↗
PubMed1985

Monoclonal antibodies to the human laminin receptor recognize structurally distinct sites.

Laminin, a glycoprotein of basement membranes, binds to a specific receptor on the surface of neoplastic and non-neoplastic cells. The laminin receptor purified from human breast carcinoma plasma membranes was used as an antigen to generate two types of monoclonal antibodies (mAbs). Both types of mAbs bind to (a) the purified receptor coated on a solid phase; (b) isolated breast carcinoma plasma membranes; and (c) the surface of cultured MCF-7 human breast carcinoma cells by immunohistology. Using immunoblotting, both types of mAbs recognize a single 67 000 Dalton protein among all the proteins extracted from breast carcinoma plasma membranes. The mAbs differed in their ability to block binding of laminin to the plasma membrane receptor. Antibody LR1 inhibited virtually 100% of the specific binding of laminin to both the isolated human breast carcinoma plasma membranes or the living MCF-7 cells. In contrast, antibody LR2 had no effect on laminin binding under identical conditions. Thus, the two types of mAbs may recognize structurally distinct sites on the laminin receptor. These mAbs should be useful to dissect the biology and the molecular genetics of the laminin receptor.

Antibodies, Monoclonal↗
PubMed1985

Laminin binds specifically to sulfated glycolipids.

Previous studies of the agglutination of erythrocytes by the basement membrane glycoprotein laminin have suggested that laminin binds to gangliosides [Kennedy, D.W., Rohrbach, D.H., Martin, G.R., Momoi, T. & Yamada, K.M. (1983) J. Cell. Physiol. 114, 257-262]. Based on the following evidence, however, we find that laminin binds specifically to sulfatides, not gangliosides. Monogalactosyl sulfatides, purified from sheep erythrocytes with a yield of 4.3 mg/kg of packed cells, bound laminin with high affinity as did authentic bovine brain sulfatide (galactosylceramide-I3-sulfate). The binding activity of these lipids and of total erythrocyte lipids was stable to alkali and neuraminidase treatment but labile to dilute acid under conditions that destroy sulfatides but not gangliosides. Of various glycolipid and phospholipid standards tested, only sulfatides bound laminin with high affinity. Sulfatide binding and agglutinating activities of proteolytic fragments of laminin indicated that the globular end regions of the 200-kDa subunits are required for both activities. Thus, monogalactosylsulfatides, and possibly other more complex sulfated glycolipids, are probably involved in the agglutination of erythrocytes. These results also suggest a physiological function of sulfatides in cell adhesion. The agglutination of erythrocytes by fibronectin is also inhibited by gangliosides [Yamada, K.M., Kennedy, D.W., Grotendorst, G.R. & Momoi, T. (1981) J. Cell. Physiol. 109, 343-351]. Fibronectin, however, did not bind to sulfatides with high affinity but rather bound with low affinity to all anionic lipids tested, including phospholipids, gangliosides, and sulfatides.

Animals↗
PubMed1985

Expression of laminin receptor in normal and carcinomatous human tissues as defined by a monoclonal antibody.

It has been hypothesized that epithelial and endothelial cells interact with the laminin component of basement membranes via a cell surface laminin receptor molecule. It has also been proposed that the expression of this molecule may be involved in the invasion of carcinoma cells from their tissue of origin and their subsequent penetration through blood vessel basement membranes. We report here the use of a monoclonal antibody, LR-3, to define the expression of laminin receptor in normal, dysplastic, and carcinomatous human tissues. Monoclonal antibody LR-3 is shown by immunoblotting to recognize the Mr 67,000 laminin receptor protein, to bind to the carcinoma cells, and to constitute approximately 0.1% of total cellular protein. Numerous normal human epithelial and endothelial cell types, as well as pulmonary macrophages, are shown to express laminin receptor to varying degrees. Selected human mammary carcinomas and colon carcinomas are shown to bind more monoclonal antibody LR-3 than normal or dysplastic counterparts. A monoclonal antibody to laminin receptor now makes possible the study of the role of laminin receptor in tumor cell metastases and in the differentiation and function of various normal human epithelial and endothelial cell types.

Animals↗
PubMed1984

Characterization of a laminin receptor from human breast carcinoma tissue.

Human breast carcinoma contains high-affinity receptors for the basement membrane glycoprotein laminin. Plasma membranes isolated from tissue samples of human breast carcinoma exhibit specific, saturable, reversible, and displaceable binding to laminin. Fibronectin, collagen, and serum proteins fail to displace this bound laminin. Scatchard analysis is linear with a Kd of 1.8 X 10(-9) M. The laminin receptor, isolated and purified 1200-fold by laminin-affinity chromatography, exhibits a molecular weight of 67 000 daltons as determined by gel electrophoresis. The receptor was verified to be located on the cell surface of the invading breast carcinoma cells by immunohistologic studies utilizing a peroxidase-conjugated fragment of the laminin molecule which contained the receptor-binding domain.

Binding, Competitive↗
PubMed1984

Laminin molecular domains which alter metastasis in a murine model.

Human and murine tumor cells contain cell surface receptors for the basement membrane glycoprotein laminin. Since a biologic role for the receptor had not previously been demonstrated, we explored the possibility that the laminin receptor may be involved in hematogenous metastases formation. Preincubation of metastatic murine melanoma cells with syngeneic whole laminin followed by tail vein injection increased tumor cell retention in the lung and strongly stimulated metastases formation. The domain of the laminin molecule responsible for stimulating metastases was identified. Laminin is a cross-shaped molecule with three short arms and one long arm. All arms have globular end regions. Purified protease-derived fragments of laminin were prepared which (a) lacked only the long arm of the molecule (alpha fragment) or, (b) lacked both the long arm and the globular end regions of the short arms (C1 fragment). Both types of fragments contained the laminin receptor binding region. The fragments had opposite effects on metastases. The alpha fragment stimulated metastases formation to the same extent as whole laminin. In contrast, the C1 fragment greatly reduced or abolished metastases formation in a dose-dependent manner. The C1 fragment also inhibited tumor cell attachment to whole amnion basement membrane in vitro. We conclude that intact globular end regions on the short arms (but not the long arm) of the cell surface receptor-bound laminin molecule are necessary for stimulating metastases by the intravenous route.

Animals↗
PubMed1983

Interactions of basement membrane components.

The binding of laminin, type IV collagen, and heparan sulfate proteoglycan to each other was assessed. Laminin binds preferentially to native type IV (basement membrane) collagen over other collagens. A fragment of laminin (Mr 600 000) containing the three short chains (Mr 200 000) but lacking the long chain (Mr 400 000) showed the same affinity for type IV collagen as the intact protein. The heparan sulfate proteoglycan binds well to laminin and to type IV collagen. These studies show that laminin, type IV collagen and heparan sulfate proteoglycan interact with each other. Such interactions in situ may determine the structure of basement membranes.

Animals↗
PubMed1983

Lectin-binding domains on laminin.

The nature and location of carbohydrate moieties on the laminin molecule were identified by studying the binding affinity of a series of lectins for purified, protease-derived fragments of laminin. Laminin is a cross-shaped molecule containing three short arms (36 nm) and one long arm (76 nm). All arms contain globular end regions by electron microscopy. Purified fragments of laminin were obtained which (a) lacked the long arm of the molecule but retained the intact short arms, or (b) lacked both the long arm and the globular end regions of the short arms. These two types of fragments differed markedly in lectin-binding capacity. Using the known sugar specificities of the lectins and hapten sugar competition for lectin-binding to laminin fragments, the following conclusions were reached: (a) alpha-D-Galactopyranosyl end groups are markedly enriched in the globular end regions of the short arms compared to the rod-shaped portions of the molecule. (b) alpha-D-Mannopyranosyl residues are present on both the globular end regions and the rod-shaped portions of the molecule. (c) Exposed N-acetyl-D-galactosaminyl end groups are absent or present in low amounts on laminin. (d) (NANA)-(2 leads to 6)-beta-D-Gal-(1 leads to 4)-beta-D-GlcNAc-(1 leads to 2)-D-Man-terminated oligosaccharide units are enriched on the rod-shaped regions of the short arms compared to the globular end regions.

Animals↗
PubMed1983

Laminin receptor on human breast carcinoma cells.

Human MCF-7 breast carcinoma cells possess a receptor-like moiety on their surface that has a high binding affinity (Kd = 2 nM) for laminin, a glycoprotein localized in basement membranes. Laminin preferentially stimulates (8-fold) MCF-7 cells to attach to type IV (basement membrane) collagen, whereas fibronectin stimulates attachment only 2-fold for these cells on type I collagen. The attachment properties of two other human breast carcinoma cell lines to type IV collagen were also studied. The attachment of ZR-75-1 cells was stimulated 4-fold by laminin and 5-fold by fibronectin, whereas T47-D cell attachment was stimulated 2-fold by laminin and 7-fold by fibronectin. By employing protease-derived fragments of laminin, the major domains of the laminin molecule that participate in MCF-7 cell attachment to type IV collagen were identified. The whole laminin molecule has the configuration of a four-armed cross with three short arms and one long arm. A major cell-binding domain was found to reside near the intersection point of the short arms, and the type IV collagen-binding domain was associated with the globular end regions of the short arms. The receptor for laminin on the surface of these tumor cells may be involved in the initial interaction of tumor cells via laminin with the vascular basement membrane to facilitate invasion and subsequent promotion of metastasis.

Breast Neoplasms↗
PubMed1983

Bioflavonoid-mediated stabilization of collagen in adjuvant-induced arthritis.

In rats with adjuvant-induced arthritis, the effect of (+)-catechin (CA) and 0-(beta-hydroxyethyl) rutosides (HR) on the crosslinking of collagen was studied. Compared with controls the arthritic group showed an increase in the reversibility of neutral salt-soluble collagen gel, solubility of acid-insoluble collagen to denaturing agents and the ratio of alpha/beta subunits of neutral salt-soluble collagen. These results suggest an impaired maturation of collagen in arthritic animals. Administration of CA or HR to the arthritic animals caused a decrease in the reversibility of collagen gel and in the solubility of acid-insoluble collagen to denaturing agents and to pronase. In addition, the electrophoretic patterns of neutral salt-soluble collagen on SDS polyacrylamide gels also showed a decrease in the alpha/beta ratio. All these results may collectively indicate that both flavonoids promote the crosslinking of collagen in arthritic animals.

Animals↗