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M S Wicha

Publications and source records attributed to M S Wicha.

At least 37 records · Page 2Linked to original sources
PubMed1989

Production of mammastatin, a tissue-specific growth inhibitor, by normal human mammary cells.

The growth of human mammary cells may be regulated by a balance between growth stimulatory and growth inhibitory pathways. Polypeptides of 47 and 65 kilodaltons (mammastatin) were isolated from conditioned medium of normal human mammary cells. Monoclonal antibodies against mammastatin were generated that blocked its activity and were used for purification and further characterization of the protein. Mammastatin inhibited the growth of 5 transformed human mammary cell lines, but had no effect on the growth of 11 transformed human cell lines derived from nonmammary tissues. Mammastatin appeared to be a heat-labile protein distinct from transforming growth factor-beta (TGF-beta). By immunoperoxidase staining it was detected in cultured normal human mammary cells, but was decreased in transformed mammary cells.

Antibodies, Monoclonal↗
PubMed1989

Laminin promotes the oxidative burst in human neutrophils via increased chemoattractant receptor expression.

The extracellular matrix component, laminin, enhances the chemotactic responsiveness of polymorphonuclear leukocytes (PMN) in vitro, and low doses of chemoattractant substances augment the expression of PMN cell surface receptors for laminin. This study determined whether laminin acts in concert with chemoattractants to activate PMN. Laminin (5 to 100 micrograms/ml) stimulated lysozyme release and superoxide production in response to the chemoattractant, FMLP by as much as 69%. These results could be explained by changes in cell surface chemoattractant receptor expression in that incubation of normal PMN with laminin (5 to 75 micrograms/ml) increased the binding of 19 nM FML[3H]P by 35 to 80%. This corresponded to as much as a 2.5-fold increase in the number of chemoattractant receptors/cells which had a lower average affinity. Laminin did not change the number or affinity of FML[3H]P receptors present on organelle-depleted PMN cytoplasts, and the laminin-induced increase in FML[3H]P receptors expressed on PMN from a patient with a specific granule deficiency was only 11 to 21% of that seen in normal PMN. These findings suggest that chemoattractants augment the expression of laminin receptors which mediate PMN attachment to basement membranes, followed by laminin-induced increases in the expression of cryptic chemoattractant receptors contained in intracellular granules, with resultant augmentation of the oxidative burst.

Adult↗
PubMed1989

Regulation of mammary differentiation by the extracellular matrix.

In multicellular organisms cell growth and differentiation are influenced by soluble factors, cell-cell interactions and cell-extracellular matrix interactions. We have used the rat mammary gland as a model system to study the role of extracellular matrix components in the regulation of milk protein gene expression. Since mammary epithelial cells differentiate on a basement membrane in vivo, we investigated the effects of basement membrane components on the expression of the milk protein genes, alpha-casein, alpha-lactalbumin, and transferrin. We have demonstrated that a basement membrane gel, as well as its major basement membrane component, laminin, induced alpha-casein and alpha-lactalbumin expression as much as 160-fold compared to tissue culture plastic. We demonstrate that laminin affects mRNA stability as well as having an effect on protein stability and secretion. Laminin interacts with mammary epithelial cells via an 68 kD cell surface receptor which is capable of interacting with the cellular cytoskeleton. In order to provide evidence that laminin affects on mammary differentiation are mediated through this receptor via the cytoskeleton, we examined the effects of cytoskeletal disrupting agents on milk protein gene expression. We demonstrate that cytochalasin D or colchicine selectively block laminin-mediated milk protein gene expression by affecting mRNA stability. Based on these experiments, we propose a model in which laminin affects mammary gene expression through interaction with cell surface receptors which interact with the cytoskeleton resulting in stabilization of mRNAs for milk protein genes.

Animals↗
PubMed1988

Role of the cytoskeleton in laminin induced mammary gene expression.

The differentiation of rat mammary epithelial cells is characterized both by morphologic changes and by the expression of a group of milk protein genes. We have previously shown that by culturing these cells on the basement membrane glycoprotein laminin, the synthesis of the milk proteins, transferrin, alpha-casein, and alpha-lactalbumin is induced. In order to determine if this effect is mediated through the cytoskeleton, we have treated these cells with cytochalasin D and colchicine. Treatment with cytochalasin D or colchicine for 24 h inhibits the accumulation of alpha-casein, transferrin, and alpha-lactalbumin without significant effect on general protein synthesis. Pulse chase studies show that cytochalasin D does not alter the intracellular turnover of alpha-casein or transferrin. Additionally, treatment with cytochalasin D causes an early (within 1 h) increase in secretion of alpha-casein and transferrin suggesting that the actin cytoskeleton provides a meshwork for secretory vesicles. The disruption of this network enhances the secretion of preformed proteins. However, long term (24 h) treatment with cytochalasin D inhibits synthesis of these milk proteins. Northern blot analysis indicates that treatment with cytochalasin D or colchicine inhibits the laminin induced increase in alpha-casein, alpha-lactalbumin, and transferrin mRNAs. These studies indicate that the major effect of the cytoskeleton on laminin induced milk protein gene expression occurs at the level of accumulation of mRNAs for these proteins. We conclude that the expression of laminin induced milk protein gene expression in primary rat mammary cultures depends on the integrity of the actin and microtubule cytoskeleton.

Animals↗
PubMed1987

Human neutrophil laminin receptors: activation-dependent receptor expression.

Activated human polymorphonuclear leukocytes (PMN) isolated from peripheral blood specifically bind 125I-laminin after stimulation with phorbol 12-myristate 13-acetate (PMA) or f-Met-Leu-Phe (FMLP) at 37 degrees C. Changes in laminin receptor expression are stimulus dose dependent at both chemotactic (10(-10) M to 10(-6) M) concentrations of FMLP, and secretory (greater than 5 ng/ml) levels of PMA. In the presence of cytochalasin B (5 micrograms/ml), 10(-7) M FMLP activation stimulates specific laminin binding, with an apparent Kd = 3.9 X 10(-9) M and 6.47 X 10(5) binding sites/cell, reaching equilibrium within 10 min at 4 degrees C. This observed activation-dependent change in laminin receptor expression is not due to interference by endogenous laminin, because no fluorescein-visualized anti-laminin antibody bound to cells without added glycoprotein, regardless of the level of activation. Levels of neutrophil lysozyme release, which show a PMA dose dependence similar to that of receptor binding activity, suggest that granule-plasma membrane fusion may be significant during increases in receptor expression. A lack of receptor stimulation by PMA from a granule-deficient patient or in granule-depleted cytoplasts from normal donors additionally supports this hypothesis. Electroblot transfer and autoradiography of subcellular fractions from unstimulated PMN reveals the presence of a 68,000 dalton laminin-binding component in the secondary/tertiary granule (beta) fraction, which may represent an intracellular laminin receptor pool.

Cell Compartmentation↗
PubMed1987

Regulation of rat mammary gene expression by extracellular matrix components.

In the mammary gland the induction and maintenance of differentiation are dependent on both lactogenic hormones and the extracellular matrix (ECM). Since mammary epithelial cells differentiate on a basement membrane in vivo we have examined the effects of basement membrane components on the expression of milk protein genes in primary rat mammary cultures. We examined the effects of a basement membrane gel derived from the Englebreth-Holm-Swarm tumor as well as its major component, laminin, on the expression of a group of milk protein genes. We demonstrate that the basement membrane gel induces alpha-casein and alpha-lactalbumin (alpha-LA) accumulation up to 160- and 70-fold, respectively, of that on tissue culture plastic. Laminin, a major component of the basement membrane, also caused significant induction of these same proteins. In order to determine whether these ECM effects occurred at a translational or post-translational level, pulse-chase experiments were performed. These experiments demonstrated that a laminin substratum selectively effects milk protein turnover and secretion. In order to demonstrate whether ECM effects occurred at the level of steady state accumulation of mRNA we performed dot blot and Northern analyses using cloned cDNA probes for alpha-, beta-, and gamma-caseins and alpha-LA. These studies demonstrated that ECM components induced alpha- and beta-caseins up to 10-fold, and alpha-LA up to 3-fold, with no significant effect on gamma-casein. These results demonstrate that milk protein genes are not coordinately regulated by ECM components. Furthermore, since the amount of induction of milk proteins exceeds the amount of induction of mRNAs for these proteins, we conclude that in our system a major effect of ECM components is at the translational and/or post-translational levels. Based on these findings we propose a model in which basement membrane components effect mammary gene expression at multiple levels.

Animals↗
PubMed1986

Clustering of cell surface laminin enhances its association with the cytoskeleton.

In order to provide evidence for an association of cell surface laminin with the cytoskeleton, we have examined the detergent extractability of cell surface laminin on murine fibrosarcoma cells. We utilized indirect immunofluorescence with affinity-purified anti-laminin antibodies to determine the distribution, mobility and detergent extractability of laminin bound to the cell surface. We demonstrate that antibody induces clustering of cell surface laminin rendering it resistant to detergent extraction. At low receptor occupancy, approx. 80% of cell surface laminin is detergent-extractable. If cell surface laminin is induced to cluster with anti-laminin antibody, IB4 isolectin from Bandeiraea simplicifolia or by high receptor occupancy, then it is rendered resistant to detergent extraction. This process is temperature-sensitive and inhibited by cytochalasin D (CD). On the basis of these findings, we propose a model in which laminin anchored in the basement membrane in vivo affects the cellular cytoskeleton by facilitating the clustering of cell surface transmembrane laminin receptors which are able to interact with cellular actin.

Animals↗
PubMed1986

Macrophages express a plasma membrane receptor for basement membrane laminin.

Laminin, a noncollagenous extracellular matrix glycoprotein, can serve as an attachment factor for epithelial and endothelial cells to basement membrane collagen. The authors have observed that inflammatory macrophages have the capacity to bind exogenous laminin in a time-, temperature- and dose-dependent manner. The laminin binds to these cells via a specific plasma membrane receptor which is detectable on several macrophage subpopulations as well as isolated macrophage membranes. The isolated receptor is a trypsin-sensitive protein with an apparent molecular weight of 70,000 which binds laminin with relatively high affinity, Kd approximately equal to 4 X 10(-9) M. The inflammatory macrophages attach preferentially to a substrate of laminin compared to Type I or Type IV collagen. Thus, the authors postulate that during tissue inflammation macrophages utilize the laminin receptor to bind to vascular basement membranes.

Animals↗
PubMed1985

A potential role for the extracellular matrix glycoprotein laminin in macrophage-tumor-cell interactions.

Although cell surface molecules are thought to be involved in macrophage (MO)-tumor-cell recognition, the nature of these molecules remains unknown. In this study we have shown that the glycoprotein laminin may facilitate macrophage-tumor-cell binding. Macrophage binding to tumor cells was assessed by measuring the adherence of radiolabelled 3-MCA2 induced malignant fibrosarcoma cells to syngeneic peritoneal MOs. Addition of exogenous laminin promoted the binding of a weakly metastatic subline of these tumor cells by 31-68%. These weakly metastatic tumor cells express negligible endogenous cell-surface laminin but display specific cell-surface receptors for binding soluble laminin. Exogenous laminin promoted MO binding of these tumor cells whether it was present during the assay or whether the tumor cells were pretreated with the laminin. This increase in binding was blocked by anti-laminin antibody. In contrast, MO binding of a strongly metastatic variant of the same tumor was not enhanced by the addition of exogenous laminin. This highly malignant fibrosarcoma line already expressed endogenous cell-surface laminin. Since the MOs were found to specifically bind 125I-laminin, the interaction between laminin-bearing tumor cells and MOs may be mediated via a specific MO plasma membrane receptor. Thus, the expression of cell-surface laminin and its receptors on both tumor cells and MOs may provide a mechanism for promoting MO-tumor-cell binding.

Animals↗
PubMed1985

Inhibition of collagen deposition in the extracellular matrix prevents the establishment of a stroma supportive of hematopoiesis in long-term murine bone marrow cultures.

Long-term production of murine hematopoietic cells in vitro is dependent on establishment of a complex microenvironment consisting of a variety of stromal cells and an extensive extracellular matrix which includes collagen, fibronectin, laminin, proteoglycans, and other undefined components adherent to the culture dishes. Cis-4-hydroxyproline (CHP), a relatively specific inhibitor of collagen secretion, was used to examine the role of extracellular collagen deposition in supporting hematopoiesis in long-term C57B1/6J mouse bone marrow cell cultures. Throughout the 10-wk culture period, all culture dishes contained either 0, 10, 25, or 50 micrograms/ml of CHP. All medium and nonadherent cells were removed at weekly intervals and replaced with fresh medium containing the previous concentrations of CHP. Nonadherent cells were assayed weekly for total cells and pluripotent, erythroid, megakaryocytic, and granulocytic-macrophage progenitor cells. Dishes were killed at selected intervals to assess protein and collagen synthesis in the adherent layer. Adherent cell numbers, as judged by microscopic examination and DNA assays, correlated inversely with CHP concentrations used and paralleled degree of collagen synthesis inhibition. The decreased hemopoietic progenitor cell production correlated closely with percent inhibition of collagen synthesis and stromal cellularity. The CHP concentrations tested were not directly toxic to hemopoietic progenitor cells. These studies demonstrate that collagen deposition in the extracellular matrix of murine bone marrow cell cultures is essential to the establishment of a functional stromal microenvironment that is supportive of long-term hematopoiesis.

Animals↗
PubMed1985

Extracellular matrix promotes the growth and differentiation of murine hematopoietic cells in vitro.

We have developed a long-term culture system in which murine marrow cells are cultured on a complex extracellular matrix (ECM) that is derived from marrow and extracted with guanidine hydrochloride and dithiothreitol. Marrow cultures were established with fresh murine marrow cells and recharged at 2 wk (week 0). Phase microscopy showed a dramatically increased adherent cell layer development on ECM compared with controls within a week after recharge. By electron microscopy, this adherent layer was composed of numerous reticular cells apparently attached to the ECM which extended cytoplasmic projections to the surrounding hematopoietic cells. Adherent cellularity on ECM-coated dishes increased to 30 times the control values by week 2. Cumulative suspension cells on ECM dishes were eight times controls. ECM influenced both hematopoietic progenitor cell proliferation and differentiation. Adherent colony-forming unit-granulocyte/macrophage and colony-forming unit-megakaryocyte were greater than 30 and 15 times the control values, respectively, by week 2 (P less than or equal to 0.05). There were more mature granulocytic and megakaryocytic cells in ECM-coated dishes than in controls at all time points. This new culture system directly demonstrates that ECM is an important component of the hematopoietic microenvironment.

Animals↗
PubMed1985

Collagen-production inhibitors evaluated as antitumor agents.

Proline analogues such as cis-4-hydroxy-L-proline (CHP) and L-azetidine-2-carboxylic acid (A2C) were tested for their antitumor activity in tissue culture and in vivo. In culture, CHP specifically inhibited those tumor cells that synthesized basement-membrane collagen. CHP appeared to selectively inhibit collagen biosynthesis with only a slight effect on protein synthesis. Culturing cells on type IV collagen matrix did not alter the antiproliferative effect of CHP. The inhibition of 450.1 mouse mammary tumor cells was fully reversible when cultures were incubated for 6 or 12 hours with 25 micrograms CHP/ml but was irreversible after 24 hours of exposure. Of the proline analogues tested against 450.1 tumor cells, A2C and CHP were the most potent inhibitors of cell growth. These two compounds were therefore tested in vivo using 3 transplantable tumors, all of which synthesized basement-membrane collagen. CHP and A2C were given twice daily to mice for 7 to 10 days at doses ranging from 50 mg/kg (body wt) to 600 mg/kg (body wt) per injection. Both CHP and A2C were completely inactive against the 450.1 mammary tumor and the EHS sarcoma. Both compounds also caused considerable liver toxicity. Against CD8F1 mammary tumors, treatment with maximum tolerated doses of CHP and A2C resulted in a slight but insignificant inhibition of tumor growth. While our studies confirmed previous findings that CHP specifically inhibited those tumor cells that synthesized basement-membrane collagen, CHP and A2C did not appear to be efficacious antitumor agents.

Animals↗
PubMed1984

Metastatic potential of murine fibrosarcoma cells is influenced by cell surface laminin.

In order to examine the role of cell surface laminin in tumor metastasis we have utilized four well-characterized murine fibrosarcoma cell lines. Two of these lines were highly metastatic when injected into syngeneic mice while the remaining two lines were significantly less metastatic. Using indirect immunofluorescence techniques, we detected cell surface laminin on the cell surface of both highly metastatic cell lines but not on the low-metastatic cell lines. Although the low-metastatic cell lines did not possess endogeneous cell surface laminin, they had the ability to specifically bind exogenous laminin to their surface in a time- and concentration-dependent manner, indicating the presence of laminin receptors on these cells. Incubation of the low-metastatic cells with exogenous laminin prior to injection into syngeneic animals significantly increased their metastatic potential. No such increase was observed when the highly metastatic lines were preincubated with exogenous laminin. On the basis of these results, we conclude that in this fibrosarcoma model, metastatic potential is influenced by cell surface laminin and that the presence of unbound laminin receptors on the cell surface is not alone sufficient to promote metastasis of these cells.

Animals↗
PubMed1984

Identification of a laminin-like substance on the surface of high-malignant murine fibrosarcoma cells.

High- and low-malignant murine fibrosarcoma cells were stained with anti-laminin antibodies using immunoperoxidase techniques and examined by electron microscopy. With the high-malignant cells, specific staining was observed along the cell surface. Use of normal rabbit serum in place of the rabbit anti-laminin or pretreatment of the anti-laminin with soluble laminin completely eliminated this staining. No immunoperoxidase staining was observed with the low-malignant cells. In additional studies, membrane fractions were prepared from the high- and low-malignant cells and used to immunize rabbits. The animals immunized with the membrane fractions from the high-malignant cells produced antibodies that reacted by enzyme-linked immunosorbent assay (ELISA) with murine laminin obtained from the EHS sarcoma. The animals immunized with membrane fractions from the low-malignant cells did not. These studies provide strong evidence that the high-malignant cells (but not the low) express on their cell surface a substance that is immunologically cross-reactive with laminin. In addition, the high-malignant cells (but not the low) secreted a material into the cell culture fluid that could be specifically immunoprecipitated with antilaminin antibodies. The immunoprecipitated material co-migrated with purified laminin when examined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis under reducing conditions. The existence of this substance associated with the surface of the high-malignant cells and its absence from that of the low-malignant cells may explain the previously noted difference between these cells in their ability to attach to type IV collagen. This difference may also contribute to the dissimilarity between these cells in their metastatic potential.

Animals↗
PubMed1984

Interaction of rat mammary epithelium with extracellular matrix components.

Primary cultures of rat mammary epithelium synthesize the basement membrane components type IV collagen and laminin and utilize these components for attachment and growth. These cells may interact with matrix components via specific cell surface receptors. However, neither laminin nor type IV collagen alone can sustain mammary differentiation or promote culture longevity in vitro. We have developed a system for the culture of mammary cells on matrices derived from rat mammary glands. This complex matrix promotes cell growth, differentiation, and hormonal responsiveness. Although the mechanisms responsible for these effects are still unknown, one possibility is that cell surface matrix receptors interact with cytoskeletal components leading to alterations in cell shape and hormone receptor configurations. The mammary gland provides an excellent model system to study the interaction of extracellular matrix components with soluble signals, e.g. hormones and growth factors, in the control of cell growth and differentiation. The way these components may interact to influence cell behavior is depicted schematically in Fig. 5.

Animals↗
PubMed1983

Macrophages express cell surface laminin.

Laminin, a non-collagenous extracellular connective tissue glycoprotein, was detected on the surface of mouse peritoneal macrophages. As determined by indirect immunofluorescence, as many as 60% of peritoneal macrophages elicited with thioglycollate expressed cell surface laminin. Only 14% of resident cells displayed detectable laminin. The expression of laminin increased with time post-injection. Concomitant with laminin expression, macrophages also displayed a receptor for the IB4 isolectin from Griffonia simplicifolia. This lectin, which binds methyl-alpha-D-galactopyranoside, may also react with the carbohydrate moeity of laminin. A small population of macrophages displayed both laminin and surface fibronectin. Unlike the difference in laminin expression between resident and thioglycollate-stimulated cells, there was no difference in cell surface fibronectin between these cell populations. Since laminin has been found to mediate cell attachment in other systems, expression of this molecule on the surface of stimulated macrophages may be important in cell-cell and cell-matrix adhesive properties of these cells.

Animals↗
PubMed1983

Connectin: cell surface protein that binds both laminin and actin.

A purified cell surface receptor protein for laminin (Mr = 70,000) isolated from mouse fibrosarcoma cells binds to actin with specificity and high affinity. This binding was demonstrated both by cosedimentation of the receptor with actin and binding of the receptor to actin immobilized on nitrocellulose filters. Specificity was demonstrated by displacement of 35S-labeled receptor by unlabeled receptor. Scatchard analysis of receptor binding to actin yielded a Kd of 6 X 10(-7) M. The receptor was observed to reduce the viscosity of actin filaments. It also caused the formation of bundles of parallel filaments. This observation and the stoichiometry of binding suggest that the receptor binds along the sides of actin filaments. Based on the ability of this receptor to bind both extracellular laminin and intracellular actin, we have named this protein "connectin." Connectin may be an example of a transmembrane protein that is capable of mediating the interaction of a cell with its extracellular matrix.

Actins↗