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M S Wicha

Publications and source records attributed to M S Wicha.

50 records · Page 3Linked to original sources
PubMed1983

Isolation of a cell surface receptor protein for laminin from murine fibrosarcoma cells.

We used affinity chromatography to isolate a specific laminin-binding protein from murine fibrosarcoma cells. These cells bind exogenous laminin to their surface with high affinity (Kd = 2 X 10(-9)M for laminin) with approximately 5 X 10(4) sites per cell. Laminin affinity chromatography of [35S]methionine-labeled cell extracts produced two distinct proteins. One was identified as Type IV (basement membrane) collagen based on its migration pattern on SDS gels and bacterial collagenase sensitivity. The other protein, which migrates as a single band or closely spaced doublet on reduced SDS gels, has a reduced molecular weight of 69,000. Using a nitrocellulose filter disk assay, we found that the latter protein specifically bound 125I-laminin with the same high affinity (Kd = 2 X 10(-9)M for laminin) as did intact fibrosarcoma cells. By iodinating intact cells, we demonstrated that this laminin-binding protein is on the cell surface. We conclude that this protein with reduced molecular weight of 69,000 is a subunit or component of a larger cell surface receptor protein for laminin in this fibrosarcoma model. This laminin receptor may mediate the interaction of the cell with its extracellular matrix.

Animals↗
PubMed1983

Extracellular matrix production by the adherent cells of long-term murine bone marrow cultures.

We have studied the deposition of extracellular matrix proteins in the adherent stroma of long-term murine bone marrow cultures. Stable hematopoiesis was maintained for greater than 12 wk. At selected intervals, culture dishes were sacrificed by removing all nonadherent cells and air drying the dishes. The adherent stromal layer was analyzed for the presence of intracellular and extracellular collagen, fibronectin, and laminin using double immunofluorescent staining with specific antisera against these matrix components. In cultures examined during the first 2 wk, large numbers of stromal cells contained collagen, fibronectin, and laminin. Over the next 2 wk, an extensive extracellular network of fibronectin, laminin, and collagen was deposited on the dishes, which persisted throughout the life of the cultures. In contrast to a previous report, we detected substantial numbers of endothelial cells by means of immunofluorescent staining of stromal cells with antisera to type IV collagen, laminin, and factor VIII antigen. Although deposition of these extracellular matrix proteins coincides with onset of active hematopoietic cell production, the relative roles of the stromal cells and the extracellular matrix in supporting hematopoiesis in murine bone marrow cell cultures remain to be determined.

Animals↗
PubMed1982

Extracellular matrix promotes mammary epithelial growth and differentiation in vitro.

The study of growth and differentiation of mammary epithelium has been hampered by the difficulty of maintaining these functions in vitro. We describe a system for the primary culture of rat mammary epithelium on an acellular matrix derived from whole rat mammary glands that maintains growth and differentiation for months. Cultures plated on this complex substratum produce 50 times the alpha-lactalbumin of those on tissue culture dishes and 5 times the alpha-lactalbumin of those on floating collagen gels as determined by radioimmunoassay. Unlike cultures grown on floating collagen gels, which rapidly lose the ability to secrete the milk sugar lactose, mammary cells on this matrix retain this ability for over 30 days in culture. The organ specificity of this mammary extracellular material is shown by the failure of extracellular matrix prepared from rat liver to support mammary differentiation. Within a given culture dish, cells on the surface of mammary extracellular matrix are more differentiated than those on the adjacent plastic. This is demonstrated by their increased alpha-lactalbumin content as shown by indirect immunofluorescence, and by their increased ability to bind fluorescein-conjugated peanut lectin. Cells on the surface of the matrix continue to synthesize DNA as determined by [3H]thymidine incorporation and autoradiography. Even when mammary epithelial cells are plated at low density, cell division continues until the matrix is covered with a confluent layer. We propose that the limited growth, differentiation, and survival of mammary cells in previously described in vitro systems may have been due to substrate that were inadequate to support these functions.

Animals↗
PubMed1981

Blocking basement membrane collagen deposition inhibits the growth of 7,12-dimethylbenzanthracene-induced rat mammary tumors.

7,12-Dimethylbenzanthracene-induced rat mammary tumors contain basement membrane collagen in vivo and in vitro in primary culture, as determined by immunofluorescence. The proline analogue dis-hydroxyproline, which selectively blocks collagen accumulation, inhibits tumor cell growth in vitro. In vivo cis-hydroxyproline blocks tumor growth and produces epithelial cell degeneration and tumor necrosis at a non-toxic dose. This compound has no effect on the growth of the mammary carcinoma cell line 64/24, which makes no basement membrane collagen. As we have previously described for the normal proliferating rat mammary gland, basement membrane collagen deposition may be necessary for the growth of some rat mammary tumors.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1981

Sensitivity of N-nitrosomethylurea-induced rat mammary tumors to cis-hydroxyproline, an inhibitor of collagen production.

The growth of primary N-nitrosomethylurea-induced rat mammary tumors was depressed by cis-hydroxyproline (CHP). This growth arrest appeared to be related to the ability of CHP to inhibit the deposition of basement membrane collagen as based on the following observations: (a) in vitro and in vivo, tumor cells synthesized type IV collagen, the collagen uniquely localized in basement membranes; (b) in vitro, the inhibition of tumor cell growth was preceded by a specific decrease in collagen accumulation with no effect on non-collagen protein synthesis; (c) a transplantable N-nitrosomethylurea-induced rat mammary tumor accumulated no type IV collagen as determined by polyacrylamide gel electrophoresis and indirect immunofluorescence. The growth of this tumor was not influenced by CHP; (d) an established human mammary tumor cell line, MCF-7, did not accumulate type IV collagen and was not inhibited by CHP. At the doses which effectively blocked the growth of primary N-nitrosomethylurea-induced mammary tumors, CHP and no toxic effects, and serum prolactin levels were not altered. The inhibitory effect was thus apparently due to the direct action of CHP upon the accumulation of collagen in cells which required type IV collagen production for continued growth.

Animals↗
PubMed1979

Hormonal requirements for basement membrane collagen deposition by cultured rat mammary epithelium.

Alveoli and ducts isolated from virgin rat mammary glands synthesize basement membrane collagen (typeIV) in primary culture. Using purified antibodies to type IV collagen, prominent intracellular and extracellular fluorescence is observed in the epithelium. No fluorescence is observed with antibodies to collagen type I and III. From quantitation of the incorporation of [14c]proline-labeled proteins, 1.5 to 2.5 per cent of the newly synthesized proteins are collagen. Type IV collagen from these cultures was biochemically identified on the basis of (1) the high ratio of labeled 3-hydroxyproline to 4-hydroxyproline (1:10), (2) the gel electrophoretic pattern of the collagenase-sensitive proteins precipitated with 1.7 M NaCl, (3)the failure of the collagen to bind to diethylaminoethyl-cellulose, and(4)the immunologic cross-reactivity with mouse tumor type IV is identical with that of type IV collagen from other sources. When the supportive hormones, insulin, prolactin, hydrocortisone, progesterone, and estradiol are removed from the cultures, there is a 90 per cent reduction in the amount of [3H]proline recovered in collagen synthesis coincides with only a 30 percentdrop in the growht rate and a 20 per cent drop in total protein synthesis of the sells over the 24-hour period without hormones. Pulse-chase experimout hormones. Pulse-chase experiments revealed an enhanced turnover of collagen following hormone withdrawal. This system may be an in vitro model of collagen turnover in mammary gland in involution.

Animals↗
PubMed1978

Unsaturated fatty acid requirements for growth and survival of a rat mammary tumor cell line.

A cell line, the growth and survival of which is markedly affected by linoleic acid, has been established from a carcinogen-induced rat mammary tumor. The cells have been continuously passaged in 5% rat serum plus 10% fetal calf serum-supplemented medium. The rat serum component was found to be indispensalbe, for when it was omitted the growth rate rapidly declined and the cells died by 5 to 7 days. Removal of the rat serum from the growth medium also resulted in a dramatic loss of Oil Red O-positive droplets in the cells, suggesting that the lipid component of rat serum might be a major growth-promoting principle in rat serum. This is likely since the total lipid fraction, but not the delipidized protein fraction, could largely supplant requirement of the cells for rat serum. Pure linoleic acid was found to be effective in maintaining the cell growth in delipidized serum or in whole fetal calf serum-supplemented medium. Fatty acid analysis revealed a 19-fold higher amount of linoleic acid in rat serum than in fetal calf serum.

9,10-Dimethyl-1,2-benzanthracene↗
PubMedDate not supplied

A bcl-x(S) adenovirus demonstrates therapeutic efficacy in an ascites model of human breast cancer.

OBJECTIVES: To determine whether a Bcl-x(S) adenoviral vector has therapeutic potential in an ascites model of human breast cancer in nude mice. METHODS: Advanced ascites were developed by injecting mice intraperitoneally (IP) with MDA-MB-231 human breast carcinoma cells. Mice received sequential IP injections of the Bcl-x(S) virus or a control lac-Z adenovirus. A third group of mice received no virus. Tumor burden and survival were monitored. Histopathology and necropsies were performed on mice. RESULTS: A single injection of the Bcl-x(S) adenovirus produced no systemic or local toxicity and no abnormal histopathology in normal mice. However, abdominal organs within these mice were transduced with the Bcl-x(S) vector. Adenoviral gene transduction efficiency in MDA-MB-231 ascites was 36+/-6.40% (n = 3). Percent weight change differences revealed that ascites bearing mice injected three times with the Bcl-x(S) vector showed a statistically significant decrease in tumor burden compared with lac-Z-injected mice (n = 7; P = .012 on days 10-15 after the first injection). Mice injected with the Bcl-x(S) vector had significantly greater survival relative to lac-Z-injected mice (n = 7; P = .0004). Bcl-x(S) protein expression was detected in aspirates of mice injected with the Bcl-x(S) vector but not the lac-Z vector. Necropsies revealed that ascites bearing mice injected with Bcl-x(S) vector lacked carcinoma in the peritoneal cavity compared with control mice. CONCLUSION: The Bcl-x(S) adenovirus can reduce tumor burden and increase survival in an ascites model of advanced stage breast cancer.

Adenoviridae↗
PubMedDate not supplied

Haemonectin, a bone marrow adhesion protein specific for cells of granulocyte lineage.

There is substantial evidence that the haematopoietic microenvironment is crucial to the growth and differentiation of haematopoietic cells. This microenvironment is composed of stromal cells, soluble factors and extracellular matrix (ECM). We have shown that a complex extract of bone marrow ECM can stimulate the growth and differentiation of haematopoietic cells in vitro. Furthermore, the use of inhibitors or stimulators of ECM synthesis in long-term marrow culture affects cell proliferation. On a molecular level, however, the interactions between ECM and haematopoietic cells are not well understood. We have investigated the adhesion between specific bone marrow ECM components and haematopoietic cells, and found a protein, 'haemonectin', of relative molecular mass 60,000 in bone marrow ECM which is a lineage- and organ-specific attachment molecule for cells of granulocyte lineage. This specificity distinguishes haemonectin from previously described adhesion proteins which have a wider tissue distribution and cell type specificity.

Antigens, Surface↗
PubMedDate not supplied

A bcl-xS adenovirus selectively induces apoptosis in transformed cells compared to normal mammary cells.

Oncogenes which drive the cell cycle, such as c-myc, can sensitize cells to apoptosis. This suggests the possibility that the expression of genes such as bcl-2 or bcl-xL is required to inhibit apoptosis induced by oncogene expression. We hypothesized that inhibition of Bcl-2/Bcl-xL by the pro-apoptotic Bcl-xS protein, would result in selective induction of apoptosis in mammary carcinoma cells compared to their nontransformed counterparts. Therefore, we compared the effects of Bcl-xS expression delivered by a bcl-xS adenovirus (bcl-xS-Adv) vector, on viability and apoptosis of nontransformed versus transformed mammary epithelial cells. We report that c-myc-transformed murine mammary cells are extremely sensitive to apoptosis induced by the bcl-xS adenovirus (bcl-xS-Adv) vector, whereas immortalized, nontransformed murine mammary cells are relatively resistant to apoptosis induced by this vector. Likewise, human mammary epithelial cells transduced with c-erbB-2 were more sensitive to apoptosis induced by the bcl-xS vector than the nontransformed parental cells. Similar results were obtained when we tested the effects of bcl-xS adenoviral infection on primary normal human mammary epithelial cells and SUM-190 PT cells, (a c-erbB-2 over-expressing human mammary carcinoma cell line) grown on Matrigel. These data are consistent with the hypothesis that inhibition of Bcl-2/Bcl-xL can result in selective killing of cancer cells compared to their nontransformed counterparts.

Adenoviridae↗