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Membrane association of the hyaluronate stimulatory factor from LX-1 human lung carcinoma cells (1988)

Knudson, Warren ; Toole, Bryan P.

New York, N.Y. : Wiley-Blackwell

Journal of Cellular Biochemistry 38 (1988), S. 165-177

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ISSN: 0730-2312

Keywords: tumor cell-fibroblast interaction ; tumor invasion ; hyaluronate synthetase ; glycosaminoglycans ; Life and Medical Sciences ; Cell & Developmental Biology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Chemistry and Pharmacology , Medicine

Notes: LX-1 human lung carcinoma cells interact with human fibroblasts in culture to cause an increase in hyaluronate production (Knudson et al: Proceedings of the National Academy of Sciences of the United States of America 81:6767, 1984). It is shown here that a similar increase in hyaluronate production also occurs when membranes derived from LX-1 cells, or detergent extracts thereof, are added to cultures of the human fibroblasts. However, no stimulation occurs when membranes or extracts from fibroblasts are added to cultures of the LX-1 cells. The hyaluronate stimulatory factor present in the detergent extracts is a heat- and trypsin-sensitive protein, requires more than 12 h for its action on fibroblasts, causes an elevation in hyaluronate synthetase activity in membranes derived from the fibroblasts, and can be reconstituted into artificial lipid vesicles. Thus, it is concluded that the stimulatory factor is a membrane-bound protein present on the surface of the LX-1 cells and that it interacts with fibroblasts to induce increased hyaluronate synthesis.

Additional Material: 6 Ill.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1002/jcb.240380304

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Stimulation of glycosaminoglycan production in murine tumors (1984)

Knudson, Warren ; Biswas, Chitra ; Toole, Bryan P.

New York, N.Y. : Wiley-Blackwell

Journal of Cellular Biochemistry 25 (1984), S. 183-196

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ISSN: 0730-2312

Keywords: glycosaminoglycans ; murine tumors ; host-tumor cell interactions ; Life and Medical Sciences ; Cell & Developmental Biology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Chemistry and Pharmacology , Medicine

Notes: Three types of murine tumors, B-16 melanoma, A-10 carcinoma, and S-180 sarcoma, were shown to contain elevated glycosaminoglycan (GAG) concentrations in vivo as compared to normal muscle or subcutaneous tissue. Hyaluronate was especially concentrated in the A-10 carcinoma, which contained approximately six times more hyaluronate than subcutaneous tissue and 18 times more than muscle. In all three tumors, chondroitin sulfates, especially chondroitin-4-sulfate, were present in higher concentrations than in the. normal tissues. In culture, however, all three tumor cell lines produced less than 5% as much GAG as mouse fibroblasts, when measured by incorporation of [3H] acetate or by chemical analysis. Varying the culture passage number or the medium composition, ie, glucose, serum, and insulin concentrations, had little effect on GAG synthesis by the tumor cells. The low GAG levels in the tumor cell cultures were not due to hyaluronidase activity in their media. In an attempt to mimic possible host-tumor cell interactions that could account for the elevated GAG levels in vivo, tumor cells were cocultured with fibroblasts, but no stimulation above the amount made by the tumor cells alone plus that by the fibroblasts alone was observed. Conditioned media from the tumor cells, either dialyzed or not against fresh complete medium, had no effect on fibroblast GAG synthesis. Tumor extracts, however, were found to stimulate synthesis of hyaluronate by fibroblasts. Stimulation by extracts of A-10 carcinoma was greater than and additive to that of serum. The above results strongly suggest that GAG production in these tumors is in pail regulated by host-tumor interactions.

Additional Material: 6 Ill.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1002/jcb.240250402

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Hyaluronate-binding protein of simian virus 40-transformed 3T3 cells: Membrane distribution and reconstitution into lipid vesicles (1987)

Chi-Rosso, Gloria ; Toole, Bryan P.

New York, N.Y. : Wiley-Blackwell

Journal of Cellular Biochemistry 33 (1987), S. 173-183

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ISSN: 0730-2312

Keywords: hyaluronate receptor ; cell-matrix interaction ; Life and Medical Sciences ; Cell & Developmental Biology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Chemistry and Pharmacology , Medicine

Notes: Hyaluronate-binding protein (HASP) has been extracted in detergent from the membranes of simian virus 40-transformed 3T3 (SV-3T3) cells (Underhill et al, J Biol Chem 258:8086-8091, 1983). When SV-3T3 cells were treated with trypsin prior to isolation and dissolution of the membranes, no hyaluronate-binding activity could be detected. This indicates that all of the detectable HABP of SV-3T3 cells is located on the external surface of the plasma membrane rather than on internal membranes, which would be inaccessible to the trypsin. The detergent-extracted HABP from SV-3T3 membranes was reconstituted into the membrane of lipid vesicles, which were formed by addition of exogenous phosphatidylcholine and cholic acid to the extracts followed by removal of detergent by dialysis against 0.02 M Tris pH 8.0 in the presence of protease inhibitors. Reconstitution was assessed by sedimentation in a discontinuous sucrose gradient and by gel filtration on Sepharose 4B in the presence and absence of detergent. The characteristics of binding of hyaluronate to the reconstituted HABP were then compared with those studied previously for the original membrane-bound HABP and the detergent-extracted HABP (Underhill et al, J Biol Chem 258:8086-8091, 1983). It was observed previously that binding of hyaluronate to HABP in the cell membranes was of higher affinity and specificity than to HABP in the detergent extracts of these membranes. It was found here that reconstitution of the extracted HABP into the membranes of lipid vesicles led to restoration of affinity of binding to the level observed in the original cell membranes. However, whereas chondroitin sulfate does not compete significantly for binding of hyaluronate to cell membrane-bound HABP, partial competition was observed for the reconstituted HABP as well as for detergent-extracted HABP. Thus, it is concluded that the high affinity of binding of hyaluronate to the plasma membrane of SV-3T3 cells is in part dependent on insertion of the HABP in the membrane, but that other interactions, not duplicated in our reconstitution experiments, must be necessary for the specificity of the HABP.

Additional Material: 5 Ill.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1002/jcb.240330304

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Transformation-dependent loss of the hyaluronate-containing coats of cultured cells (1982)

Underhill, Charles B. ; Toole, Bryan P.

New York, NY [u.a.] : Wiley-Blackwell

Journal of Cellular Physiology 110 (1982), S. 123-128

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ISSN: 0021-9541

Keywords: Life and Medical Sciences ; Cell & Developmental Biology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Medicine

Notes: The sizes of hyaluronate-containing coats on the surfaces of parent and virus-transformed cell lines (3T3 vs. SV-3T3; BHK vs. PY-BHK) were compared according to the method of Clarris and Fraser (1968, Exp. Cell Res., 49: 181-193) in which fixed red blood cells were allowed to settle slowly on the surface of culture dishes containing the cells. The coats were seen as areas devoid of red blood cells surrounding each of the cultured cells and could be destroyed by the addition of small amounts of streptomyces hyaluronidase, an enzyme specific for hyaluronate. In the case of the parent cell lines (3T3 and BHK), the coats were clearly visible, whereas for their virus-transformed counterparts (SV-3T3 and PY-BHK), the coats were either greatly reduced or absent. To confirm these observations, the amount of hyaluronate associated with each of the cell lines was measured using a direct chemical assay and shown to be significantly greater for the parent cell lines than for their virus-transformed counterparts. In addition, the parent cell lines secreted greater amounts of hyaluronate into the medium and retained a larger fraction of the total amount of hyaluronate at the cell surface than the virus-transformed cells. Thus the larger amount of hyaluronate on the surfaces of the parent cell types may be the result of both a faster rate of production and a decreased rate of release.

Additional Material: 1 Ill.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1002/jcp.1041100204

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Membrane-associated hyaluronate-binding activity of chondrosarcoma chondrocytes (1989)

McCarthy, Mary T. ; Toole, Bryan P.

New York, NY [u.a.] : Wiley-Blackwell

Journal of Cellular Physiology 141 (1989), S. 191-202

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ISSN: 0021-9541

Keywords: Life and Medical Sciences ; Cell & Developmental Biology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Medicine

Notes: The association of hyaluronate with the surface of chondrocytes was examined by several approaches using primary cultures of chondrocytes derived from the Swarm rat chondrosarcoma. In culture, chondrosarcoma chondrocytes produced large pericellular coats, which can be visualized by particle exclusion, and which can be removed by Streptomyceshyaluronidase. Exposure of chondrocytes, which had been metabolically labelled with 3H-acetate, to exogenous hyaluro-nate or to Streptomyceshyaluronidase resulted in the release of 36-38% of the endogenous, labelled chondroitin sulfate from the cell layer into the incubation solution. These results imply that at least 37% of the cell layer chondroitin sulfate proteoglycan is retained there by an interaction with hyaluronate. Thus membranes were prepared from cultured chondrocytes and examined for sites which bind3H-hyaluronate. Binding was observed and found to be saturable, specific for hyaluronate, of high affinity (Kd =∼10-10M),and destroyed by treating the membranes with trypsin. The 3 H-hyaluronate-binding activity was inhibited competitively by hyaluronate decasaccharides but not by hexasaccharides or octasaccharides, indicating that the binding sites recognize a sequence of hyalu-ronate composed of five disaccharide repeats. The binding activity was partially purified from a detergent extract of chondrocyte membranes by ion exchange chromatography on DEAE-cellulose, followed by affinity chromatography on wheat germ agglutinin-agarose. Analysis of the partially purified binding activity by SDS-PAGE revealed five protein bands of 48,000-66,000 daltons in silver-stained gels. SDS-PAGE followed by Western blotting and exposure to mono-clonal antibodies which recognize epitopes present in link protein and in the hyaluronate-binding region of cartilage proteoglycan revealed no immunoreac-tive protein bands in the partially purified material. We conclude that one mechanism by which hyaluronate associates with the chondrocyte surface may be via interaction with a membrane-bound hyaluronate-binding protein which is distinct from link protein and proteoglycan.

Additional Material: 11 Ill.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1002/jcp.1041410127

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