Anne P. Sherblom

Cell surface properties of ascites sublines of the 13762 rat mammary adenocarcinoma: Relationship of the major sialoglycoprotein to xenotransplantability

Abstract The relationship between cell surface sialoglycoprotein and xenotransplantation has been investigated in ascites sublines of the 13762 rat mammary adenocarcinoma. Two of the five sublines (MAT-C and MAT-C1) can be transplanted into mice. These two sublines also have the greatest amounts of total, trypsin-releasable and neuraminidase-releasable sialic acid. Chemical labeling using periodate treatment followed by [ 3 H]borohydride reduction indicates that most of the protein-bound sialic acid is associated with a single major sialoglycoprotein (or family of glycoproteins) with a low mobility on polyacrylamide gels in dodecyl sulfate (SDS). This glycoprotein, denoted ASGP-1, is also labeled by lactoperoxidase and 125 I, indicating its presence at the cell surface. Metabolic labeling with [ 3 H]glucosamine shows that ASGP-1 is the major glycosylated protein in both xenotransplantable (MAT-C1) and non-xenotransplantable (MAT-B1) sublines, representing >70% of the protein-bound label in each. The labeling studies indicate that the non-xenotransplantable subline does not have a substantially greater amount of ASGP-1 on its cell surface. Likewise cationized ferritin labeling and transmission electron microscopy (TEM) do not show substantially greater amounts of negatively charged groups distributed along the cell surfaces of MAT-C1 than of MAT-B1 cells. The results indicate that the transplantation differences between these sublines cannot be explained solely by the presence of a major sialoglycoprotein at the cell surface.

Shedding of the major plasma membrane sialoglycoprotein from the surface of 13762 rat ascites mammary adenocarcinoma cells

Abstract ASGP-1 (ascites Sialoglycoprotein 1) the major sialoglycoprotein of 13762 rat ascites mammary adenocarcinoma cells, is shed from MAT-B1 (nonxenotransplantable) and MAT-C1 (xenotransplantable) sublines when incubated in vitro after labeling in vivo with [3H]glucosamine. The rates of shedding of label in both particulate and soluble form are similar for the two sublines, but the turnover of label in the cells is 80% greater for MAT-C1 cells ( t 1 2 2.4 days) than for MAT-B1 cells ( t 1 2 4.1 days). Shed soluble ASGP-1 was smaller than ASGP-1 in the particulate fraction by gel filtration in dodecyl sulfate. By CsCl density gradient centrifugation, gel filtration, and sucrose density gradient centrifugation, all in 4 m guanidine hydrochloride, the shed soluble ASGP-1 was found to be slightly more dense and smaller than ASGP-1 purified from membranes. No differences in sialic acid or oligosaccharides released by alkaline borohydride treatment were found between the shed soluble ASGP-1 and purified ASGP-1. These results suggest that the shed soluble ASGP-1 is released from the membrane by a proteolytic cleavage. This mechanism is supported by the inhibition of the release of soluble shed ASGP-1 by aprotinin, a protease inhibitor. Soluble ASGP-1 in ascites fluid is also smaller by gel filtration, but is more heterogeneous, suggesting a similar release mechanism in vivo followed by more extensive degradation in the ascites fluid.

Sialoglycoprotein differences between xenotransplantable and nonxenotransplantable ascites sublines of the 13762 rat mammary adenocarcinoma

Abstract MAT-B1 and MAT-CI rat ascites mammary adenocarcinoma cells differ in morphology, lectin receptor mobility, and xenotransplantability. Since these properties may be related to cell surface organization, the predominant sialoglycoproteins of these sublines have been investigated by chemical labeling, proteolysis, and alkaline borohydride elimination. Treatment of both sublines with periodate and tritiated borohydride labels one major sialoglycoprotein (ASGP-1) with a low electrophoretic mobility on polyacrylamide gels in dodecyl sulfate. Treatment of labeled or unlabeled cells with trypsin releases about 30% of the total cell sialic acid without significant decrease in cell viability. Gel filtration in pyridine-acetate buffer or in dodecyl sulfate indicates that the released materials are very heterogeneous, and that most of the MAT-C1 sialoglycopeptides are larger than sialoglycopeptides of MAT-B1. Amino acid compositions are quite similar for the released material from the two sublines, but they differ substantially in sialic acid. Further degradation of trypsin-released material with Pronase gives products which are included in a column of mixed Bio-Gel P-10 and P-30 and which also indicate a larger average size for MAT-C1 sialoglyco-peptides. Oligosaccharides from the sialoglycopeptides were obtained by alkaline borohydride treatment of trypsin-released, labeled material and fractionated by chromatography on Bio-Gel P-2. The oligosaccharide(s) comprising the major peak from MAT-C1 cells was larger in size than most of the material from MAT-B1 cells and contained galactosaminitol, galactose, glucosamine, sialic acid, and fucose. These results suggest that MAT-C1 ASGP-1 has more complex oligosaccharides than MAT-B1 ASGP-1, a difference which may play an important role in the differences in cell behavior between the sublines, including transplantability. Regardless of whether the ASGP-1 plays a role in transplantation, investigations of the sialoglycoproteins of these sublines provide a potentially valuable tool for understanding some of the mechanisms by which tumor cells control their cell surface properties.

Changes in expression of a major sialoglycoprotein associated with ascites forms of a mammary adenocarcinoma

Glycoproteins of a cultured form (MR) of the 13762 rat mammary adenocarcinoma and its variants have been studied by analyses for peanut agglutinin receptors, [3H]glucosamine labeling, lactoperoxidase labeling and CsCl density gradient centrifugation. The 13762 MR cells, derived from 13762 MAT-B ascites cells, do not contain detectable ASGP-1, the predominant cell surface sialoglycoprotein of the ascites forms of the 13762 tumor. Transplantation and continued passage as ascites cells of MR cells or clonal lines derived from MR results in abrupt expression of ASGP-1 at about passage 16; it is absent in early passages of the ascites tumor. When these ascites cells are transferred to culture, ASGP-1 is again lost. No ASGP-1 is found in solid tumors derived from subcutaneous transplantation of the 13762 MR cells. The results suggest modulation of ASGP-1 content of the 13762 tumor cells.

Gelation of sarcoma 180 extracts. Membrane involvement and the elimination of the temperature dependence by proteolysis.

Abstract The gelation reaction of 100 000 g supernatants of sarcoma 180 homogenates has been investigated in an attempt to delineate the factors which are important in the reaction. Gel filtration and sucrose density gradient centrifugation indicate that actin-binding protein is in the form of a membrane-bound complex in the 100 000 g supernatants prior to gelation. When the sarcoma 100 000 g supernatants are warmed to room temperature, gelation occurs. Three major proteins are concentrated in the gel: actin, ABP and a component (E) which barely penetrates dodecyl sulfate polyacrylamide gels. Proteolysis of the 100 000 g supernatants enhances the rate of gelation and eliminates the temperature dependence. At 4 °C the enhancement of gelation by protease occurs without substantial cleavage of ABP or actin. Proteolysis does not enhance actin polymerization under the same conditions. The combined results of these experiments suggest that the temperature and proteolysis effects do not occur directly on the gelation reaction, but rather on factors controlling the ABP or actin interactions necessary for gelation.