Differences in surface expression of WGA-binding proteins of cells from a lymphosarcoma and its liver metastases

Abstract

The protein and glycoprotein compositions of a subcutaneous lymphosarcoma (1 degree) and its metastatic deposits in the liver (2 degrees) have been investigated in Triton X-100 extracts obtained from tissue, single cells and membrane preparations. No consistent differences in the electrophoretic patterns for 1 degree and 2 degrees tissue or cells were observed for separations visualized with the protein stain Coomassie Blue. Substantial and consistent reductions in the glycoprotein content of extracts from 2 degrees tissue or cells were observed if the separated proteins were treated with the radioiodinated lectin, Wheat Germ Agglutinin (WGA). Densitometric scans of autoradiographs indicated that WGA binding occurred in 4 major areas; the approximate mol. wts of these were 180,000, 102,000, 84,000 and 23,000 daltons. All these components except the 23,000 component were shown to be located in the cell membrane and to be reduced in 2 degrees preparations. Possible sources of host contamination were also investigated, but these did not show WGA binding patterns that were similar to that obtained for the tumour. If 2 degrees tumour was transplanted into the 1 degree site, the resultant growth exhibited a WGA-binding pattern normally shown by a 1 degree tumour growing in this site. Conversely, if 1 degree tumour was transplanted into the liver, the WGA binding of the resultant growth was substantially reduced. The results suggest that local and metastatic tumours do contain cells that express different glycoproteins on their surfaces but that the site of tumour growth is a very important factor in determining this difference in surface expression. Images Figure 1 Figure 2 Figure 4 Figure 5

Variations in WGA-Binding Proteins in Tumours Growing in Different Implantation Sites

“…The degree of reduction varied with the tumour line and the site of the primary; generally, it was less for the BL6 and LL than for the ML and M5. This finding has been previously reported for the ML [9]. Figure 5 shows the WGA binding to ex tracts of normal muscle, spleen, kidney, liver and lungs.…”

“…After centrifugation at 12,000g, supernatants were stored at -7 0 °C and their protein contents were estimated by the Lowry method. Triton removes mainly membrane glycopro teins from cells [II] and other studies have shown that these are very similar to those obtained from purified membranes [9,12]. It was also found in pilot experiments that centrifugation of extracts at higher speeds than 12,000 g ( 100,000 g for 1 h) did not have a noticeable effect on the protein patterns of the supernatants.…”

“…Tumour cells (10s cells in 3 pi medium 199) were injected into the left portion of the median lobe of the liver, the spleen, the left kidney, the peri toneal cavity, the thorax, the right thigh muscle and subcutaneously. The preparation and implantation of cells, and the assessment of tumour growth and me tastasis were as previously described [9][10][11]. After 14-21 days, primary tumour tissue was excised, transferred to phosphate-buffered saline (PBS A, pH 7.4, Ca-and Mg-free; Flow Laboratories), dissected free of host material, washed extensively in PBS A and either extracted fresh or transferred to -7 0°C until required.…”

“…Prior to electrophoresis, supernatants were di luted 1:2 with Tris-HCl buffer (pH = 8.4, 10 mmol/1) Extracts (60 pg protein per sample) were subjected to slab gel electrophoresis, the gels fixed and stained with Coomassie Blue (CB), and the separated proteins treated with radio-iodinated Concanavalin (Con A) or wheat germ agglutinin (WGA) as previously described [9,14). Bound lectin was visualised by autoradiogra phy.…”

Analyses of protein extracts of human breast cancers: changes in glycoprotein content linked to the malignant phenotype

“…Chan et al (1984) in which glycoproteins extracted by Triton Xl 00 from various animal tumours were found to be virtually identical to those extracted from corresponding isolated membranes.…”