Transformed cells sialyltransferase

Hematoside and Sialyltransferase in Newborn Rat Kidney Cells. Another transformed cell system that we have investigated has shown a positive correlation between expression of the transforming virus and glycolipid content. Newborn rat kidney cells (NRK) transformed by either the Kirsten isolate of murine sarcoma virus (Ki-MSV) or a temperature-sensitive mutant were analyzed for glycolipid composition. The major sialylglycolipid in NRK cells is Gm3 When the cells were grown at the permissive temperature for transformation (31°C), there was substantially less hematoside in both transformed lines than in the control cells (unpublished observation of P. H. Fishman, S. A. Aaron-son, and R. O. Brady). At the nonpermissive temperature (39 ), the cells transformed by the mutant had more hematoside than the control cells. Lactosylceramide CMP-NANA sialyltransferase activity was deter-... 

A. Sialic Acid and Sialyltransferase Activity in Transformed Cells... 

Total sialic acid content was found to be lower in several SV40-transformed clones of Swiss 3T3 mouse cells (Ohta et aL, 1968). The plasma membrane as well as other membrane fractions of similar cell lines had reduced amounts of sialic acid (Wu et aL, 1969). Isolated plasma membranes from Py-BHK cells had 60% less sialic acid than membranes from normal BHK cells (Makita and Seyama, 1971). These changes appear to be associated with reduced sialyltransferase activities in the transformed cells. Grimes (1970) determined the levels of sialic acid and sialyltransferase activity in normal and SV40-transformed Swiss and Balb 3T3 mouse cells. There was a close correlation between sialic acid content and sialyltransferase activity in each cell line even though enzyme levels were measured with exogenous acceptors such as desialylated fetuin or bovine submaxillary mucin. This correlation has been extended to a number of other cell lines (Grimes, 1973). 

Following treatment of the fucose-labeled glycopeptides derived from the surfaces of RSV-BHK cells with neuraminidase, the radioactive products appeared similar to glycopeptides from normal cells (Warren et al., 19726). These results suggested that increased sialic acid content may account for the higher molecular weight of the glycopeptides from transformed cells. When sialyltransferase activity was as-... 

We are only beginning to understand the regulation of the expression of sialyltransferase activity. The higher level of sialylation of many tumor cells and increased sialyltransferase activities are well known (see chapter 3 of Vol. 30 and section 10.5 below]. Transformation of FR3T3 cells with the c-Ha-ras oncogene resulted in a marked increase of the expression of 3-galactoside a-2,6-sialyltransferase activity and. 

At present little detailed evidence is available for sialyltransferases specifically involved in membrane glycoprotein biosynthesis. It is probable that the membrane-bound sialyltransferase specificities described for glycoproteins (sections III. 3 and III.4) and glycolipids (section III. 5) will be similar, as the structures of membrane complex carbohydrates show the same features found in soluble molecules (e.g. Kobata 1979, Rauvala and Finne 1979, Montreuil 1980). Much indirect evidence has come from cell culture studies, frequently in studies on normal and transformed or malignant cells (Hughes 1976, Harmon 1978, Kottgen et al. 1979). 

Table 2. Alteration of sialyltransferase activities in transformed and neoplastic cells...

Hematoside in Hamster Cells. Hakomori and Murakami (1968) reported that the levels of hematoside, the only ganglioside in BHK, was reduced in spontaneous and Py-transformed BHK cells. Similar results were observed with BHK cells transformed by two strains of Rous sarcoma virus (RSV) (Hakomori et aL, 1968). The reduced hematoside content in at least the Py-BHK cells was attributed to decreased activity of lactosylceramide CMP-NANA sialyltransferase, reaction 1 in Figure... 

Den et aL, 1971). Experiments in our own laboratory on normal and Py-BHK cells obtained from Dr. Walter Eckhart of the Salk Institute did not show a difference in the Gms content of these cell lines. The quantity of hematoside and level of sialyltransferase activity were similar in both cell lines and not affected by cell density in culture (Table IV). Further studies in another polyoma transformant as well as two lines transformed by temperature-sensitive mutants of the virus gave similar results. 

Sialyltransferase activities in normal and virally transformed mouse 3T3 cells have also been measured with specific endogenous acceptors (Bosmann, 1972a). The acceptors were prepared by incubating the intact cells with neuraminidase and then trypsin. The desialylated material released from the cells was used to measure sialyltransferase activity in crude detergent extracts of the cells. Cells transformed by MSV, RSV, or Py had more acceptor and transferase activity than normal 3T3 cells. Surface sialyltransferase activity as measured by the ability of intact cells to transfer sialic acid- C from CMP-NANA- C to undefined surface acceptors was also higher in the transformed lines (Bosmann, 1972 ). 

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