Neuraminidase activity and cell surface sialic acid turnover in Rous sarcoma virus transformed chick embryo fibroblasts.

Neuraminidase activity of Rous sarcoma virus transformed chick embryo fibroblasts (RSV-CEF) was assayed using an exogenous substrate, neuraminlactitol-[3H], and endogenous, cell surface [14C]-N]-acetyl-neuraminic acid. RSV-CEF had higher neuraminidase activity toward both substrates than did chick embryo fibroblasts (CEF) or nontransformed, Rous associated virus infected CEF (RAV-CEF). The total sialic acid content of RSV-CEF was lower than CEF or RAV-CEF, and more of the total sialic acid was accessible to extracellular Clostridium perfringens neuraminidase. Activity of the enzymes synthesizing and degrading the substrate for sialyltransferase, cytidine-5'-monophosphate-N-acetyl-neuraminic acid (CMP-AcNeu) was measured in order to determine whether control of substrate levels for sialyltransferase might contribute to the decreased levels of glycoprotein bound sialic acid. No change in activity of these enzymes was found in RSV-CEF as compared to CEF or RAV-CEF.

1-beta-D-arabinofuranosylcytosine nucleotide inhibition of sialic acid metabolism in WI-38 cells.

Because cytidine nucleotides have been demonstrated to affect activity of sialytransferases of both normal and malignant cells, we have investigated the effects of nucleotides of 1-beta-D-arabinofuranosylcytosine (ara-C) [1-beta-D-arabinofuranosylcytosine 5'-monophosphate and 1-beta-D-arabinofuranosycytosine 5'-triphosphate (ara-CTP)] on synthesis of sialoglycoproteins. Normal human diploid fibroblasts (WI-38 cells) were used in culture at confluency, when fewer than 1% of the cells were synthesizing DNA. 1-beta-D-Arabinofuranosylcytosine 5'-monophosphate was inhibitory to both sialytransferase activity of the intact cell and total cell homogenate transferase activity. The enzymes which synthesize and degrade the substrate of sialyltransferases, cytidine 5'-monophosphate-N-acetylneuraminic acid (CMP-AcNeu), were also tested for inhibition by nucleotides of ara-C. Synthesis of CMP-AcNeu was competitively inhibited by ara-CTP; however, formation of CMP-AcNeu when ara-CTP was supplied as substrate could not be detected. Hydrolysis of CMP-AcNeu was inhibited more severely by cytidine 5'-triphosphate than by ara-CTP or 1-beta-D-arabinofuranosylcytosine 5'-monophosphate. Confluent cultures of WI-38 cells exposed to ara-C have decreased amounts of glycoprotein sialic acid, suggesting that ara-C nucleotides may reach sufficient intracellular concentrations to affect the enzyme systems described.

Sialyltransferase activities of aging diploid fibroblasts.

Sialyltransferase activity and cell-cell adhesion rates of aging WI-38 cells were studied to determine the possible basis for a previously described decrease in membrane bound sialic acid and loss of proliferation of senescent cells. Ectosialyltransferase was demonstrated on the surface of both young and old WI-38 cells. The sialyltransferase assays consist of an enzyme source which is either the surface of intact cells (ectoenzyme) or a Triton X-100 cell homogenate, the nucleotide sialic acid donor (cytidine monophosphate-N-acetylneuraminic acid), and an asialo-acceptor which may be endogenous to the enzyme preparation or may be added exogenously. When sialyltransferase activity is measured in the absence of exogenous acceptors, there is a greater amount of sialic acid transferred by odl cells. However, when exogenous acceptors are provided, the amount of transfer is stimulated to a greater extent in young cells equalizing the amount of sialic acid incorporated into young and old cells. This suggests that there are fewer asialoglycoproteins and that acceptor concentration is a limiting factor in assays of young cell sialyltransferase. The end result of this may be the previously described decreased amount of membrane-bound sialic acid of old cells. A change in the adhesiveness of old cells is described which may be related to the altered cell surface.

Neutral protease activity of Rous sarcoma (RSV) transformed chick embryo fibroblasts.

The proteolytic activities of normal, Schmidt-Ruppin Rous sarcoma virus (SR-RSV) transformed, and infected (RAV) chick embryo fibroblasts (CEF) have been measured by a highly sensitive technique using 3H-acetylated haemoglobin as a substrate. When all 3 types of CEF cells were maintained in serumless media, no differences were detected in the amount of pH 3-4 protease activity released into the media over a 24-h period, and only negligible amounts of pH 7-6 proteolytic activity were found. When normal, transformed, and infected cells were maintained in serumless media and later incubated with 3H-acetylate haemoglobin, a significant proteolysis of the haemoglobin, a 6-fold increase compared to the normal CEF cells, was associated only with plates containing SR-RSV-CEF cells. A fluorescent assay for peptides confirmed that SR-RSV-CEF cells have increased cell-associated proteolytic activity. The net surface charge of the transformed CEF cells was unchanged by maintenance in serumless media but the net surface negativity of the normal and RAV-CEF cells was significantly increased by incubation in media minus serum for 24 h. This suggests that normal CEF cells, maintained in media plus serum, have a substance masking their surface charge which is absent from the surface of transformed cells, possibly because of proteolytic degradation.

Serum and host liver activities of glycosidases and sialyltransferases in animals bearing transplantable tumors.

Rats bearing Reuber H-35 or Novikoff hepatomas and mice bearing L1210 or L5178Y murine leukemias exhibited elevated serum levels of fetuin : N-acetylneuraminic acid transferase (EC 2.4.99.1) activity. The serum transferase activity could be correlated with the growth rate of the tumor; in animals bearing the more rapidly growing Novikoff hepatoma, activity was higher than in animals bearing the Reuber H-35 hepatoma. Higher transferase levels were also found in L1210 leukemic mice than in mice with the slightly slower growing L5178Y leukemia. Serum from rats bearing Reuber H-35 hepatoma and mice bearing L1210 murine leukemia had elevated levels of alpha- and beta-glucosidase (EC 3.2.1.20 and EC 3.2.1.21), alpha- and beta-galactosidase (EC 3.2.1.22 and (3.2.1.23), beta mannosidase (EC 3.2.1.25), alpha- and beta-fucosidase (EC 3.2.1.- and EC 3.2.1.38), beta-N-acetylglucosaminidase (EC 3.2.1.30) and acid phosphatase (EC 3.1.3.2); alpha-mannosidase (EC 3.2.1.24), beta-N-acetylgalactosaminidase (EC 3.2.2.-) and beta-xylosidase (EC 3.2.1.37) were not elevated. In animals bearing Reuber H-35 hepatoma, host liver levels of glycosidases, beta-glucuronidase (EC 3.2.1.31) and acid phosphatase were elevated over both the control and the hepatoma values. The data are interpreted to mean that the tumors or various host tissues release large quantities of enzymes into the serum and that enzyme levels in host organs may also be affected by the tumor.

Cell surface changes accompanying viral transformation: N-acetylneuraminic acid ectotransferase system activity.

The activity of the sialyl ectotransferase system of normal chick embryo fibroblasts (CEF) and chick embryo fibroblasts transformed with the Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV) have been compared. Neuraminidase treatment of the intact cells increased the sialyl ectotransferase system activity of control and transformed cells two to three times. The ectotransferase system activity increased as the pH was decreased from 7.8 to 6.0. The temperature optimum for both systems was 40 degrees C. Approximately 60% of the 14C-sialic acid incorporated at pH 6.5 or above could be removed with neuraminidase. The activity of the transformed cell system with or without neuraminidase treatment was more stimulated by addition of Mn2+ ions, particularly above pH 7.0. This difference in ion sensitivity indicates that a different cell surface phenomenon is being studied after transformation.