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Release of Glycosyltransferase and Glycosidase Activities from Normal and Transformed Cell Lines1

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Release of Glycosyltransferase and Glycosidase Activities from Normal and Transformed Cell Lines1 [CANCER RESEARCH 41, 2611-2615, July 1981J 0008-5472/81 /0041-OOOOS02.00 Release of Glycosyltransferase and Glycosidase Activities from Normal and Transformed Cell Lines1 Wayne D. Klohs,2 Ralph Mastrangelo, and Milton M. Weiser Division of Gastroenterology and Nutrition, Department of Medicine, State University of New York at Buffalo, Buffalo, New York 14215 ABSTRACT Indeed, a cancer-associated isoenzyme of serum galactosyl transferase has been reported in humans and animals with The release of galactosyltransferase, sialyltransferase, and certain malignant cancers (24, 26). Bernacki and Kim (2) and several glycosidase activities into the growth media from sev Weiser and Podolsky (34) have suggested that such increases eral normal and transformed cell lines was examined. Six of in serum glycosyltransferase levels may be the consequence the seven cell lines released galactosyltransferase into their of both an increased production and release from the tumor culture media. Only the human leukemia CCRF-CEM cells cells, perhaps through cell surface shedding of the enzymes, failed to release demonstrable galactosyltransferase activity. but the validity of this supposition has yet to be demonstrated. Release of galactosyltransferase activity into the media closely It is also not clear whether the elevated levels of circulating paralleled the growth curves for all but the BHKpy cells. These glycosyltransferases perform any molecular or physiological cells continued to release peak levels of galactosyltransferase function relative to the malignant condition. activity into the culture media after their growth had plateaued. In the present study, we examined the release of glycosyl Media galactosyltransferase activity was unaffected by Triton transferase and glycosidase activities from several normal and X-100 treatment and remained in the supernatant fraction of a transformed cell lines as part of an investigation into the nature 100,000 x g, 12-hr centrifugation, suggesting that the cells of these unbound enzymes, the mechanism by which these release galactosyltransferase in a soluble form. In contrast to enzymes are released into the medium or bloodstream, and galactosyltransferase activity, only one of the cell lines (L1210) their possible biological significance. released sialyltransferase activity in appreciable amounts. Even this level of activity was 20-fold less than that observed for galactosyltransferase in the media from L1210 cells. Of the MATERIALS AND METHODS nine glycosidase activities assayed, only N-acetylglucosamini- Cell Cultures. WI38 human embryonic lung fibroblasts (pas dase was observed in significant amounts in the media from all sage 16) (obtained from the Human Cell Culture Bank, Mason but the CCRF-CEM cells. However, W-acetylglucosaminidase Research Institute, Rockville, Md.) and HTC rat hepatoma cells release did not correlate closely with cell growth. These find (29) (generously provided by Dr. Darrell Doyle, Roswell Park ings suggest a relatively specific release of galactosyltransfer Memorial Institute, Buffalo, N. Y.) were grown in DMEM3 (As ase and A/-acetylglucosaminidase activities by cells in tissue sociated Biomedic Systems, Inc., Buffalo, N. Y.) supplemented culture. Moreover, the release of galactosyltransferase closely with 10% HI-FCS (Grand Island Biological Co., Grand Island, parallels cell growth. The significance of these released en N. Y.), 16 m.M HEPES, and 8 mw MOPS, pH 7.0. BHK-21 (C- zymes, especially to cell growth, has yet to be determined. 13) BHK fibroblasts (obtained from the American Type Culture Collection, Rockville, Md.) and the polyoma virus-transformed INTRODUCTION counterpart of BHK, BHKpy (generously provided by Dr. George Poste, Roswell Park Memorial Institute), were grown in Glycosyltransferases are enzymes that catalyze the transfer DMEM supplemented with 10% HI-FCS, 10% tryptose phos of monosaccharides from nucleotide sugars to oligosaccharide phate broth, 16 mM HEPES, and 8 mM MOPS. MDA-MB-231 chains of glycoproteins or glycolipids. They appear to be human breast tumor cell line (8) (obtained from the Human Cell located in the smooth endoplasmic reticulum (7, 31) and the Culture Bank, Mason Research Institute) was grown in DMEM Golgi apparatus (16, 32) where they participate in the biosyn supplemented with 10% HI-FCS, insulin (lO^g/ml), and corti- thesis of various glycoconjugates. A number of reports (17,19, sol (5 jug/ml). Murine leukemia L1210 cells and human leuke 20, 27, 33) have demonstrated recently the presence of gly- mia CCRF-CEM cells (12) (generously provided by Dr. Gerald cosyltransferases on the plasma membrane of several different Grindey, Roswell Park Memorial Institute) were grown in Ros cell types, and it is thought that these cell surface enzymes well Park Memorial Institute Tissue Culture Medium 1640 sup may mediate cellular recognition and adhesion (13, 28). Ele plemented with 10% HI-FCS, 16 mM HEPES, and 8 mM MOPS. vated levels of glycosyltransferases have also been observed All cells were grown in 35- x 10-mm polystyrene tissue culture in a number of animal and human tumors as compared to plates (Corning) at 37°.Cells were harvested in either 0.54 mM normal tissue or cells (2, 5), as well as in the bloodstream of EDTA or 0.25% trypsin (Grand Island Biological Co.) at various both animals and humans bearing metastatic tumors (2, 6, 15). times after plating for determination of cell number. Cells were ' This work was supported in part by American Cancer Society Grant PDT-88 counted with the use of a Model ZF Coulter Counter (Coulter and National Cancer Institute Grant CA25074 from NIH. Electronics, Inc., Hialeah, Fla.). Viability was determined by 2 To whom requests for reprints should be addressed, at Division of Gastro enterology and Nutrition, Department of Medicine, State University of New York 3 The abbreviations used are: DMEM, Dulbecco's modified Eagle's minimal at Buffalo, Clinical Center Annex, CC186, 462 Grider Street, Buffalo, N. Y. essential medium; HI-FCS, heat-inactivated fetal calf serum; HEPES, N-2-hy- 14215. droxyethylpiperazine-W-2-ethanesulfonic acid; MOPS, morpholinopropane sul- Received April 2. 1980; accepted March 24, 1981. fonic acid; BHK, baby hamster kidney. JULY 1981 2611 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1981 American Association for Cancer Research. W. D. Klohs et al. trypan blue exclusion. Media were collected and centrifuged 2 L1210, and CCRF-CEM cells was determined over a 4-day times at 1500 x g prior to measurement of enzyme activities. growth period (Tables 1 and 2). All cell lines released significant All cell lines were routinely screened for Mycoplasma contam galactosyltransferase into their media over 4 days of growth ination. except for the human leukemia cell line CCRF-CEM (Table 1). Glycosyltransferase Assays. Sialyltransferase assays were Galactosyltransferase activity was barely detectable in the performed according to procedures described previously by media from these leukemic cells which contrasts with the Klohs et al. (18). A typical assay medium (total volume, 100 mouse leukemic cell line, L1210, where substantial galacto jul) contained 50 fi\ of cell culture medium, 20 /il of desialylated syltransferase activity was observed. With the exception of fetuin (15 mg/ml), 10 /il of 0.1 M MgCI2, 10 ¡i\of 1.0 M BHKpy cells (and CCRF-CEM cells), release of galactosyltrans cacodylate buffer (pH 7.2), and 10 ¿ilof82.2 /ivi CMP-A/-acetyl- ferase from the various cells closely paralleled the growth ["*C]neuraminic acid (specific radioactivity, 304 mCi/mmol; curve for these cell types (Chart 1). BHK cells released the Amersham Corp., Arlington Heights, III.). The assay procedure greatest amount of galactosyltransferase of all the cells tested for galactosyltransferase activities was similar to that described (Table 1). This was true not only in regard to the total galacto by Podolsky er al. (25). A stock solution of 714 juM UDP- syltransferase activity detected in the culture media, but when galactose was prepared to a final specific activity of 6.1 Ci/ expressed on a per cell basis, each BHK cell was shown to mmol by using unlabeled UDP-galactose (Sigma Chemical Co., release more galactosyltransferase activity than any of the St. Louis, Mol.) and UDP-[3H]galactose (specific radioactivity, other cell types studied (Table 3). The human breast tumor 12.3 Ci/mmol; New England Nuclear, Boston, Mass.). A typical cells, MDA-MB-231, as well as BHKpy cells, also released assay medium (total volume, 100 /il) contained 50 /il of culture substantial galactosyltransferase activity into the culture media medium, 20 /il of fetuin minus sialic acid and galactose (15 (Tables 1 and 3). The L1210, W138, and HTC cells, however, mg/ml), 10/il of 0.1 M MnCI2, 10/tl of 1.0 M cacodylate buffer, discharged one-fourth to one-fifth the galactosyltransferase pH 7.2, and 10 /il of stock UDP-galactose. Incubations for both activity as that observed for the BHK or MDA-MB-231 cells glycosyltransferase activities were carried out at 37° in a (Table 3). The release of galactosyltransferase activity for all shaker bath for 1 hr. The reaction was terminated by the the cells studied was relatively constant between 48 and 96 hr addition of 2.0 ml of 1% phosphotungstic acid in 0.5 N HCI. with the exception of the BHKpy cells (Table 3). The growth of Samples were washed twice in 10% trichloroacetic acid and these cells plateaued between 72 and 96 hr, yet the level of once in 95% ethanol:ether (2:1, v/v) and counted in a Packard media galactosyltransferase from these cells continued to in Tri-Carb liquid scintillation counter. Enzyme activities are cal crease. This observation is reflected in the data from Table 3 culated as the difference between the exogenous and endog enous activities. Table 1 Pyrophosphatase and Hydrolase Activities. To determine Release of galactosyltransferase activity from cultured normal and transformed the breakdown of CMP-sialic acid and UDP-galactose during cell lines transferase assays, incubation mixtures were deproteinized by Enzyme activity was determined in a medium (total volume.
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