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

Home , Glycosyltransferase, Sialyltransferase

[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 37Â°.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

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 Â¡i\of 1.0 M BHKpy cells (and CCRF-CEM cells), release of galactosyltrans cacodylate buffer (pH 7.2), and 10 Â¿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 37Â° 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. 100 fil) containing the addition of 1.0 ml of 95% ethanol. The precipitate was 50 fil of culture medium, 20 /il of fetuin minus sialic acid and galactose (15 mg/ ml), 10/il of 0.1 M MnCI2, 10/il of 0.1 M cacodylate buffer (pH 7.2), and 10 (il of removed by centrifugation, and the supernatant material was stock UDP-galactose. Enzyme assays were performed on samples of media chromatographed on S & S orange ribbon paper (Schleicher taken at 5. 24, 48, 72, and 96 hr during cell growth. Results are the average of & Schuell, Keene, N. H.) using a solvent system composed of 2 experiments, each performed in triplicate. 95% ethanol: 1 M ammonium acetate (7:3, v/v). Radioactivity Specific activity (pmol galactose/50 fil media/hr) was located on the chromatogram using a Packard Model CelltypeBHKBHKpyMDA-MB-231HTCW138L1210CCRF-CEM5hr201010024hr1035040.6048hr4310202.58130.572hr10555236.51323196hr15011025818.5261 7201 radiochromatogram scanner. Areas on the chromato gram showing radioactivity were removed and counted as described earlier. Glycosidase Assays. Enzyme incubation mixtures consisted of 0.05 M citrate buffer (pH 4.5), 3.3 ITIMp-nitrophenylglyco- side, and 200 jul of culture medium in a total volume of 1.0 ml. The reactions were performed at 37Â°for 90 min and terminated

by the addition of 0.5 ml of 50 mw glycine, pH 10.5. The Table 2 amount of product formed was measured in a Beckman Model Release of Sialyltransferase activity from cultured normal and transformed cell 25 spectrophotometer at a wavelength of 400 nm. lines Determination of Cellular Breakdown. As a measure of Enzyme activity was determined in media (total volume, 100 /il) containing 50 jil of cell culture medium. 20^1 of desialylated fetuin (15 mg/ml), 10 /il of 0.1 M cellular disruption, culture media were assayed for succinic- MgCI2, 10 fil of 1.0 M cacodylate buffer (pH 7.2), and 10 Â¿ilof82.2 /JM CMP-W- INT-reductase and lactic dehydrogenase activities following acetyl['4C]neuraminic acid. Samples of media were taken at 5. 24, 48, 72, and procedures described by MorrÃ©(22) for the reducÃase and 96 hr. Wroblewski and LaDue (38) for lactic dehydrogenase. Specific activity (pmol sialic acid/50 /il media/hr) All experiments were performed at least twice, and individual CelltypeBHKBHKpyMDA-MB-231HTCW138L1210CCRF-CEM5hr000000.15024hr0000.0300.42048hr0000.090.050.75072hr0.2000.140.051.0096hr0.400.200.230.200.051.40.02 assays were performed in triplicate.

RESULTS

The release of galactosyltransferase and Sialyltransferase into the media from BHK, BHKpy, MDA-MB-231, HTC, W138,

2612 CANCER RESEARCH VOL. 41

to media-soluble galactosyltransferase and sialyltransferase activities, all media were also assayed for lactic dehydrogenase and succinic-INT-reductase enzyme activities. Neither enzyme I could be detected in the samples tested. In addition, no inhi 140 bition of either enzyme activity was found when cellular (ho- mogenate) enzyme activity was mixed with growth media.

100 g In view of recent studies reporting elevations in glycosidase activities in the sera from patients with certain cancers (10-

oa 1.4 Â«i 12, 14), samples from the media of both normal and tumor cell < 4. 60 Â» KO types were assayed for glycosidase activities over the 96-hr 0.4 eÃ§m cell growth period. Of the 9 glycosidase activities studied (a- "II D-galactosidase, ÃŸ-o-galactosidase, a-D-glucosidase, /3-o-glu- 0.2 20 cosidase, a-D-mannosidase, a-L-fucosidase, /S-u-fucosidase, O N-acetylglucosaminidase, and N-acetylgalactosaminidase), 5 24 48 72 96 HOURS only mannosidase, N-acetylgalactosaminidase, and N-acetyl- Chart 1. The release of galactosyltransferase (â€¢ â€¢¿)andsialyltransferase glucosaminidase were detected in the media during the 4-day (â€¢ â€¢¿)activitiesinto the medium during growth of BHK cells. Both enzyme growth of these cells. Elevations in the former 2 enzyme activities were determined as described in Tables 1 and 2. Cell growth and the release of galactosyltransferase as exemplified with BHK cells were also ob activities over 96 hr, however, appeared insignificant. However, served with MDA-MB-231, HTC, L1210, and W138 cells. a definite increase in N-acetylglucosaminidase activity was observed in the culture media during the 96-hr growth of 6 of Table 3 the 7 cell lines (Table 4). Only in the growth media from the Media galactosyltransferase activity per cell human leukemic CCRF-CEM cells were we unable to detect N- Enzyme activity was determined as described in Table 1. Enzyme activity acetylglucosaminidase activity (as well as any other glycosi represents the specific activity divided by the number of cells at each time point. Results are the average of 2 experiments, each performed in triplicate. dase activity determined). Specific activity (pmol galactose x 10~Vcell/hr) DISCUSSION CelltypeBHKBHKpyMDA-MB-231HTCW138L1210CCRF-CEM48hr11.943.01012.11.80.1772hr9.724.788.771.43.252.30.2596hr12.59.681.142.82.60.25 The mechanism by which glycosyltransferases, especially those elevated activities observed in the sera of cancer pa tients, enter the bloodstream is not understood, and even less is known about any metabolic role which these transferase enzymes may play, particularly in metastasis. Moreover, it is still uncertain that the tumor cells contribute to the reported elevations in serum glycosyltransferases observed in cancer in that the galactosyltransferase activity released per cell in patients (14). The work reported in this study uses tissue creases between 48 and 96 hr. culture cells to determine if cells release glycosyltransferase It is interesting that murine leukemic L1210 cells were the activities, and if they do, how and what form. With the exception of the human leukemia CCRF-CEM cells, only cells to release sialyltransferase into the culture medium in significant quantities (Table 2). Media sialyltransferase activ all cell lines in this study released soluble galactosyltransferase ities were barely detectable in all other cell types including the into the tissue culture media. A number of investigations (3, human leukemic CCRF-CEM cells (Table 2). L1210 media 11, 19, 23) have demonstrated increased levels of cellular sialyltransferase, however, was still approximately one-twen galactosyltransferase in transformed cells when compared to tieth that of the galactosyltransferase activity released from normal ones, but we found no consistent differences in the these same cells. No inhibition of sialyltransferase activity was release of this enzyme from the limited number of in vitro cells observed in mixing experiments in which solubilized sialyltrans in the present study. Perhaps, the shedding or secretion of ferase from several cell types was added to their growth media. galactosyltransferase is not correlated with whether or not the Both galactosyltransferase and sialyltransferase activities cell is cancerous as cellular galactosyltransferase seems to be. appear to be released into the media in soluble form. The In support of this, Whitehead ef al. (35) reported recently that addition of Triton X-100 (0.1%) to sialyltransferase or galac Table 4 tosyltransferase assay media had no effect on either enzyme Release of N-acetylglucosaminidase activity from cultured normal and activity. Furthermore, all of the glycosyltransferase activities transformed cell fines remained in the supernatant fraction after a 100,000 x g Activities were assayed in a medium (total volume, 1.0 ml) containing 0.05 M citrate buffer, pH 4.5, 3.3 mm p-nitrophenyl-N-acetylglucosamine, and 200 Â¡i\of centrifugation (for 12 hr) of the growth media of the cells. culture medium. Samples of media were taken at 5, 24, 48, 72, and 96 hr. To determine if either CMP-sialic acid hydrolase or UDP- Specific activity (nmol/ml/hr) galactose pyrophosphatase was interfering with the assay of sialyl- or galactosyltransferases, media from all cells and at all CelltypeBHKBHKpyMDA-MB-231HTCW138L12105hr15516516021060017524hr20021517530561523048hr28023518531068531572hr48028025067071045096hr540425330975725525 sample times were tested for both hydrolytic activities under the identical conditions used in the glycosyltransferase assays. Neither CMP-sialic acid hydrolase nor UDP-galactose pyro phosphatase could be detected in the media from any of the 7 cell lines. In addition, to assess the contribution of broken cells

JULY 1981 2613

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1981 American Association for Cancer Research. W. D. Klohs et al. greater glycosyltransferase activities were found in the media the amount of sialyltransferase activity in the media from normal from cultured human fetal colonie cell lines than from several (Chang) liver cells more closely resembled that which we colonie tumor cell types. In addition, while LaMont et al. (19) observed in all our cell lines except L1210 cells. found greater galactosyltransferase activity in the media from In both the normal and transformed cells, we failed to detect the transformed cells, NILpy, than in the media from normal galactosidase, glucosidase, and fucosidase, and relatively min NIL cells, we observed higher galactosyltransferase activity in imal mannosidase and N-acetylgalactosaminidase activities the media from normal BHK cells than in the media from the were found in their culture media. In accord with previous transformed BHKpy cells. findings for the mouse leukemic L5178Y cell line (4), we In view of our present understanding of glycoconjugate bio observed appreciable /V-acetylglucosaminidase activity in the synthesis, it is intriguing that only one (L1210) (Chart 6) of the growth media for all of the cell lines except CCRF-CEM cells, 7 cell lines studied released significant amounts of sialyltrans- although there appears to be no correlation between transfor ferase into the culture media. If the addition of galactose to the mation and the amount of enzyme in the media. Recent studies growing end of an oligosaccharide chain is followed by attach (36, 37) have suggested that, under in vitro conditions, extra ment of sialic acid to the galactose, one might expect the cellular glycosidase activity from certian cells may be (at least glycosyltransferases responsible for these reactions to be in in part) of nonlysosomal origin. Our findings with A/-acetylglu- close proximity to one another and represent part of a complex cosaminidase lend support to this notion. The high levels of N- of enzymes collectively referred to as a multiglycosyltransfer- acetylglucosaminidase activity (relative to other glycosidase ase system (28). If such a complex of glycosyltransferases activities) found in the culture media suggest that this lysosomal does occur within the cell, the selective release of galactosyl enzyme may be secreted into the media (at least in part) by a transferase into the growth media by 5 of 7 cell lines suggests pathway such as that described for more traditionally associ that a different pathway for galactosyltransferase release may ated secretory enzymes (9). exist. Perhaps, in certain cells, the galactosyltransferase may In conclusion, we have demonstrated a selective release of be more loosely bound to the membranes of the cells than is galactosyltransferase by several different normal and trans sialyltransferase, rendering it easier for dissociation at the cell formed tissue culture cell lines. We have also demonstrated surface. This might explain the soluble nature of the galacto significantly greater N-acetylglucosaminidase activity in these syltransferase found in the culture media. Alternatively, the fate culture media than other glycosidase activities tested. The of these enzymes may be different due to a selective reabsorp- release of these enzymes in this limited study does not appear tion or recycling of sialyltransferase by these cells. to be correlated with neoplastic transformation. As yet, it is A distinctive feature of the human leukemia CCRF-CEM cells unclear how these enzymes are released from these cells. was their inability to release into their growth media significant Studies are presently in progress to determine if these enzymes sialyltransferase, galactosyltransferase, or A/-acetylglucosa- are secreted by these cells or are shed from the cell surface. minidase activitÃ©s. These were the only cells in our studies Finally, in view of numerous reports demonstrating elevated unable to shed or secrete the latter 2 enzymes. The significance sialyltransferase activities in patients with cancer, the failure to of this observation, however, remains unclear. The murine detect sialyltransferase activity in the culture media of 6 of the leukemic cell line, L1210, not only released galactosyltransfer 7 cell lines is important. Perhaps in vitro conditions inhibit ase and N-acetylglucosaminidase activity, but this was the only sialyltransferase activity release by these cells. We believe that cell line to release appreciable levels of sialyltransferase activ these results do emphasize the need to more critically examine ity into the culture media, although this activity was one-twen the nature of serum sialyltransferase activity in an effort to tieth the galactosyltransferase activity we found in the same identify the source of these elevations in activity observed in media. Other cells have been reported recently (35) to release many cancer patients. sialyltransferase activity into their culture media, and like L1210 cells, the sialyltransferase activity was considerably less than that activity observed for galactosyltransferase in the ACKNOWLEDGMENTS same culture media (35). In view of recent studies (2, 6, 15) We are very thankful to Judith Rabin for her excellent technical assistance. reporting that the elevations of serum sialyltransferase activity observed in animals and patients with various cancers may originate from the tumor tissue, we found it surprising that the REFERENCES L1210 cells were the only transformed cell line in our study to 1. Bernacki. R. J. Plasma membrane ectoglycosyltransferase activity of L1210 release sialyltransferase activity. We have observed, for in murine leukemic cells. J. Cell Physiol.. 83: 457-466, 1974. stance, no significant sialyltransferase activity in the media of 2. Bernacki, R. J., and Kim. U. Concomitant elevations in serum sialyltransfer the human breast tumor-derived MDA-MB-231 cells (Table 2) ase activity and sialic acid content in rats with metastasizing mammary tumors. Science (Wash. D. C.). Â»95.577-580, 1977. or a human breast tumor-derived cell line, MCF-7 (data not 3. Bosmann. H. B. Cell surface glycosyltransferases and acceptors in normal shown), yet increases in sialyltransferase activity have been and RNA and DMA virus transformed fibroblasts. Biochem. Biophys. Res. demonstrated in the sera from animals and patients with malig Commun., 48: 523-529, 1972. 4. Bosmann, H. B.. and Bernacki, R. J. Glycosidase activity in L5178Y mouse nant mammary tumors (2, 15). Conceivably, as has been sug leukemic cells and the activity of acid phosphatase, /i-galactosidase, and gested previously (14, 15, 17), these observed increases in /5-N-acetylgalactosaminidase and /3-W-acetylglucosaminidase in synchro nous L5178Ycell population. Exp. Cell Res., 67. 379-386, 1970. serum sialyltransferase activity from cancer patients may be 5. Bosmann, H. B., and Hall. T. C. Enzyme activity in invasive tumors of human due to liver involvement in the diseased state. In contrast to breast and colon. Proc. Nati. Acad. Sei. U. S. A., 77. 1833-1837, 1974. our findings for HTC cells, a recent report by Liu ef a/. (21) 6. Bosmann, H. B., and Hilf, R. Elevations in serum glycoprotein: W-acetylneuraminic acid transferases in rats bearing mammary tumors. FEBS Lett., observed that 2 human hepatoma cell lines released significant 44: 313-316. 1974. levels of sialyltransferase activity into the culture media, and 7. Bouchilloux, S., Chabaud, O., Michel-Bechet. M., Ferrand, M., and AthonÃ«l-

2614 CANCER RESEARCH VOL. 41

Haon, A. M. Differential localization in thyroid microsomal subfractions of a 23. Patt, L. M., and Grimes, W. J. Cell surface glycolipid and glycoprotein mannosyltransferase, two N-acetylglucosaminyltransferases, and a galac- glycosyltransferases of normal and transformed cells. J. Biol. Chem.. 249. tosyltransferase. Biochem. Biophys. Res. Commun., 40: 314-320, 1970. 4157-4165, 1974. 8. Caillean, R., Young, R., Olive, M., and Reeves, W. J. Breast tumor cell lines 24. Podolsky, D. K., and Weiser, M. M. Galactosyltransferase activities in human from pleural effusions. J. Nati. Cancer Inst., 53. 661-674. 1974. sera: detection of a cancer-associated isoenzyme. Biochem. Biophys. Res. 9. Case. R. M. Synthesis, intracellular transport, and discharge of exportable Commun., 65: 545-551, 1975. proteins in the pancreatic acinar cell and other cells. Biol. Rev. Camb. 25. Podolsky. D. K., Weiser, M. M., and Isselbacher, K. J. Inhibition of growth Philos. Soc., 53. 211-354, 1978. of transformed cells and tumors by an endogenous acceptor of galactosyl 10. Chatterjee, S. K., Bhattacharya, M., and Barlow, J. J. Elevated activity of transferase. Proc. Nati. Acad. Sei. U. S. A., 75. 4426-4430. 1978. cytidine 5' monophospho-N-acetylneuraminic acid hydrolase in serum of 26. Podolsky, D. K., Weiser, M. M., Westwood. J. C., and Gammon, M. Cancer- ovarian cancer patients as a possible indicator of malignancy. Biochem. associated serum galactosyltransferase activity. Demonstration in an animal Biophys. Res., Commun., 80. 826-832, 1978. model system. J. Biol. Chem., 252. 1807-1813, 1977. 11. Chatterjee, S. K., Bhattacharya, M., and Barlow, J. J. Glycosyltransferase 27. Porter, C. W., and Bernacki, R. J. Ultrastructural evidence for ectoglycosyl- and glycosidase activities in ovarian cancer patients. Cancer Res., 39. transferase systems. Nature (Lond.), 256. 648-650, 1975. 1943-1951, 1979. 28. Roseman, S. The synthesis of complex carbohydrates by multiglycosyltrans- 12. Foley, G. E., Lazarus, H., Farber, S., Uzman, B. E., Boone, B. A., and ferase systems and their potential function in intercellular adhesion. Chem. McCarthy, R. E. Continuous culture of human lymphocytes from peripheral Phys. Lipids, 5:270, 1970. blood of a child with acute leukemia. Cancer (Phila.), 78: 522-529, 1965. 29. Thompson, E. B., Tomkins, G. M., and Curran. J. F. Induction of tyrosine 13. Hughes. R. C. The complex carbohydrates of mammalian cell surfaces and a-ketoglutarate transaminase by steroid hormones in a newly established their biological roles. Essays Biochem., 11: 1-36. 1975. tissue culture cell line. Proc. Nati. Acad. Sei. U. S. A., 56. 296-303, 1966. 14. Ip, C., and Dao, T. L. Increase in serum and tissue glycosyltransferases and 30. Verbert, A., Cacan, R., and Montreuil, J. Ectogalactosyltransferase. Pres glycosidases in tumor-bearing rats. Cancer Res., 37. 3442-3447, 1977. ence of enzyme and acceptors on the rat lymphocyte cell surface. Eur. J. 15. Ip, C., and Dao, T. Alterations in serum glycosyltransferases and 5'-nucle- Biochem., 70: 49-53. 1976. otidase in breast cancer patients. Cancer Res., 38. 723-728, 1978. 31. Wagner, R. R., Pettersson, E., andDallner. G. Association of the two glycosyl 16. Keenan, T. W., Moore, D. J., and Basu, S. Ganglioside biosynthesis: con transferase activities of glycoprotein synthesis with low equilibrium density centration of glycosphingolipid glycosyltransferases in Golgi apparatus. J. smooth microsomes. J. Cell Sci., 12: 603-615, 1973. Biol. Chem., 249. 310-315, 1974. 32. Warley, A., and Cook, G. M. W. Isolation of a Golgi-apparatus-enriched 17. Kessel, D., Sykes, E., and Henderson, M. Glycosyltransferase levels in fraction from leukemic cells. Biochem. J., 756. 245-251, 1976. tumor metastatic to liver and in uninvolved liver tissue. J. Nati. Cancer Inst.. 33. Weiser, M. M. Intestinal epithelial cell surface membrane glycoprotein syn 59. 29-32. 1977. thesis. II. Glycosyltransferases and endogenous acceptors of the undiffer- 18. Klohs, W. D.. Bernacki, R. J., and Korytnyk, W. Effects of nucleotides and entiated cell surface membrane. J. Biol. Chem., 248. 2542-2548, 1973. nucleotide: analogs on human serum sialyltransferase. Cancer Res.. 39. 34. Weiser, M. M., and Podolsky, D. K. Cell surface galactosyltransferase in 1231-1238, 1979. mitosis, differentiation, and neoplastic transformation and mÃ©tastases.In: R. 19. LaMont, J. T., Gammon, M. T., and Isselbacher, K. J. Cell-surface glycosyl E. Harmon (ed.), Cell Surface Carbohydrate Chemistry, pp. 67-82. New transferases in cultured fibroblasts: increased activity and release during York: Academic Press, Inc., 1978. serum stimulation of growth. Proc. Nati. Acad. Sei. U. S. A., 74. 1086- 35. Whitehead, J. S., Fearney. F. J.. and Kim, Y. S. Glycosyltransferase and 1090, 1977. glycosidase activities in cultured human fetal and colonie adenocarcinoma 20. LaMont, J. T., Perrotto, J. L., Weiser. M. M., and Isselbacher. K. J. Cell cell lines. Cancer Res., 39: 1259-1263, 1979. surface galactosyltransferase and lectin agglutination of thymus and spleen 36. Willcox, P. Secretion of 0-N-acetylglucosaminidase isoenzymes by normal lymphocytes. Proc. Nati. Acad. Sei. U. S. A., 71: 3726-3730, 1974. human fibroblasts. Biochem. J., 773. 433-439, 1978. 21. Liu, C. K., Schmied, R., and Waxman, S. The specific release of sialyltrans 37. Willcox, P., and Rattray, S Secretion and uptake of /i-W-acetylglucosamin- ferase activity by human hepatoma cell lines. Clin. Chim. Acta, 98: 225- idase by fibroblasts: effect of chloroquine and mannose-6-phosphate. 233, 1979. Biochim. Biophys. Acta, 586: 442-452, 1979. 22. MorrÃ©,D. J. Isolation of Golgi apparatus. Methods Enzymol., 22. 130-148, 38. Wroblewski. F., and LaDue. J. S. Lactic dehydrogenase activity in blood. 1971. Proc. Soc. Exp. Biol. Med., 90: 210-213, 1955.

JULY 1981 2615

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1981 American Association for Cancer Research. Release of Glycosyltransferase and Glycosidase Activities from Normal and Transformed Cell Lines

Wayne D. Klohs, Ralph Mastrangelo and Milton M. Weiser

Cancer Res 1981;41:2611-2615.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/41/7/2611

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/41/7/2611. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.