Printed in Sweden Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any tom reserved 0014-4827/78/I I I I-0047$02.00/O
Experimental
CHANGES GROWTH
Cell Research 111 (1978) 47-53
IN GLYCOSIDASE OF NORMAL J. E. THOMPSON, Departments
of Biology Waterloo,
ENZYME AND
ACTIVITY
TRANSFORMED
DURING CELLS
M. Y. GRUBER and J. KRUUV and Physics, Ont., Canada
University N2L 3G1
of Waterloo,
SUMMARY Homogenate specific activities of four glycosidases-N-acetyl-/3+galactosaminidase, N-acetylp-Dglucosaminidase, /3+galactosidase and a-n-mannosidase-were determined at daily intervals during preconfluent growth and after confluence for BHK and pyBHK cells. Regression lines were determined and the regressions were subjected to analysis of covariance in order to compare slopes and elevations. The results indicate that levels of glycosidase activity are correlated with growth; activity increased in growing cells but remained essentially constant in quiescent cells. Thus by extrapolation, in the in vivo state tumour cells would tend to have higher levels of glycosidase activity than their normal counterparts by reason of their inability to achieve growth control. The data are consistent with previous reports of elevated glycosidase and protease activity in oncogenicrdly transformed cells and provide further evidence for a role of lysosomal enzymes in mediating the uncontrolled growth of transformed cells.
There is evidence that molecular moditication of the cell periphery by lysosomal enzymes, a process termed sublethal autolysis, contributes to loss of growth control and maintenance of the neoplastic state [l-3]. Protease action on the cell surface endues normal cells with a sufficiently altered surface architecture to enable escape from contact inhibition of growth [4], although exceptions to this have been noted [5]. Breakdown of glycoproteins and glycolipids on the surface membrane of density-inhibited normal cells by neuraminidase has been shown to stimulate cell division [6]. Protease action renders normal cells as agglutinable by plant lectins as their transformed counterparts [7]. Bosmann & Hall [8] have reported higher levels of P-galactosidase, cY-mannosidase, neur4-771807
aminidase and acid protease in homogenates of malignant human heart and colon tissue than in corresponding normal tissue. In general viral transformation of established cell lines leads to enhanced glycosidase and protease activities [9, lo] as does primary infection with Rous sarcoma virus [ll]. However, the enhancements are not of comparable magnitude from cell line to cell line and there have been some exceptions to this finding. For example, the enzymes P-Dgalactosidase, N-acetyl+ D-glucosaminidase, N-acetyl-&D-galactosaminidase and a-Dmannosidase of 3T3 cells showed enhancements ranging from 2to 27-fold after transformation by RNA tumour viruses [lo], whereas the same enzyme activities are only 20-55% higher in pyBHK cells than in BHK cells [ 121. GangExp Cell
Res I I I (1978)
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Thompson
et al.
lioside neuraminidase levels were reported elevated in transformed hamster cells [13], but in mouse cell lines no differences were found between normal and transformed cells [ 141. There are also reports of more extensive leakage of glycosidases and proteases from transformed cells than from normal cells [15-171. Higher digestive enzyme activity in transformed cells is consistent with the prospect that sublethal autolysis contributes to maintenance of the neoplastic state. However, levels of lysosomal enzyme activity are sensitive to factors other than transformation, including type of culture medium, pH, frequency of medium change and passage number [18-201. The activities of digestive enzymes also change with growth and are sensitive to subculture shock, showing a decline in activity during the lag phase in growth that immediately follows subculturing [20, 211. Growth-associated modulation of lysosomal enzymes could well influence the relative levels of digestive enzyme activities in normal and tumour cells. This would be particularly true if changes in enzyme activity in contact inhibited cells were different and independent of those incurred during exponential phase, for in vivo, normal cells are for the most part in GO and tumour cells are actively growing. In the present study we have attempted to clarify this question by examining changes in glycosidase activity ofgrowing BHK and pyBHK cells up to and beyond confluence. MATERIALS
AND METHODS
Cell culturing BHK and polyoma-transformed (pyBHK) cells were routinely cultured at 37°C in 30 oz. glass prescription bottles in Minimal Eagle’s Medium (MEM) supplemented with L-glutamine, penicillin-streptomycin and 10% fetal calf serum (FCS). Stocks were subcultured at split ratios of 1 : 10 to 1: 20 every 3 or 4 days to prevent selection of spontaneous transformants. Cells Exp Cell Res III
(1978)
1. Abscissa: time (hours); ordinate: cell no./cm2 (log scale). Growth curves for (A) BHK; (II) pyBHK cells.
Fig.
were removed from the glass surfaces by treatment for 5-10 min at 37°C with a solution of 0.25% trypsin and 0.10% disodium ethylene diamine tetraacetic acid (EDTA) in Cae+- and MgP+-free phosphate-buffered saline (CMS). For experiments in which glycosidases were being measured as a function of cell density, stock bottles were seeded at 1 x 1W cells/cm2 and grown for 48 h. These cells were then seeded at a density of 2.5~ 103 cells/cm2 in large glass Petri dishes (15 cm diameter). The medium was changed 2 days after seeding and every 3 days thereafter. Duplicate cultures were harvested at daily intervals through the period 1-14 davs after seedinz. The cells were removed with trvnsin-EDTA as described above and diluted to 30 ml &h ohosuhate-buffered saline containina 2% FCS. Ali&rot; were counted with a Celloscope electronic cell counter and the remainder of the suspension was pelleted by centrifugation at 800 g for 15 min. The cells were then washed twice by resuspension in 30 ml of 0.15 M NaCl and centrifugation. Pellets of washed cells were resuspended in 3-10 ml of 0.03 M NaHCO,, pH 7.5. These suspensions were stored at -2O”c, usually overnight but never for longer than 18 h, and then homogenized and assayed for protein and enzyme activity.
Enzyme and protein measurements Suspensions of washed cells were homogenized with 30 strokes of a Potter Elvehjem homogenizer (0.20 mm clearance) rotating at 850 rpm. Homogenates were treated with 0.1% Triton X-100 as described previously r21, 221 in order to destroy latencv of lvsosomal enzymes. Giycosidase activities of the homogenates were measured as described by Bosmann f121. The following substrates obtained from Sigma Chemical Co. were used: p-nitrophenyl-N-acetyl-fi-o-glucosamide for N-acetyl-P-Dglucosaminidase (EC3.2.1.29); p-nitrophenyl-N-acetyl-/3-D-galactosaminide for Nacetyl-/3-o-galactosaminidase (EC3.2.1.53); p-nitrophenyl-/3-D-galactoside for p-o-galactosidase (EC3. 2.1.23); p-nitrophenyl-a-p-mannoside for a-n-mannosidase (EC3.2.1.24). p-Nitrophenol, pH 10.5, was
Changes in glycosidase activity I
Fig. 2. Abscissa: time (hours); ordinate: nmoles pnitrophenol released/h/mg protein. Regression lines showing changes in N-acetyl-/S-r+ galactosaminidase specific activity before and after confluence for (A) BHK; (If) pyBHK cells.
used as a standard. Each assay was run in duplicate and parallel enzyme and substrate blanks were determined with each estimation. Protein was assayed as described by Lowry et al. [23] using bovine serum albumin as a standard.
RESULTS When seeded at 2.5~ 103 cells/cm2 BHK cells grew exponentially with a doubling time in the order of 14.5 h before reaching confluence and displaying contact inhibition at about 168 h (fig. 1A). Cultures of pyBHK cells seeded at the same cell density also achieved confluence after about 168 h but thereafter, in keeping with their transformed nature, continued to grow (fig. 1B).
49
The tendency for the growth profile of pyBHK cells to plateau at contluence reflects a high incidence of cells falling off into the medium. Such cells were not included in the totals used to compute the growth curves. Homogenate specific activities of four glycosidases-N-acetyl-~-D-galactosaminidase, N-acetyl-/3-D-glucosaminidase, P-Dgalactosidase and cr-D-mannosidase-were determined at daily intervals before and after confluence for both cell lines. Regression lines for changes in the activity of N-acetyl-P-D-galactosidase are illustrated in fig. 2. For BHK cells the enzyme showed an essentially linear increase in activity during exponential growth, but the rate of increase greatly decreased once the cells became contact inhibited (fig. 2A). However, for pyBHK cells the rates of increase were comparable during preconfluent and postconfluent growth (fig. 2B). By contrast Nacetyl+-Dglucosaminidase showed a greatly decreased rate of increase in both normal and transformed cells after confluence (figs 3 A, B) . p-D-Galactosidase and cy-D-mannosidase showed only slight increases in activity during preconfluent growth and
Table 1. Comparison of correlation coefficients for regression lines depicting changes in glycosidase activity before and after confluence Correlation coefficients Enzyme N-Acetyl-/I?-D-galactosaminidase N-Acetyl-B-D-glucosaminidase p-u-Galactosidase a-o-Mannosidase
BHK Before confluence After confluence Before confluence After confluence Before confluence After confluence Before confluence After confluence
0.85” 0.14 0.80” 0.12 0.48” 0.04 0.75” 0.20
PYBHK (14) (17) (32) (19) (38) (15) (14) (14)
0.78” 0.60” 0.63” 0.17 0.48” 0.17 0.65’ 0.07
(15) (20) (30) (22) (23) (21) (14) (21)
Regression lines in figs 2-5 were used for these analyses. Degrees of freedom are indicated in parentheses. a Significance at the 5 % level. Exp CellRes
111 (1978)
50
Thompson
et al.
Fig. 3. Abscissa: time (hours); ordinate: nmoles pnitrophenol released/h/mg protein. Regression lines showing changes in N-acetyl-B-D glucosaminidase specific activity before and after confluence for (A) BHK; (B) pyBHK cells.
Fig. 4. Abscissa: time (hours); or&rule: nmoles pnitrophenol released/h/mg protein. Regression lines showing changes in /3-o-galactosidase specific activity before and after confluence for (A) BHK; (B) pyBHK cells.
tended to level off for both cell lines once confluence was achieved (figs 4,5). Correlation coeffkients and regression coeffkients for the regression lines are compared in tables 1 and 2. For BHK cells the correlation coefficients for exponential growth were all significant at the 5 % level (table 1). However the rates at which the four enzymes increased in activity varied. The regression coefficients for N-acetylP-Dglucosaminidase and N-acetyl-g-Dgalactosaminidase were 0.293 and 0.067, respectively, reflecting quite steep rates of change, whereas those for /3-D-galactosidase and cw-D-mannosidase were very low by comparison (table 2). For pyBHK cells the correlation coefficients of the regressions for preconfluent growth were also significant at the 5% level for all of the enzymes (table 1). The regression coefficients for preconfluent growth, although tending to be lower for transformed cells than for normal cells (table 2), did not prove significantly different at the 5 % level between the two cell lines for any of the enzymes when tested by analysis of covariance as outlined by Snedecor & Cochran [24]. Nor were the elevations of corresponding regression lines, which reflect the magnitude of increase, significantly different between the two cell lines during preconfluent growth when tested by analysis of covariance.
Thus the rates of change for each of these enzymes are essentially the same for BHK and pyBHK cells during exponential growth. Changes in the enzyme activities following contact inhibition of normal cell growth ah showed non-significant correlation coefficients, indicating that there was no significant increase with time in the contactinhibited state (table 1). Regression coefftcients were all reduced by f&lo-fold relative to those for exponential growth, and with the exception of N-acetyl-@-D-ghrcosaminidase were essentially zero (table 2). For pyBHK cells, only IV-acetyl+D galactosaminidase showed a significant correlation coeffkient during post-confluent growth (table 1). However, both N-acetylp-D-galactosaminidase and N-acetyl-/I-Dglucosaminidase continued to rise after confluence with regression coefficients of 0.033
Exp Cell Res I I1 (1978)
Fig. 5. Abscissa: time (hours); ordinate: nmoles pnitrophenol released/h/mg protein. Regression lines showing changes in o-Dmannosidase specific activity before and after confluence for (A) BHK; (E) pyBHK cells.
Changes
Table 2. Comparison glycosidase
activity
in glycosidase
of regression coefficients for regression before and after confluence
activity
lines depicting
changes
51 in
Regression coeffkients Enzyme
BHK
N-Acetyl-/3-n-galactosaminidase N-Acetyl-P+glucosaminidase /3-n-Galactosidase a-n-Mannosidase
Before confluence After confluence Before confluence After confluence Before confluence After confluence Before confluence After confluence
0.067 0.008 0.293 0.034 0.017 -0.001 0.008 0.001
PYBHK 0.045 0.033 0.163 0.045 0.017 0.008 8:E
Regression lines in figs 2-5 were used for these analyses.
and 0.045, respectively (table 2). The regression coefficients for P-D-galactosidase and a-Dmannosidase were very low after confluence, but these enzymes showed only slow rates of increase during preconfluent growth (table 2). DISCUSSION Elevated levels of lysosomal enzymes in transformed cells lend support to the view that sublethal autolysis at the cell surface contributes to loss of growth control [g-12]. However, there are also reports that the activities of these enzymes change with cell growth [W211, suggesting that the distinctive growth behaviours of normal and transformed cells could influence their relative levels of digestive enzyme activity. To further clarify this possibility regression lines for changes in glycosidase activity both before and after confluence have been examined in normal and transformed cells. Glycosidases are capable of cleaving bonds between monosaccharides or between a monosaccharide and some other moiety in glycoproteins, glycolipids, glycosaminoglycans and oligo- and polysaccharides, and thus could significantly alter cell surfaces.
For BHK cells each of the enzymes showed a positive and significant regression during exponential growth, but once the cultures reached confluence and became density-inhibited, none of the regressions showed a significant correlation with time. Calculated slopes (regression coefftcients) were also less following contact inhibition, dropping by 8-lo-fold. In fact for three of the enzymes-N-acetyl-/3-D-galactosaminidase, /3-D-galactosidase and a-Dmannosidase-the regression coefficients during the post-confluent period were essentially zero. Thus, while there may have been some tendency for the activity to increase following contact inhibition, it is clear that the rates of increase were markedly less than those observed during exponential growth. Indeed, the very low correlation coefftcients indicate that changes in activity after the cultures became confluent were simply variation which was independent of time. Such variation may well reflect the tendency of refeeding to stimulate cell division, for it has been shown in studies with synchronous cell populations that glycosidase specific activity increases over the cell cycle [25]. Digestive enzyme activity is also known to be affected by celExp Cell Res 11 I (1978)
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et al.
lular senescence [26], but in these experiments any such effects were minimized by regular refeeding of the cultures. Heukels-Dully & Niermeijer [20] have reported that the specific activity of lysosomal enzymes increased steadily during growth of cultured skin fibroblasts, but they also observed a decrease in activity immediately following trypsinization and subculturing which they attributed to subculture shock. A slight decline in glycosidase specific activity was apparent immediately after subculturing BHK and pyBHK cells as well (figs 2-5). Horvat & Acs [21] in a study with 3T3 cells noted a very dramatic decline in the specific activities of three lysosomal enzymes following subculture which persisted for up to 72 h, and no substantial increase in activity was apparent until the cells were beginning to reach confluence. They interpreted their data as suggesting that digestive enzyme activity does not increase appreciably during the growth phase. However, the apparently high sensitivity of these cells to an effect of subculturing on lysosomal enzymes together with the fact that the cells were seeded at a relatively high density (8x lo”/cm2) may have precluded accurate measurement of activity during the exponential phase. Glycosidase activity showed a positive and significant regression during exponential growth of pyBHK cells as well, and for one of the enzymes--N-acetyl-/3D-galactosaminidase-the regression remained significant during post-confluent growth. The rates of increase (regression coefficients) were also comparable before and after confluence for this enzyme; in fact they were not significantly different at the 5 % level when tested by analysis of covariance. The other three enzymes showed reduced rates of increase once the pyBHK cultures became confluent, and the acExp Cell
Res I I I (1978)
tivities were variable. This is perhaps surprising since the cells were still growing. However, the rate of growth was slower, and it is conceivable that the constant sloughing off of cells into the medium during this period caused fluctuations in activity similar to those brought on by subculturing, which masked a pattern of change attributable to growth. This would be particularly true for p-D-galactosidase and C-P D-mannosidase which showed only small rates of increase even before the cultures became confluent. Moreover, pyBHK cells are especially prone to sloughing off as they pile up to form multilayers during postconlluent growth. Granted the assumption that technical difficulties have precluded detection of a true pattern of change during post-confluent growth of the transformed cell line, the data are consistent with the contention that levels of glycosidase activity are correlated with growth; activity increases in growing cells but remains essentially constant in quiescent cells. Thus by extrapolation, in the in vivo state tumour cells would tend to have higher levels of glycosidase activity than their normal counterparts by reason of their inability to achieve growth control. This, coupled with a more extensive leakage of glycosidases and proteases [U-17], could in part account for cell surface alterations characterizing the malignant phenotype as well as the invasive nature of malignancies. Transformationspecific enhancement of digestive enzyme activity, which is apparently independent of any increase attributable to the distinctive growth modes of normal and transformed cells, is also well documented [8121. It is perhaps noteworthy that in the present study the elevations of the enzyme regression lines for exponential phase pyBHK cells were not significantly dif-
Changes in glycosidase activity ferent from those for exponential phase BHK cells. Nor were the slopes significantly different. The enzymes selected for the study were those previously shown to have higher activity in pyBHK cell homogenates than in those for BHK cells [12]. However, these differences were not large; they ranged from 20-55 % and could perhaps reflect differences in growth status of the cultures at the time of harvest. The authors gratefully acknowledge a grant-in-aid this research from the MRC of Canada.
of
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Em Cell
Res 111 (1978)