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Growth of baby hamster kidney cells in media containing neuraminidase
Printed in Sweden Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved
Experimental
GROWTH
Cell Research 8.5 (1974) 362-366
OF BABY HAMSTER CONTAINING
KIDNEY
CELLS IN MEDIA
NEURAMINIDASE
R. C. HUGHES and JULIE CLARK’
Aktional Institute for Medical Research, Mill Hill, London NW7 IAA, UK
SUMMARY An established line of baby hamster kidney cells, BHK 21/C13 grows in media containing high concentrations of Vibrio cholerae neuraminidase at the same initial exponential rate as control cells grown in the absence of the enzyme. Glycoprotein fractions removed from the surface of cells grown with neuraminidase contain less than 4 % of the sialic acid present in similar fractions removed from control cells, The significance of these results are discussed in relation to the role suggested for sial glycoproteins in growth control. A small but significant increase was observed in the density of confluent cells in media containing neuraminidase compared with control cultures.
Recent suggestions [l] as to the importance of cell surface carbohydrates, and in particular sialic acid residues, in growth control of cells in culture have led us to carry out the experiments described in this paper. An established line [2] of baby hamster kidney cells, BHK 21/C13 has been grown in media containing Vibrio cholerae neuraminidase and the kinetics of growth have been compared to control cells incubated in media lacking the enzyme. MATERIALS AND METHODS Neuraminidase from Vibrio cholerae was purchased from Hoechst Pharmaceuticals, Kew Bridge, London. Growth of cells. Baby hamster kidney fibroblasts BHK 2l/C13 were grown at 35°C in Eagle minimal essential medium containing 10 % tryptose phosphate broth, penicillin (50 units/ml), streptomycin (100 pug/ ml) and 10 % fetal calf serum. At confluency the cells were removed from the surface by treatment at 35°C for a few minutes with 0.125 % trypsin in phosphate-
1 Present address: University of Cambridge, Cambridge, UK. Exptl Cell Res 85 (1974)
buffered saline (pH 7.8) containing 0.3 mM EDTA. The cells were replated onto 35 mM Falcon plastic plates at approx. IO5 cells/plate. After incubation for 2 to 3 h in medium as above, most of the cells had attached to the Dlastic surface. The incubationmedium (1.5 ml per plate) was removed and replaced either with medium A lackinn neuraminidase or medium B containing 37.5 units of Vibrio cholevae neuraminidase. Daily thereafter the content of viable cells/plate was counted in a hemocytometer after release at room temperature for a few minutes with isotonic saline containing 0.125 % trypsin and 0.3 mM EDTA, pH 7.8. Duplicate plates were usually taken for each time and at least 8 independent counts were made per plate.
of fetal calf serum. Fetal calf serum contains relatively large quantities of covalently bound sialic acid. Analysis by the method of Svennerholm [3], showed a value of 1.5 mg/ml of serum. This was released by treatment of fetal calf serum (4 ml) at 35” for 24 h with Vibrio cholerae neuraminidase (1 ml, 500 units). The incubation mixture was then dialysed overnight at 2°C against water to remove free sialic acid. The non-diffusible material was sterilized by passage through a Millipore filter (0.45 pm pore size) and added to Eagle minimal medium supplemented with 10 % tryptose phosphate broth and antibiotics to give 40 ml finally. Fresh neuraminidase (500 units, 1 ml) was added to this medium iust before use (Medium B). Control medium (A) lacking neuraminidase contained fetal calf serum (10 %) that had been incubated at 35°C and dialysed with&t the addition of neuraminidase. Preparation
Incovpovation experiments. Ceils growing exponentially on 3.5 mM Falcon plastic plates either in control medium A or medium B containing neuraminidase were incubated with [3H]glucosamine (20 &i/plate, spec. act. 526 ,uCi//Lmole). After 17 h of labelling, culture media (1.5 ml/plate) were removed from each plate, the cells were rapidly rinsed with cold 0.125 % trypsin and 0.3 mM EDTA in balanced salts solution nH 7.8 (1 ml) and finally detached bv incubation at loom temperature for a few minutes in the same trypsin solution (1 ml). Froteolysis was stopped by addition of fetal calf serum (0.2 ml) and the volume of the single cell suspension was adjusted to 2 ml. Portions (0.5 ml) were removed for cell counting and the remaining cell suspensions were centrifuged at 1 000 g at room temperature for 3 min. The trvpsinate supernatants (IS-ml) were keot for analysis and the-cell pellets were resuspended&in 10 mM Tris-HCI buffer (pH 7.4) containing 10 mM NaCl and 1.5 mM MgCI,. Estimation of sidic acid. Sialic acid was isolated from culture fluids, trypsinates or cell pellets either directly or after mild acid hydrolysis, by chromatography on Dowex 2, x 8 (acetate form) as described by Svennerholm [3j. To release sialic acid, fractions (1.5 ml) were diluted with N-HCl (0.2 ml) and water (0.3 ml) and heated at 80°C for 1 h. Each sample was then supplemented with carrier N-acetyl neuraminic acid (500 /Ag) and applied separately to columns (1.2 cm x 18 cm) containing resin. After elution with 0.1 N acetic acid (IO0 ml) to remove large quantities of unidentified radioactive compounds probably consisting largely of free glucosamine and desialylated glycoproteins. sialic acid was removed from the resin with i M so&m acetate buffer, pH 4.6 [3]. Column fractions were each 4.5 to 5.0 ml. Portions (2 ml) were analysed for carrier sialic acid by the ‘resorcinol method of Svennerholm [3]. Total recoveries were usually greater than 85 %. Other portions (0.5 ml) were diluted with a toluene-triton based scintillation cocktail for radioactive counting.
56 52 I 48/
?52
148 i
163
Fig. 1. Abscissa: time in culture (hours); or&ate: (left) I_, ceils/plate x 1OV; (right) ---* log cell no. -BHK 2I/CI 3 hamster fib&blasts, harvested at confluency by trypsinization were seeded onto 35 mM Falcon plastic plates (IO5 cells approx. per p]a?e). After 2-3 h incubation when most of the cells bad attached to the plastic surface, the incubation ,medium (1.5 ml/plate) was removed and replaced either with medium A lacking neuraminidase or medium B containing 31.5 units/plate of Vibrio ckolerae neuraminidase. The detailed compositions of these media are given in the text. Daily thereafter the content of viable cells per plate was counted in a hemocytometer after trypsinization. Each point represents at least eight separate determinations. 0. cells in medium A lacking ‘neuraminidase; , cells ‘m medium B containing neuraminidase. Growth was at 35 i-0.25”C and the medium of each dish was renewed each day untii the end of the experiment. The vertical range bars represent the range of cell densities obtained in 3 independent growth experiments; o ) weighted means calculated using all of the values determined experimentally at each time point.
ESULTS It is found that the initial rates of exponential growth of BHK cells are identical, regardless of the absence or presence of neuraminidase (fig. 1). The cell doubling time is about 14 h in each case. There is a small but significant increase in the cell numbers of cultures at confluency in media containing neuraminidase compared with control cultures. The question then arises whether in media containing neuraminidase, the complement of siahc acid at the surface of growing cells is much reduced compared with control cells. The experiments described in fig. 2 suggest
that there is a drastic reduction in these residues compared with cells grown in medium lacking neuraminidase. Table I summarizes the data. A substantial proportion (59 %) of t radioactive siahc acid of BNK. 21/C13 cehs cultured in medium containing ~e~ra~i~~dase and 3H-glucosamine was recovered in the culture fluids as the free sugar (fig. 2~3,ta 1). Presumably this material largely represents siahc acid residues accessible to neuraminidase at the surface of intact cells, since greater than 95 76 of the cehs were imperExpti Celi Res 85 (1974)
364
Hughes and Clark
Fig. 2. Abscissa: fraction no.; ordinate:
(left) o, cpm/fraction; (right) O, carrier sialic acid yglfraction. Radiolabelling and isolation of sialic acid residues of BHK 21/C13 cells growing in medium containing neuraminidase and control medium lacking the enzyme. Cells growing exponentially (see fig. 1) at 22 h either in medium B containing neuraminidase or in control medium A were incubated with 3H-glucosamine for 17 h. Culture media (1.5 ml each) were removed from each plate and cells were detached by incubation in trypsin solution. Proteolysis was stopped by addition of fetal calf serum. The single cell suspension was centrifuged at 1 000 g at room temperature for 3 min. The trypsinate supernatants were kept for analysis and the cell pellets were resuspended in 10 mM Tris-NC1 buffer (pH 7.4) containing 10 mM NaCl and 1.5 mM MgC&. Sialic acid was isolated from the culture fluids, trypsinates or cell pellets either directly or after mild acid hydrolysis, by chromatography on Dowex 2, x 8 (acetate form) as described in the text. Authentic N-acetyl neuraminic acid (500 ,ug) was added as carrier to each sample prior to chromatography. Fractions (4.5-5 ml) were analysed for radioactivity ( 0) or calorimetrically for sialic acid content (e). Only the latter parts of typical chromatographic profiles are shown: (a) culture fluids from control cells; (b) culture fluids from cells grown in media containing neuraminidase; (c) trypsinate from control cells; (d) trypsinate from cells grown in the presence of neuraminidase. The culture fluids were examined directly for their content of free sialic acid. The trypsinates were first hydrolysed in dilute acid to release bound sialic acid residues.
meable to trypan blue. No free radioactive sialic acid was found in the extracellular fluids of cells cultured in medium A (fig. 2a, table 1). A small quantity of covalently bound sialic acid was released after mild acid hydrolysis, perhaps originating from a few cells present in the medium or alternatively from glycoproteins that accumulate in extracellular fluids through membrane turnover during culture [4, 51. Sialic acid deriving from this material by neuraminidase action can account however at most to only 16% of the free radioactive sialic acid recovered from culture fluids of cells growing in medium B. The amount (59 %) of radioactive sialic acid present as the free sugar in extracellular fluids Exptl Cell Res 85 (1974)
containing neuraminidase is similar to other estimates (50 and 66 %) [6, 71, of the proportion of total cellular sialic acid that is released by prolonged treatment of BHK 211 Cl3 cells with neuraminidase, but is lower than that (78 % of total) reported by Ohta et al. [S]. Supporting evidence that the surface of BHK 21/C13 cells cultured in medium B (fig. 1) carried very low amounts of sialic acid residues comesfrom analysis of glycopeptides releasedfrom intact viable cells by crystalline trypsin. Since the mild conditions used to detach cells from the plastic plates retained almost complete cellular viability, the glycopeptides unquestionably reflect the chemical
T&k 1. Recovery of radioactive sialic acid from BHK 21/C13 cells grown in the presence or absence of neuraminidase Cells were grown and labelled as described in fig. 2. Sialic acid was isolated by chromatography on Dowex 50, either without (free sialic acid) or after (total siahc acid) mild acid hydrolysis. The amounts of radioactivity, corrected for losses, in the total sialic acid fractions recovered from culture fluids, trypsinates and cell pellets of cells removed from a plate are added together and the amounts of radioactive sialic acid in each fraction are expressed as percentages of the total. In the culture fluids of cells grown in the presence of neuraminidase it is assumed that the amount of free sialic acid is equivalent to the total amount. ND, not determined
BHM 2ljCl3 cells by trypsin together with that present on the purified surface membrane fraction of trypsinized ceils amounted to only 50 % of the total ce?lu.larsiahc acid. ISCUSSION
The exact role [I, a6, 18, 21, 221of cell surface sialic acid in the control of cellular unknown, but the following facts are relevant: (I) increased
cell electrophoretic
mobilitgi
is
associated with cell division 112, 131. Neuraminidase pretreatment of premitotic ceils - Neur-I-Neurlower their mobility. (2) The total levels of aminidase aminidase siahc acid in several cell types, including BHK Sialic % % 21/C13 relate to growth in culture. S&&c acid acid cpm total total cm increased just before division and decreased immediately after the onset of mitoses [14. Culture fluids Free 29 720 59 Total 4 560 12 ND 1.5,171.(3) Neuraminidase added TOcultures Trypsinates Total 2 215 6 120 0.25 of cells at confluency stimuiate a limited Cell associated Total 29 920 82 20400 40 round of division [16, 22, 231. Recovery of Our results suggest that the cyclic expressialic acid 36 695 50 240 sion at the cell surface of a large proportion of sialic acid residues is not an obligatory refor orderly cell division. The quirement nature of the total glycoprotein components of the outer cell surface. The glycopeptide approximate residence time of a siahc acid fraction released by trypsin from the surface residue appearing at the surface of a cell cuiof cells cultured in medium B with neur- tured (fig. 1) in medium containing neuraminidase contained less than 4 % (fig. 2c, aminidase (25.0 units/ml) n be calcuiated as total cellular content of sialic table 1) of the radioactive sialic acid associated follows. K 21/C13 has been variously with the comparable fraction from control acid in estimated as 0.46 pg [6], 0.60 pg [7] and cells cultured in medium A. adioactive sialic acid (40% of the total) 0.83 pug 971 per IO6 cells. A unit of neurthat remains associated with cells cultured in aminidase is defined as the amount of enzyme (fig. 2d, table 1) presumably required to release I ,ug of N-acetylneurer intracellularly, on membranes aminic acid from human E-acid glycoprotein 1 allowing [9] and in soluble nucleotide pools, or alter- at pH 5.5 and 37°C in 15 min. and temnatively at the cell surface but in components for the changed conditions of that are resistant to attack by trypsin, for perature and the unknown nature of the subexample glycoproteins in cryptic membrane strates, it seems unlike!y that a sites [I] or ganghosides [lo, 111. Chemical residue, newly appearing at the data [6] argues against the latter interpreta- periphery, remains in situ for longer than a tion of the results in table 1. The total sialic few minutes. Several i~~er~retat~o~s of our results are acid content in material released from intact Exptl
Ceil Res 85 (1974)
366
Hughes and Clark
possible: (1) The hypothetical important regulatory molecules contain the very small proportion (4 %) of sialic acid that can still be demonstrated as exposed at the periphery of BHK cells grown in neuraminidase containing media. It may be relevant that the reduced amount of sialic acid present at the periphery of mitotic cells in culture is distributed differently to the higher levels present in interphase cells [17, 181.In particular a glycopeptide enriched in sialic acid is present transiently in large amount in dividing BHK cells. The activity of a specific sialyl transferase catalysing the appearance of the transient sialoglycopeptide is increased in rapidly growing cells compared to stationary phasecells. Other, lessspecific sialyl transferase activities may be reduced in rapidly dividing cells or in transformed cell lines [18, 19, 211. However, the sialoglycopeptide is released from BHK cells by trypsin treatment [17, 181 suggesting an exposed site on the membrane, and the sialic acid residues are readily released from the trypsin solubilized material by neuraminidase [18]. There is no reason therefore why neuraminidase should not release sialic acid from this material during its transient appearance at the surface of mitotic BHK cells. (2) The hypothetical regulatory signals accompanying fluctuations in cellular sialic acid content may operate intracellularly. Chemical changes seenin surface membranes are found in internal membranes (mitochondria, nucleus and endoplasmic reticulum) as well [20, 211. In this caseexternally added neuraminidase would of course not show an effect on cellular control mechanisms. (3) Adjustment of cellular sialic acid levels may be required to re-direct important regulatory glycoproteins from intracellular sites to the cell surface. Once arrived at, it is of little consequence that the sialyl residues are present for the proper ‘functioning of these macromolecules. Exptl Cell Res 85 (1974)
The role of sialic acid residues in regulation of growth may best be resolved by isolation of stable genetic variants of BHK cells blocked in sialic acid metabolism, in which the levels of both intracellular as well as surface sialic acid contents can be stringently controlled. Such a class of mutants should be resistant to myxovirus infection since the specific receptor required for virus adsorption [9] is lacking, and their isolation and physiological characterization would be of great interest.
REFERENCES 1. Burger, M M, Curr top cell regul 3 (1971) 13.5. 2. Stoker. M & Macoherson. 1. Nature 203 (1964) 1355. ’ 3. Svennerhofm L, Acta them Stand 12 (1958) 547. 4. Warren, L, Curr top dev bio14 (1969) 197. 5. Hughes, R C, Sanford, B & Jeanloz, R W, Proc natl acad xi US 69 (1972) 942. 6. Buck, C A, Glick,. M C & Warren, L, Biochemistry 9 (1970) 4567. 7. Kraemer, P M, J cell physiol 67 (1966) 23. 8. Ohta, N, Pardee, A B, McAuslan, B R & Burger, M M, Biochim biophys acta 158 (1968) 98. 9. Hughes, R C, Prog biophys mol bio126 (1973) 189. 10. Klenk, E, The chemistry and biology of mucopolysaccharides (ed G E Wolstenholme & M O’Connor) p. 296. Churchill, London (1958). 11. Weinstein, D B, Marsh, J B & Glick, M C, J biol them 245 (1970) 3928. 12. Mayhew, E, J gen physio149 (1966) 717. 13. Brent, T P & Forrester, J A, Nature 215 (1967) 92. 14. Glick, M C, Comstock, C A & Warren, L, Biochim biophys acta 219 (1970) 290. 15. Glick, M C, Gerner, E W & Warren, L, J cell physiol 77 (1971) 1. 16. Vaheri, A, Rouslahti, E & Nordling, S, Nature new biol 238 (1972) 211. 17. Glick, M C & Buck ,C A, Biochemistry 12 (1972) 85. 18. Warren, L, Fuhrer, J P & Buck, C A, Proc natl acad sci US 69 (1972) 1838. 19. Grimes, W J, Biochemistry 9 (1970) 5083. 20. Meezan, E, Wu, H C, Black, P H & Robbins, P W, Biochemistry 8 (1969) 2518. 21. Tu, S-H, Nordquist, R E & Griffin, M Y, Biochim biophys acta 290 (1972) 92. 22. Fisher, H W & Yeh, J, Science 155 (1967) 581. 23. Casa, L V, Anat ret 172 (1971) 551. I
I
Received November 9, 1973 Revised version received December 17, 1973
.
,
Experimental
GROWTH
Cell Research 8.5 (1974) 362-366
OF BABY HAMSTER CONTAINING
KIDNEY
CELLS IN MEDIA
NEURAMINIDASE
R. C. HUGHES and JULIE CLARK’
Aktional Institute for Medical Research, Mill Hill, London NW7 IAA, UK
SUMMARY An established line of baby hamster kidney cells, BHK 21/C13 grows in media containing high concentrations of Vibrio cholerae neuraminidase at the same initial exponential rate as control cells grown in the absence of the enzyme. Glycoprotein fractions removed from the surface of cells grown with neuraminidase contain less than 4 % of the sialic acid present in similar fractions removed from control cells, The significance of these results are discussed in relation to the role suggested for sial glycoproteins in growth control. A small but significant increase was observed in the density of confluent cells in media containing neuraminidase compared with control cultures.
Recent suggestions [l] as to the importance of cell surface carbohydrates, and in particular sialic acid residues, in growth control of cells in culture have led us to carry out the experiments described in this paper. An established line [2] of baby hamster kidney cells, BHK 21/C13 has been grown in media containing Vibrio cholerae neuraminidase and the kinetics of growth have been compared to control cells incubated in media lacking the enzyme. MATERIALS AND METHODS Neuraminidase from Vibrio cholerae was purchased from Hoechst Pharmaceuticals, Kew Bridge, London. Growth of cells. Baby hamster kidney fibroblasts BHK 2l/C13 were grown at 35°C in Eagle minimal essential medium containing 10 % tryptose phosphate broth, penicillin (50 units/ml), streptomycin (100 pug/ ml) and 10 % fetal calf serum. At confluency the cells were removed from the surface by treatment at 35°C for a few minutes with 0.125 % trypsin in phosphate-
1 Present address: University of Cambridge, Cambridge, UK. Exptl Cell Res 85 (1974)
buffered saline (pH 7.8) containing 0.3 mM EDTA. The cells were replated onto 35 mM Falcon plastic plates at approx. IO5 cells/plate. After incubation for 2 to 3 h in medium as above, most of the cells had attached to the Dlastic surface. The incubationmedium (1.5 ml per plate) was removed and replaced either with medium A lackinn neuraminidase or medium B containing 37.5 units of Vibrio cholevae neuraminidase. Daily thereafter the content of viable cells/plate was counted in a hemocytometer after release at room temperature for a few minutes with isotonic saline containing 0.125 % trypsin and 0.3 mM EDTA, pH 7.8. Duplicate plates were usually taken for each time and at least 8 independent counts were made per plate.
of fetal calf serum. Fetal calf serum contains relatively large quantities of covalently bound sialic acid. Analysis by the method of Svennerholm [3], showed a value of 1.5 mg/ml of serum. This was released by treatment of fetal calf serum (4 ml) at 35” for 24 h with Vibrio cholerae neuraminidase (1 ml, 500 units). The incubation mixture was then dialysed overnight at 2°C against water to remove free sialic acid. The non-diffusible material was sterilized by passage through a Millipore filter (0.45 pm pore size) and added to Eagle minimal medium supplemented with 10 % tryptose phosphate broth and antibiotics to give 40 ml finally. Fresh neuraminidase (500 units, 1 ml) was added to this medium iust before use (Medium B). Control medium (A) lacking neuraminidase contained fetal calf serum (10 %) that had been incubated at 35°C and dialysed with&t the addition of neuraminidase. Preparation
Incovpovation experiments. Ceils growing exponentially on 3.5 mM Falcon plastic plates either in control medium A or medium B containing neuraminidase were incubated with [3H]glucosamine (20 &i/plate, spec. act. 526 ,uCi//Lmole). After 17 h of labelling, culture media (1.5 ml/plate) were removed from each plate, the cells were rapidly rinsed with cold 0.125 % trypsin and 0.3 mM EDTA in balanced salts solution nH 7.8 (1 ml) and finally detached bv incubation at loom temperature for a few minutes in the same trypsin solution (1 ml). Froteolysis was stopped by addition of fetal calf serum (0.2 ml) and the volume of the single cell suspension was adjusted to 2 ml. Portions (0.5 ml) were removed for cell counting and the remaining cell suspensions were centrifuged at 1 000 g at room temperature for 3 min. The trvpsinate supernatants (IS-ml) were keot for analysis and the-cell pellets were resuspended&in 10 mM Tris-HCI buffer (pH 7.4) containing 10 mM NaCl and 1.5 mM MgCI,. Estimation of sidic acid. Sialic acid was isolated from culture fluids, trypsinates or cell pellets either directly or after mild acid hydrolysis, by chromatography on Dowex 2, x 8 (acetate form) as described by Svennerholm [3j. To release sialic acid, fractions (1.5 ml) were diluted with N-HCl (0.2 ml) and water (0.3 ml) and heated at 80°C for 1 h. Each sample was then supplemented with carrier N-acetyl neuraminic acid (500 /Ag) and applied separately to columns (1.2 cm x 18 cm) containing resin. After elution with 0.1 N acetic acid (IO0 ml) to remove large quantities of unidentified radioactive compounds probably consisting largely of free glucosamine and desialylated glycoproteins. sialic acid was removed from the resin with i M so&m acetate buffer, pH 4.6 [3]. Column fractions were each 4.5 to 5.0 ml. Portions (2 ml) were analysed for carrier sialic acid by the ‘resorcinol method of Svennerholm [3]. Total recoveries were usually greater than 85 %. Other portions (0.5 ml) were diluted with a toluene-triton based scintillation cocktail for radioactive counting.
56 52 I 48/
?52
148 i
163
Fig. 1. Abscissa: time in culture (hours); or&ate: (left) I_, ceils/plate x 1OV; (right) ---* log cell no. -BHK 2I/CI 3 hamster fib&blasts, harvested at confluency by trypsinization were seeded onto 35 mM Falcon plastic plates (IO5 cells approx. per p]a?e). After 2-3 h incubation when most of the cells bad attached to the plastic surface, the incubation ,medium (1.5 ml/plate) was removed and replaced either with medium A lacking neuraminidase or medium B containing 31.5 units/plate of Vibrio ckolerae neuraminidase. The detailed compositions of these media are given in the text. Daily thereafter the content of viable cells per plate was counted in a hemocytometer after trypsinization. Each point represents at least eight separate determinations. 0. cells in medium A lacking ‘neuraminidase; , cells ‘m medium B containing neuraminidase. Growth was at 35 i-0.25”C and the medium of each dish was renewed each day untii the end of the experiment. The vertical range bars represent the range of cell densities obtained in 3 independent growth experiments; o ) weighted means calculated using all of the values determined experimentally at each time point.
ESULTS It is found that the initial rates of exponential growth of BHK cells are identical, regardless of the absence or presence of neuraminidase (fig. 1). The cell doubling time is about 14 h in each case. There is a small but significant increase in the cell numbers of cultures at confluency in media containing neuraminidase compared with control cultures. The question then arises whether in media containing neuraminidase, the complement of siahc acid at the surface of growing cells is much reduced compared with control cells. The experiments described in fig. 2 suggest
that there is a drastic reduction in these residues compared with cells grown in medium lacking neuraminidase. Table I summarizes the data. A substantial proportion (59 %) of t radioactive siahc acid of BNK. 21/C13 cehs cultured in medium containing ~e~ra~i~~dase and 3H-glucosamine was recovered in the culture fluids as the free sugar (fig. 2~3,ta 1). Presumably this material largely represents siahc acid residues accessible to neuraminidase at the surface of intact cells, since greater than 95 76 of the cehs were imperExpti Celi Res 85 (1974)
364
Hughes and Clark
Fig. 2. Abscissa: fraction no.; ordinate:
(left) o, cpm/fraction; (right) O, carrier sialic acid yglfraction. Radiolabelling and isolation of sialic acid residues of BHK 21/C13 cells growing in medium containing neuraminidase and control medium lacking the enzyme. Cells growing exponentially (see fig. 1) at 22 h either in medium B containing neuraminidase or in control medium A were incubated with 3H-glucosamine for 17 h. Culture media (1.5 ml each) were removed from each plate and cells were detached by incubation in trypsin solution. Proteolysis was stopped by addition of fetal calf serum. The single cell suspension was centrifuged at 1 000 g at room temperature for 3 min. The trypsinate supernatants were kept for analysis and the cell pellets were resuspended in 10 mM Tris-NC1 buffer (pH 7.4) containing 10 mM NaCl and 1.5 mM MgC&. Sialic acid was isolated from the culture fluids, trypsinates or cell pellets either directly or after mild acid hydrolysis, by chromatography on Dowex 2, x 8 (acetate form) as described in the text. Authentic N-acetyl neuraminic acid (500 ,ug) was added as carrier to each sample prior to chromatography. Fractions (4.5-5 ml) were analysed for radioactivity ( 0) or calorimetrically for sialic acid content (e). Only the latter parts of typical chromatographic profiles are shown: (a) culture fluids from control cells; (b) culture fluids from cells grown in media containing neuraminidase; (c) trypsinate from control cells; (d) trypsinate from cells grown in the presence of neuraminidase. The culture fluids were examined directly for their content of free sialic acid. The trypsinates were first hydrolysed in dilute acid to release bound sialic acid residues.
meable to trypan blue. No free radioactive sialic acid was found in the extracellular fluids of cells cultured in medium A (fig. 2a, table 1). A small quantity of covalently bound sialic acid was released after mild acid hydrolysis, perhaps originating from a few cells present in the medium or alternatively from glycoproteins that accumulate in extracellular fluids through membrane turnover during culture [4, 51. Sialic acid deriving from this material by neuraminidase action can account however at most to only 16% of the free radioactive sialic acid recovered from culture fluids of cells growing in medium B. The amount (59 %) of radioactive sialic acid present as the free sugar in extracellular fluids Exptl Cell Res 85 (1974)
containing neuraminidase is similar to other estimates (50 and 66 %) [6, 71, of the proportion of total cellular sialic acid that is released by prolonged treatment of BHK 211 Cl3 cells with neuraminidase, but is lower than that (78 % of total) reported by Ohta et al. [S]. Supporting evidence that the surface of BHK 21/C13 cells cultured in medium B (fig. 1) carried very low amounts of sialic acid residues comesfrom analysis of glycopeptides releasedfrom intact viable cells by crystalline trypsin. Since the mild conditions used to detach cells from the plastic plates retained almost complete cellular viability, the glycopeptides unquestionably reflect the chemical
T&k 1. Recovery of radioactive sialic acid from BHK 21/C13 cells grown in the presence or absence of neuraminidase Cells were grown and labelled as described in fig. 2. Sialic acid was isolated by chromatography on Dowex 50, either without (free sialic acid) or after (total siahc acid) mild acid hydrolysis. The amounts of radioactivity, corrected for losses, in the total sialic acid fractions recovered from culture fluids, trypsinates and cell pellets of cells removed from a plate are added together and the amounts of radioactive sialic acid in each fraction are expressed as percentages of the total. In the culture fluids of cells grown in the presence of neuraminidase it is assumed that the amount of free sialic acid is equivalent to the total amount. ND, not determined
BHM 2ljCl3 cells by trypsin together with that present on the purified surface membrane fraction of trypsinized ceils amounted to only 50 % of the total ce?lu.larsiahc acid. ISCUSSION
The exact role [I, a6, 18, 21, 221of cell surface sialic acid in the control of cellular unknown, but the following facts are relevant: (I) increased
cell electrophoretic
mobilitgi
is
associated with cell division 112, 131. Neuraminidase pretreatment of premitotic ceils - Neur-I-Neurlower their mobility. (2) The total levels of aminidase aminidase siahc acid in several cell types, including BHK Sialic % % 21/C13 relate to growth in culture. S&&c acid acid cpm total total cm increased just before division and decreased immediately after the onset of mitoses [14. Culture fluids Free 29 720 59 Total 4 560 12 ND 1.5,171.(3) Neuraminidase added TOcultures Trypsinates Total 2 215 6 120 0.25 of cells at confluency stimuiate a limited Cell associated Total 29 920 82 20400 40 round of division [16, 22, 231. Recovery of Our results suggest that the cyclic expressialic acid 36 695 50 240 sion at the cell surface of a large proportion of sialic acid residues is not an obligatory refor orderly cell division. The quirement nature of the total glycoprotein components of the outer cell surface. The glycopeptide approximate residence time of a siahc acid fraction released by trypsin from the surface residue appearing at the surface of a cell cuiof cells cultured in medium B with neur- tured (fig. 1) in medium containing neuraminidase contained less than 4 % (fig. 2c, aminidase (25.0 units/ml) n be calcuiated as total cellular content of sialic table 1) of the radioactive sialic acid associated follows. K 21/C13 has been variously with the comparable fraction from control acid in estimated as 0.46 pg [6], 0.60 pg [7] and cells cultured in medium A. adioactive sialic acid (40% of the total) 0.83 pug 971 per IO6 cells. A unit of neurthat remains associated with cells cultured in aminidase is defined as the amount of enzyme (fig. 2d, table 1) presumably required to release I ,ug of N-acetylneurer intracellularly, on membranes aminic acid from human E-acid glycoprotein 1 allowing [9] and in soluble nucleotide pools, or alter- at pH 5.5 and 37°C in 15 min. and temnatively at the cell surface but in components for the changed conditions of that are resistant to attack by trypsin, for perature and the unknown nature of the subexample glycoproteins in cryptic membrane strates, it seems unlike!y that a sites [I] or ganghosides [lo, 111. Chemical residue, newly appearing at the data [6] argues against the latter interpreta- periphery, remains in situ for longer than a tion of the results in table 1. The total sialic few minutes. Several i~~er~retat~o~s of our results are acid content in material released from intact Exptl
Ceil Res 85 (1974)
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Hughes and Clark
possible: (1) The hypothetical important regulatory molecules contain the very small proportion (4 %) of sialic acid that can still be demonstrated as exposed at the periphery of BHK cells grown in neuraminidase containing media. It may be relevant that the reduced amount of sialic acid present at the periphery of mitotic cells in culture is distributed differently to the higher levels present in interphase cells [17, 181.In particular a glycopeptide enriched in sialic acid is present transiently in large amount in dividing BHK cells. The activity of a specific sialyl transferase catalysing the appearance of the transient sialoglycopeptide is increased in rapidly growing cells compared to stationary phasecells. Other, lessspecific sialyl transferase activities may be reduced in rapidly dividing cells or in transformed cell lines [18, 19, 211. However, the sialoglycopeptide is released from BHK cells by trypsin treatment [17, 181 suggesting an exposed site on the membrane, and the sialic acid residues are readily released from the trypsin solubilized material by neuraminidase [18]. There is no reason therefore why neuraminidase should not release sialic acid from this material during its transient appearance at the surface of mitotic BHK cells. (2) The hypothetical regulatory signals accompanying fluctuations in cellular sialic acid content may operate intracellularly. Chemical changes seenin surface membranes are found in internal membranes (mitochondria, nucleus and endoplasmic reticulum) as well [20, 211. In this caseexternally added neuraminidase would of course not show an effect on cellular control mechanisms. (3) Adjustment of cellular sialic acid levels may be required to re-direct important regulatory glycoproteins from intracellular sites to the cell surface. Once arrived at, it is of little consequence that the sialyl residues are present for the proper ‘functioning of these macromolecules. Exptl Cell Res 85 (1974)
The role of sialic acid residues in regulation of growth may best be resolved by isolation of stable genetic variants of BHK cells blocked in sialic acid metabolism, in which the levels of both intracellular as well as surface sialic acid contents can be stringently controlled. Such a class of mutants should be resistant to myxovirus infection since the specific receptor required for virus adsorption [9] is lacking, and their isolation and physiological characterization would be of great interest.
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Received November 9, 1973 Revised version received December 17, 1973
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