Synthesis of complex saccharides by synchronized NIL-8 hamster cells

Experimental

SYNTHESIS

OF

COMPLEX

SYNCHRONIZED

NIL-8

E. A. DAVIDSON’ Imperial

Cancer

9.5 (1975) 218-222

Cell Research

Research

Fund

SACCHARIDES HAMSTER

BY

CELLS

and I. MacPHERSON Laboratories,

London

WC2A

3PX,

UK

SUMMARY Complex saccharide synthesis by synchronized NIL-8 cells was studied by metabolic labeling with [3H]glucosamine. Hyaluronic acid, a chondroitin sulfate and heparan sulfate are produced during G 1, S and G 2/M but the latter is absent or altered in media during G 2/M. Glucosamine is the sole amino sugar in cetylpyridinium bromide precipitable glycopeptides except for G 1 cell associated material; CPB-soluble glycopeptides contained label in both glucosamine and galactosamine in contrast to products of NIL-8 cells transformed by hamster sarcoma virus (HSV) in which galactosamine was absent from the glycopeptide fractions. The transformed cells synthesized hvaluronic acid, chondroitin sulfate and heparan sulfate in amounts comparable to those found in the NIL-8 line.

The role of cell surface saccharides

in a wide variety of biological events such as adhesion, reception and recognition of stimuli has been extensively investigated. In addition, several reports have noted the presence of sulfated polysaccharides in cultured cells, their production by a variety of cell types and their probable association with the plasma membrane [ 1, 23. Here we describe the synthesis of several polysaccharides as a function of the cell cycle in cultured NIL cells. MATERIALS

AND METHODS

The NIL-8 line is a clone derived from the hamster tibroblast line NIL-2E [3, 41. It is blocked in the G 1 phase of the cell cycle in medium containing small amounts of serum. The cells were routinely maintained of Biological 1 Present address: Department Chemistry, The Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, PA 17033, USA. Exptl

Cell

Res 95 (1975)

in Dulbecco’s modification of Eagle’s medium with 10% calf serum. The NIL-8 HSV cells were obtained after transformation of NIL-8 cells with hamster sarcoma virus [5] and cultured in a similar fashion. The NIL-8 cells were svnchronized bv arowth in low serum followed by hydroxyurea according to the procedure of Critchley 161. Data are summarized in tig. 1. Based on [3H]thymidine incorporation data and mitotic indices, cells synchronized at a densitv of 3x I@/5 cm dish were exposed to [3H]glucosamine (5 &i/ml) and 35,,=(30 pCi/ml) for the periods O-18 h (2 mM hydroxyurea, 10% serum), 18-24 h and 24-30 h (regular medium) following release from low serum. These periods approximately correspond to G 1, S and G 2/M, respectively. Attempts to synchronize a line of hamster sarcoma virus transformed NIL cells (NIL-8 HSV) utilizing the low serum-hydroxyurea technique described above were not successful due in part to the insensitivity of these cells to the hydroxyurea concentration employed (E. A. Davidson, unpublished observations). However, sequential exposure to cytochalasin B (2 pg/ml, 48 h [7]) and hydroxyurea (5 mM, 18 h) both in 10% serum medium, followed by release into regular medium resulted in reasonablv effective svnchronization as indicated both by thymidine incorporation and by mitotic indices (fig. 2). Since the results obtained with these cells were somewhat less well defined than those for the NIL cells, detailed analyses were carried out during S phase only, O-6 h follovving release from hydroxyurea. Following the appropriate incubation period, media

Saccharides

of synchronized

NIL-8

cells

Table 2. Distribution of r3H]label 0.4 M NaCl eluate fractions

‘4 72

\ b

219 in the

C&r-NH,”

Gal-NH,

Sialic acidb

98t 98+ 98+

<1
9.9 11.6 19.4

87 98+ 98+

13
6.3

Media

I

Gl :2,M CeN associated:

I

h!!~-*Icy

\

1

I

I

I

0 I 2 3 4 5 6 7 8 9 IO Figs I, 2. Abscissa: time post hydroxyurea (hours); ordinate: (left) mitotic counts per field (O--O); (right) [aH]TdRX lO-3 (@a). Fig. I. Synchrony data for NIL-8 cells following exposure to low serum (0.3 %) and 2 mM hydroxyurea in 10% serum medium. Mitotic counts were performed every 2 h until hour 6 and hourly thereafter. [3H]Thymidine incorporation into acid-precipitable material was measured in 1 h windows as indicated. Following incubation of 3X 105 cells in 2.5 ml of medium containing 0.1 &i/ml of [3H]thymidine, the media was removed by aspiration, the cells washed 2X with phosphate-buffered saline and Iysed with I % sodium dodecyl sulfate. After addition of 2 mg of bovine serum albumin, the lysates were mixed with an equal volume of 20% trichloracetic acid, allowed to stand at 0°C for 1 h and centrifuged. The precipitate was washed 2x with cold 10% trichloracetic acid and once with ethanol. After drying in air the pellets were dissolved in base and aliquots counted. Values are the average of at least duplicates; in no instance did duplicated samples differ by more than 20%.

Table 1. Distribution of [3H]label and cell associated fractions

CPB soluble” r3H] (X lO-B)

in media

Gl :2,M

::2

(2 The figures for glucosamine and galactosamine are as percentages of label in the combined amino sugar fraction. b The sialic acid figure is as percentage of total radioactivity in the salt eluate. This was determined by acid hydrolysis and separation of the released sialic acid on an anion exchange column according to the procedure of Horvat $ Touster [II]. Substantially identical results were obtained by assay of neuraminidase sensitive radioactivity.

was removed from the cells by decantation and saved. The cells were harvested by mechanical means and washed with phosphate-buffered saline; material remaining with the cell pellets following the saline wash is termed ‘cell associated. The media and cell associated saccharides were separated by salt elution from cetylpyridium bromide (CPB) precipitates following the general procedures described by Satoh et al. [I]. Labeled material not precipitated by the cationic detergent is almost exclusively glycopeptide in nature and contained about 85% of the tritium label but negligible sulfate. This fraction was examined for amino sugar composition and sialic acid content but not further characterized.

Salt eluate fractions r3H] (X10-a) 0.4 M

0.8 M

1.2 M

824

356

178

7.29 8.43

566 534

209 135

108 95

1.57 0.82 1.00

89 73 76

58 62 58

30 30

Media

Gl :2/M

13.3

Cell associated

Gl :2/M

u Figures represent total non-dialysable tritium counts from twelve 50 mm dishes (3.5~105 cells/dish) for each phase of the cell cycle examined. Negligible radioactivity was present in the 2.0 M salt extract. 15-i51813

2. Synchrony data for NIL-8 HSV cells after exposure to cytochalasin B and hydroxyurea. Experimental details are as described for fig. 1.

Fig.

Exprl

Cell

Res 95 (1975)

220

Davidson

and MacPherson HA

CSA

GA

4

i

4

Table 4. Distribution soluble glycopeptides

4-

of [“HIlabel

in CPB

Glu-NHZb Gal-NH,

Sialic acid”

78 75 65

22 25 35

5.3 5.5 7.9

21 26 >lO

6.3 5.9 3.6

Media”

Gl Z2,M

Cell associated Gl

S G 2/M IO

30

20

40

50

fraction no.; ordinate: [3H]cpmx IF. Chromatography of the 0.4 M salt eluate fraction from G 1 media obtained from NIL-8 cells on a 0.7~70 cm column of CPG 10-240 porous glass beads. The eluting solvent was 0.5 M CaCI,; 0.8 ml fractions were collected at a flow rate of 14 ml/h. The marker saccharides (HA, hyaluronic acid from vitreous humor; CSA, chondroitin+sulfate from porcine costal cartilage and GA, glucuronic acid) were detected by the orcinol reaction [ 121; peak tubes are indicated. Fig. 3. Abscissa:

Table 3. Distribution

of [3H]label in polysaccharide components Hyaluronic acidb

HeparanSO,e

Chondroitinsop

Media” Gl

41.7

9.2 (7.6)

26.7 (23.0)

:2/M

30.5 30.6

Cl 14.1 (12) (1.2)

22.1 21.1 (24.6) (21.4)

11.1 (8.9) 16.7 (15.1) 11.3+ (16.2)

21.5 (25.3) 10 (9.1) 12.6-t (14.7)

Cell associated

Gl S G 2/M

30.4 38.2 37.5

(1 Data are for synchronized NIL-8 cells. * Figures for hyaluronic acid are based on hyaluronidase sensitive, glucosamine containing high molecular weight material. c Figures for heparan sulfate are based on heparitinase sensitive, glucosamine containing material. d Figures for chondroitin sulfate are based on chondroitinase sensitive galactosamine containing material. For the latter two fractions, the percentage of dialysable [YS]O, counts following enzymatic digestion is indicated in parentheses. fipti

Cell

Res 95 (1975)

79 74 90+

1. Data are for synchronized NIL-8 cells. b The figures for glucosamine and galactosamine are as percentages of label in the combined amino sugar fraction. c The sialic acid figure is as percentage of total radioactivity in the salt eluate. This was determined as described for table 2. Substantially identical results were obtained by assay of neuraminidase sensitive radioactivity. Amino sugars were fractionated on a 0.9~50 cm column of cation exchange resin following hydrolysis for 18 h in vacua with 4 N HCI. After removal of the HCI in vacua, 5 PM each of glucosamine and galactosamine were added, the samples transferred to the column and eluted with 0.3 N HCI [8]. Aliquots of 0.75 ml were neutralized with NaOH and analysed for amino sugar by the Elson-Morgan reaction [9]. Fractions of the appropriate tubes were assayed for radioactivity in a liquid scintillation counter. The susceptibility of individual polysaccharide fractions to enzymatic digestion was assessed by measuring the loss in labeled material after incubation with the appropriate enzyme and dialysis against water. Testicular hyaluronidase was obtained from Worthington Inc.; chondroitinase ABC from Miles Laboratories; Vibrio cholerae neuraminidase from Calbiochem. Heparitinase was a gift from Dr Alfred Linker to whom we are indebted. Glass bead exclusion columns were calibrated with polysaccharide standards of known molecular weight. Chemicals and solvents were reagent grade. Polysaccharide identification was based on several criteria including amino sugar composition, charge properties, molecular size and enzymatic susceptibility.

RESULTS The total incorporation of [3H]glucosamine into the salt eluate fractions of the NIL cells is summarized in table 1. As observed in other systems, the bulk of the anionic saccharides precipitated by cetylpyridinium bromide appear in the media frac-

Saccharides

Table 5. Distribution

of [3H]label

in saccharides

of NIL-HSV

CPB soluble

Mediaa Cell associated

of synchronized

NIL-8

221

cells

cells

0.4 M NaCI eluate

[3H]total (X 10-B)

Glu-NH: cw

Gal-NH, cm

Sialic’ acid

pHJtotal (X 1W)

Glu-NH, (%I

Gal-NH, (%I

Sialic acid

23 16.6

98+ 98+


7.1 8.0

2.67 3.06

98+ 98-t


8.0 10.0

LI Data are for S phase ‘synchronized’ NIL-8 HSV cells. ’ The figures for glucosamine and galactosamine are as percentages of label in the combined amino sugar fraction. c The sialic acid figure is a percentage of total radioactivity in the salt eluate. This was determined as described for table 2. Substantially identical results were obtained by assay of neuraminidase sensitive radioactivity.

tions (82-88 %) with the proportion of the CPB-precipitable material ranging from 10 to 13 % of the non-dialysable radioactivity. There seems to be little difference in the incorporation of [3H]glucosamine label between S and G 2/M but during these phases of the cell cycle apparently more glycoprotein/proteoglycan is synthesized per cell unit time than during G 1. A typical fractionation of the 0.4 M salt eluate fraction by porous glass bead exclusion chromatography is illustrated in fig. 3. The high molecular weight peak present in the 0.4 M fraction accounts for about 40% of the glucosamine label and was characterized as hyaluronic acid on the basis of its molecular size, complete susceptibility to hyaluronidase digestion and identification of glucosamine as the sole amino sugar present (table 2). The lower molecular weight peak contained all of the sialic acid present in the 0.4 M fraction and represents mixed glycopeptide components . In general, the precipitated glycopeptides contained 20-40 % of the glucosamine label; the remainder was distributed between hyaluronic acid, chondroitin sulfate and heparan sulfate (table 3). Approx. 25% of the label present in the 1.2 M media salt eluate fraction G 1 and S is found in heparan

sulfate but the corresponding G2/M fraction does not contain any label in this polysaccharide. Since some heparan sulfate is present in the 0.8 M fraction during all phases of the cell cycle, this observation may reflect a difference in degree of sulfation or proteoglycan molecular size of material lost from the cells during G2/M. In general, the cell associated sulfated polysaccharides contained a higher proportion of heparan sulfate than is found in the media although the latter fraction is greater in magnitude. The distribution of label in amino sugar and sialic acid for the soluble glycopeptides is summarized in table 4. The NIL-HSV cells exhibited several interesting features insofar as glycoprotein and proteoglycan synthesis are concerned. As indicated in table 5, nearly comparable amounts of label were found in the media and cell associated soluble glycopeptide fraction and 0.4 M salt eluate. This is in sharp contrast to the distribution observed for NIL-8 cells described above. In addition, galactosamine appeared to be absent in both the soluble and 0.4 M salt eluate fractions suggesting the complete absence of glycoproteins containing this amino sugar. This is not due to a failure to synthesize galactosamine itself since the higher salt eluates contain predominantly chonEkptl

Cell Res 95 (1975)

m

Davidson

2L2

and MacPherson

droitin sulfate. Hyaluronic acid, heparan sulfate and a chondroitin sulfate were all produced by the transformed line. The proportion of cell associated material was higher for the NIL-HSV line in all fractions except the 2.0 M salt eluate which contained only chondroitin sulfate. DISCUSSION It is of interest that glucosamine is the sole amino sugar present in the 0.4 M glycopeptide fraction except for the cellassociated G 1 material which contains 13 % galactosamine. The macromolecule which contains the galactosamine is either degraded to fragments too small to be recovered by the isolation procedure employed or loses sufficient sialic acid to be soluble in the presence of the cationic detergent precipitant. In either case, it seems clear that a sialylated glycoprotein is synthesized during G 1 that is not produced during either S or G2/M. Further identification of the galactosamine containing component was not attempted due primarily to the limited amounts of material available. It may be concluded from the data in tables 2 and 4 that the more highly sialylated glycoproteins contain exclusively glucosamine as the amino sugar component (with the exception noted above) but that significant galactosamine is present in the soluble glycopeptide fraction. The low percentage of label found in the galactosamine of the cell associated G2/M soluble

Exprl

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Res 95 (1975)

glycopeptide fraction may reflect an altered synthetic capacity during this phase of the cell cycle. It is tempting to speculate that the galactosamine containing glycoprotein synthesized during G 1 by NIL cells is related to the transformation sensitive protein described by Hynes [ lo]. This macromolecule is either not synthesized by transformed cells or is degraded to very small fragments at a sufficiently high rate so as to escape detection by the methods employed. Any possible relationship of this material to the initiation of S phase biosynthetic activities and phenomena such as contact inhibition awaits further study. This work was supported in part by USPHS Grant CAl5483.

REFERENCES I. Satoh, C, Banks, J, Horst, P, Kreider, J W & Davidson, E A, Biochemistry 13 (1974) 1233. 2. Kraemer, P M, J cell biol 55 (1972) 713. 3. Diamond, L, Int j cancer 2 (1967) i43. 4. McAllister, R M & MacPherson. I A, J gen virol 2 (1968) 99. 5. Zavada, J & MacPherson, I A, Nature 225 (1970) 24. 6. Critchley, D R, Chandrabose, K A, Graham, J M & MacPherson, I A, Cold Spring Harbor symp Grant biol (ed B Clarkson & R Baserea). New York (1974). 7. Westermark, B, Exp cell res 82 (1973) 341. 8. Gardell, S, Acta them Stand 7 (1953) 207. 9. Elson, L A & Morgan, W T J, Biochem j 27 (1933) 1824. 10. Hynes, R 0, Proc natl acad sci US 70 (1973) 3170. 11. Horvat, A & Touster, 0, J biol them 243 (1968) 4380. 12. Brown, A H, Arch biochem I 1 (1946) 269. Received February 18, 1975 Revised version received April 25, 1975