Inr. J. Biochem. Vol. 22, No. 8, pp. 889-893, Printed in Great Britain. Ail rights reserved
0020-711X/90 $3.00+0.00 Copyright C 1990PergamonPressplc
EFFECT OF RETINOIC ACID ON TWO GLYCOSYLTRANSFERASE ACTIVITIES IN C6 CULTURED GLIOMA CELLS PASCALREBOUL,PIERREBR~U~T,* PASCALGEORGE and PIERRE LOWSOTS INSERM U. 189, Laboratoire de Biochimie G&&ale et Midicale, Faculti de M&de&e Lyon-Sud, B.P. 12. 69921 Ouliins, France [Tel. 7851-08-26: Fax 7850-71-521 (Received 10 January 1990)
Abstract-l. Activity of two glycosyltransferases was studied in retinoic acid-treated C6 cultured glioma cells. 2. The fi-galactoside a 2,3+ialyltransferase transferring N-acetylneuramin onto the O-glycans residues of glycoproteins was activated up to twice after chronic treatment (from 24 to 96 hr) with all-trans retinoic acid. 3. No effect was observed for shorter treatments. 4. On the opposite, the N-glycan galactosyltransferase activity remained unchanged whatever the length of retinoic acid treatment was. 5. The activatory effect was not dependent on isomery, as all-trans and 13-cis retinoic acid isomers were both activators of the C6 glioma cell sialyltransferase. 6. Measurement of adhesion of retinoic acid-treated cells using labelled plasma membranes showed an enhancement of adhesion in correlation with enhancement of sialyltransferase activity.
melanoma cell lines both to enhancement of gp160 and to activity of an asialofetuin sialyltransferase. In previous studies, we have demonstrated the existence in nervous tissues of two sialyltransferases transfering N-acetylneuraminic acid (NeuAc) from CMP-NeuAc onto O-glycans, namely a /I-galactoside ~2,3_sialyltransferase and a a-N-acetylgalactosaminide or2,&sialyltransferase in addition to N-glycan sialyltransferase (Baubichon-Cortay er al., 1986a, 1989a,b). In cultured C6 glioma cells, the a-N-acetylgalactosaminide r2,6-sialyltransferase was not present, while under definite conditions, we were able to measure solely the activity of the /?-galactoside ct2,3_sialyltransferase. Retinoic acid has been demonstrated to have some effects onto C6 glioma cells, inhibiting growth and inducing morphological changes (Fischer et al., 1987). However, these effects have not been related to biochemical changes. In the present paper, we have studied the P-galactoside a2,3+ialyltransferase activity in retinoic-treated C6 cells. The activity of a N-glycan glycosyltransferase, the ovomucoid galactosyltransferase was also performed as a control.
acid and synthetic analogs of vitamin A can affect the proliferation of many cultured cells, including embryonic carcinoma, melanoma, leukemia, epithelial and neuroblastoma cells (Lotan, 1980). In addition, retinoids often induce cellular differentiation, accompanied by the suppression of cellular properties associated with the transformed phenotype, such as anchorage-inde~ndent growth (Dion et al., 1978). Such properties of retinoids have been used for prevention and treatment of various cancers (Hill and Grubbs, 1982; Sporn, 1977). Retinoids’ mechanism(s) of action is still unclear. Their ability to modulate cellular differentiation indicates that they act at the level of the nucleus (Takahashi and Breitman, 1989). Pioneer work of Lotan et al. (1980) have demonstrated in HeLa cells changes in surface glycoproteins induced by retinoic acid. These authors have also demonstrated specific changes in cell surface glycoconjugates of S91 murine melanoma retinoic acid-treated cells (Lotan et al., 1983) and enhancement of a N-glycan glycoprotein with M, equal to 160,000 (“gpl60”). More recently, Lotan et al. (1988) have correlated the growth inhibition of two murine Retinoic
MATERIALS AND METHODS
*Charge de Recherche au Centre National de la Recherche Scientifique. tTo whom all correspondence should be addressed. Abbreuiufions: NeuAc, N-acetylneuraminic acid; Gal, galactose; GalNac, N-acetyl-galactosamine; DMEM, Dulbecco-modified Eagle Minimum Essential Medium; FCS, fetal calf serum; CMF, calcium and magnesiumfree Hank’s solution buffered with 20 mM Heoes 14-(2hydroxyethyi)-l-piperazineethanesulfonic acidj, pI? 714; CMF-A, the same solution containing 5 mgjml of lipidfree serum albumin.
All reagents were of analytical grade. Most of reagents such as fetuin (grade III), all-trans and 134s j?-retinoic acid and retinyl acetate were obtained from Sigma (St Louis, U.S.A.; CMP-[‘%]NeuAc (sp. act. 300 Ci/mol, Il. 1 GBq/mmol) and UDP-[14C]galactose (UDP-[‘4C]Gal) (sp. act. 273 Ci/mol, IO.IGBq/mmol) were from New England Nuclear (Boston, U.S.A.); [‘Hlthymidine (sp. act. 5 Ci/mol, 0.18 TBq/mol) and [3H]leucine (140 Ci/mol, O.l8TBq/mmol) from the Commissariat ri 1’Energie Atomique (CEA). Asialofetuin was obtained by mild acid hydrolysis of feutin (50 mM H,SO,, 80°C 60 min).
C6 glioma cells obtained from Dr G. Rebel (Centre de Neurochimie du CNRS, Strasbourg, France) were routinely cultivated in Costar 75 cm* flasks at 36.K in 95% O,-5% CO,. The medium was Dulbecco-modified Eagle minrmum essential medium (DMEM, Flow Laboratories) containing 200 U/ml of penicillin and 2OOpg/ml of streptomycin (G&co) and supplemented with 10% heat inactivated fetal calf serum (I.B.F., France). Cultures at SO-100% confluency were incubated in the dark for various periods of time with retinoic acid in DMEM medium without fetal calf serum (FCS). The cells were then rinsed with Tris-HCl lOmM, pH 7.0, NaCl 8.4 g/l, scraped off with a rubber policeman, and washed again with same buffer by pelleting 10min at 12OOg. Enzymatic assays were performed on-the pellets susnended in 2 ml of 50 mM Tris-HCl. oH 7.2 (Tris buffer) with a Potter--Elvehjem homogenizer. viability‘of the cell$ was routinely controlled by Trypan blue exclusion. ~~aly~tran~~rase (EC 24.99.4) assaq
The standard assay medium contained (BaubichonCortay, 1986a) in a total volume of 250~1:200~1 of Tris buffer containing cell homogenate, 400 pg of asialofetuin, 0.5% Triton X-100, 5 mM MnCl,, 40 mM Mes pH 6.0 and CMP[‘4C]NeuAc (50 nCi). The incubation was performed at 2O’C for 120 min, and the reaction stopped with 2 ml of a mixture of trichloroa~tic acid (10% w/v) and phosphotungstic acid (5% w/v) (2 ml). The precipitate was filtered on GF-B Whatman filters and radioactivity counted as described previously (Broquet et al., 1975). Assays were performed in duplicates and results expressed as pmol of [‘“C]NeuAc transferred after 120min related to I mg of cellular proteins or to 10’ cells. Galactosyltransferase (EC 126.96.36.199) assay
The standard assay medium contained in a total volume of 250 it I : 200 p 1 of Tris buffer containing cell homogenate, 4OOpg of ovomucoid, 0.5% Triton X-100, 5mM MnCl,, 40mM Mes pH 6.0, AMP 4.8mM and UDP-[‘4C]Gal (50 nCi). The incubation was performed at 25°C for 30 min, and the reaction stopped as described for the sialyltransferase. Pr~pararion qf ~abeiled membranes fur bjndj~g assays
Cell membranes were prepared according to Santala (I/ a/. (1977). Briefly, the cells (l-2 x IO8cells) were labelled with [H]le&ine (jpCi/75cmi flask) for 48’hr. Cells were then carefully collected using 4 ml/flask of Ca*+ Mg2+-free Hank’s solution buffered with Hepes 20mM pH 7.4 (“CMF”) cooled to 4-C, pelleted at ISOg for Smin and washed once with 1Oml of CMF. The cells were then resuspended in CMF containing 5 mg/ml bovine serumalbumin (“CMF-A”) plus 0.1 mg/ml of DNAse and were disrupted with a Dounce homogenizer. The homogenate was pelleted at 39,OOOnfor 20 min. The pellet was layered at the bottom of a di~ontinuous gradient and centrifuged as described (Santala er al., 1977). The layer at 2540% sucrose interface was collected and used as the labelledmembrane source. Membrane binding assays
The membrane binding assays were performed according to Santala er ul. (1977)and Santala and Glaser (1977). Cells treated or not with retinoic acid were collected and carefully resuspended in 3 ml of CMF-A. An aliquot was taken out for glycosyltransferase assays. To 0.5 ml of the suspension were added 10~1 of the labelled membranes (60,OOOdpm) and incubation was monitored at 37°C for 0, 15 and 30 min. Reaction was stopped by addition of 0.75 ml of cold CMFA and pelleting 5 min at 1508. The pellet, rinsed without stirring with 1 ml of CMF-A was solubilized in I ml of 1%
Triton X-100. The radioactivity was determined in a scintillation counter using Packard E299 ~intillation medium. High performance liquid chromatography analysis qf retinoic acid isomers
Retinoic acid isomers were analysed according to Annesley et al. (1984) using a Hypersil ODS column (5 pm, I/4” x 150 mm). Elution was performed with solution A (0.5% acetic acid in acetronitrile) and B (0.5% acetic acid in water). The elution gradient was as following: from 0 to 4 min, 70% solution A; from 4 to 8 min. linear change from 70% to 90% solution A; from 8 to I5 min 90% solution A; from 15 to 16min, linear change from 90 to 70% solution A, all at constant flow (1 ml/min). Detection was performed at 360 nm with a Lambda max Waters detector. The preparation of samples was performed in the dark. To I ml of culture medium, was added 50 ~1 of retinyl acetate (lOmg/ml in acetonitrile) as standard. After shaking, 2.5ml of phosphate buffer I M, pH 6.0 and 6.0ml of ethylic ether were added. After shaking for I5 min and centrifugation, the ether layer was taken out, dried under nitrogen stream and resuspended in 100~1 of solution A. Standards were prepared with 13-cis and all-trans retinoic acid isomers. Other assays
Nucleoside diphosphate pyrophosphatases and CMPNeuAc hydrolase were assayed as described by Mookerjea and Yung (1975). Protein was measured by the bicinchoninic acid method (Smith et al., 1985). RESULTS
The sialyltransferase studied in this work transfers NeuAc from CMP-NeuAc onto desialylated O-glycans of glycoproteins. Comparison with sialyltransferases from rat brain indicates that this enzyme is similar to the j?-galactoside ~2,3-sialyltransferase described by Baubichon-Cortay et al. (1989b) which transfers NeuAc in ct2-3 linkage on Gal of the Gal-GalNAc 0-glycannic structure of asialofetuin giving a monosialotrisaccharide. The galactosyltransferase transfers galactose from UDP-Gal onto free terminal ~-acetyIg~ucosaminy1 residues of N-glycans of ovomucoid. We have studied the effect of the retinoic acid when added to the culture medium of C6 glioma cells. Previous experiments were conducted with different concentrations of all-rrans retinoic acid for 96 hr. Sialyltransferase activity was increased for retinoic acid concentrations > lo-’ M. Subsequent experiments were then conducted at retinoic acid concentration equal to 1O-5 M. Under the same conditions, no effect was observed with galactosyltransferase whatever the concentration of retinoic acid was. From preliminary studies, it had been demonstrated that hydrolysis of the UDP-Gal substrate was important without addition of a pyrophosphate inhibitor such as AMP which was then added to each galactosyltransferase assay. Additional controls (data not shown) gave evidence that CMP-NeuAc and UDPGal hydrolysis by phosphatases and/or nucleoside pyrophosphatases was at the same level in control and retinoic acid-treated cells. This indicates that the activation
for the sialyltrans-
ferase activity was not the result of hydrolysis of CMP-NeuAc substrate by inhibition of either a CMP-NeuAc hydrolase or a phosphatase by retinoic acid.
Effect of retinoic acid on C6 glioma sialyltransferase
I 46 Time
Fig. I. Time course effect of retinoic acid on C6 glioma sialyltransferase and galactosyltransferase activities in DMEM medium for times up to 24 hr. Retinoic acid concentration was 10e5 M and specific activities (refered to concentration of proteins) were related to specific activity of control (without retinoic acid) at the same treatment time. Results are means from two separate experiments. q, Sialyltransferase; 0, galactosyltransferase.
Fig. 3. Time course effect of retinoic acid on C6 glioma sialyltransferase activities in DMEM medium for times up to 96 hr. Retinoic ,acid concentration was IO-‘M and specific activities (refered to concentration of proteins) were related to specific activity of control (without retinoic acid) at treatment time 0. Results are means from four separate experiments. n , Control; 0, retinoic acid-treated cells.
Time course effect of retinoic acid treatment on glycosyltransferase activity in C6 glioma cells
at confluence. When all sialyltransferase activities are referred to activity of control cells at time 24 hr, decrease was observed both for control and retinoic-treated cells, and speed of decrease was the same (Fig. 3). But anyway, sialyltransferase activities were always higher in retinoic acid-treated cells. As retinoic acid might act on cell proliferation, specific activities were also related to cellular density, giving roughly similar patterns. On the opposite, galactosyltransferase activity patterns were the same in control and retinoic acid-treated cells.
Figure 1 shows effect of retinoic acid upon the two glycosyltransferase activities for times up to 24 hr. Activities were related to activities of control cells collected at the same time. Activation effect was observed only for sialyltransferase activity and only after 24 hr-treatment. This result is corroborated by results of Fig. 2; if treatment was performed for longer times, sialyltransferase activation was higher (up to twice the control) for time treatments of 72 and 96 hr. Under the same conditions, no activation of galactosyltransferase was observed. However, one must focus on one important point: in cultured C6 glioma cells, glycosyltransferase activities are decreasing with time, activities being maximal a few hours after subculture and minimal
160 2 ‘0 $ s ”
Comparison of effect of retinoic acid isomers Table 1 gives results of the effect of the 13-cis and all-rrans isomers of retinoic acid onto the sialyltransferase activity. Both isomers led to an activation, which was however slightly higher with the all-trans isomer. Isomerization of retinoic acid in the culture medium during the treatment was postulated, leading to only one active isomer. To control this fact, we performed HPLC analysis of retinoids derivatives in the culture medium after treatment with each isomer. No significative isomerization of all-trans to 13-cis retinoic acid or reverse was detected if C6 glioma cell cultures and extraction of retinoic acid were performed in the dark.
Table 1. Comparison
of C6 glioma cell sialyltransferase different retinoic acid isomers Retinoic
Fig. 2. Time course effect of retinoic acid on C6 glioma sialyltransferase and galactosyltransferase activities in DMEM medium for times up to 96 hr. Results were expressed as in Fig. I. Means were from four separate experiments.
Time treatment 24 48 72
1.68 f 0.02 1.56 + 0.08 2.00 + 0.10
1.25 i 0.03 1.39fO.12 1.66kO.12
Results were specific activities (refered to concentration of protein) related to specific activity of control (without retinoic acid) at the same treatment time. Means and SEM are for three separate experiments.
PASCAL REBOGL et al.
Cellular adhesion of C6 glioma cells treated with retinoic acid Labelled C6 glioma cell membranes were prepared as described by Santala et al. (1977). These membranes were incubated for 15 and 30 min with control cells and cells treated with either 13-cis or all-trans retinoic acid. As shown in Table 2, adhesion was significantly increased in retinoic acid-treated cells both with the two retinoic isomers, and this increase was the same than activation of the sialyltransferase.
Retinoic acid treatment led to a significant increase of P-galactoside cr2,3 sialyltransferase in C6 glioma cells. This increase of activity of a sialyltransferase after retinoic acid treatment is not specific of C6 glioma cells as Deutsch and Lotan (1983) have described such an activation in melanoma cells. However, this effect has not been observed with all cell lines as the chondrosarcoma Hs705 sialyltransferase activity was not activated. Moreover, response to retinoic acid treatment is not the same for all glycosyltransferases as we have demonstrated that in C6 glioma cells, the galactosyltransferase was not activated by retinoic acid treatment. The activation of sialyltransferase is not the result of an increase of the concentration of the substrate CMP-NeuAc after inhibition of a CMP-NeuAc hydrolase by retinoic acid as in the incubation medium, CMP-NeuAc hydrolysis remained low and the CMP-NeuAc concentration at the same level in control and treated cells. We have also demonstrated that the configuration of retinoic acid is not essential as all-trans and 13-c& isomers led together to slightly different but comparable activation of the sialyltransferase. The activation of the sialyltransferase is not dependent of a second messenger effect as it had not been demonstrated for treatment times lower than 24 hr. In addition, nobody has been able to report an increase of CAMP or cGMP after retinoic acid treatment (Lotan, 1980). On the opposite, increase of activation after chronic exposure (96 hr) suggests heavily that the effect was linked to increase of sialyltransferase biosynthesis. As a control, no activation was observed with in vitro experiments, with retinoic acid directly added to the sialyltransferase assay medium, indicating that the phenomenon is not a direct activation of the enzyme. Table 2. En~ncement
of adhesion of retinoic acid-treated C6 glioma cells
As shown in Fig. 3, the sialyltransferase activity decreased as cellular density increased. Other studies from us have demonstrated a correlation between these two phenomenons. One explanation for the retinoic acid effect would then be through reducing of the decrease of sialyltransferase related to an inhibition of cellular proliferation and therefore lower cellular density in treated cells. Fischer et al. (1987) have noted such an inhibition in proliferation in retinoic acid treated C6 glioma cells, with inhibition up to 60% with IO-’ M retinoic acid. However, these authors indicated that the growth inhibition they observed depend heavily on serum concentration, giving “conflicting regulatory signals”. They indicated also that different C6 glioma clones may be more or less responsive to inhibition by retinoic acid as it had been previously shown for human neuroblastoma clones. Anyway, under our experimental conditions, we never observed a significant difference in cellular density or cellular proliferation between control and retinoic acid-treated C6 glioma cells. So, from our results, it is clear that whatever the cell density was, sialyltransferase activity in the retinoic acid treated cells was higher that in control cells. We then have measured adhesion of cells treated with retinoic acid under the conditions where sialyltransferase was activated. This was performed as described by Santala et al. (1977), using labelled membranes from C6 glioma cells as a probe for adhesion. According to these authors, use of the same cell strain for labelled membrane preparation and for cells which have to be tested, is the best way, being close to physiological conditions. Under these conditions, the adhesion was clearly increased (x 2) in treated cells and increase of SiaIyltransferase was at the same extent. This is in agreement with the hypothesis that the increase in sialyltransferase activity led to an increase in biosynthesis of some membrane glycoproteins responsible for adhesion. This is consistent with the observation from Deutsch and Lotan (1983) who have previously demonstrated in melanoma strain that retinoic acid treatment led to the increase of the sialylation of a specific membrane glycoprotein (gp 160). Identification of such a glycoprotein in C6 glioma cells is presently under investigation. Acknowledgements-Dr Gerard Rebel from the Centre de Neurochimie du CNRS (Strasbourg, France) is gratefully acknowledged for his help in initiation of this work, gift of C6 strain and fruitful discussions. This work was supported by the Institut National de la Sante et de la Recherche Medicale, the Centre National de ia Recherche Scientifique and the University of Lyon (Fact&t de Mtdecine LyonSud).
Time of contract 15 min
Percent of adhesion stimulation ~ For IO6 cells Factor Control All-rrons i3-&
1.63 i: 0.34 3.20 + 0.56 3.33 IO.23
Percent of adhesion stimulation
-For IO’ cells
I .96 2.02
3.53 5 0.23 4.13 * 0.26
The labelled plasma membrane fraction was incubated for IS or 30 min with C6 glioma cells treated either with iO-‘M all-trans or 134s retinoic acid or with untreated cells. Binded radioactivity was then reported to IO’ cells.
Annesley T., Giarcherio D., Wilkerson K., Grekin R. and Ellis C. (I 984) Analysis of retinoids by high-performance liquid chromatography using programmed gradient separation. J. Chromat. 305, 199-203. Baubichon-Cortay H., Serres-Guillaumond M., Louisot P. and Brosuet P. (1986a) A brain sialvltransferase having a marrow-s~ci~c~ty for ‘O-glycosicall~-links oiigosaccharide chains. Carbohyd. Res. 146, 209-223. Baubichon-Cortay H., Serres-Guillaumond M., Broquet P. and Louisot P. (1986b) Different reactivity to lysophos-
Effect of retinoic
acid on C6 glioma
phatidylcholine, DIDS, and trypsin of two brain sialyltransferases specific for 0-glycans: a consequence of their topography in the endoplasmic membrane. Biochim. biophys. Acia 862, 243-253. Baubichon-Cortay H., Broquet P., George G. and Louisot P. (1989a) Different reactivity of two-brain sialyltransferases towards sulfhydryl reagents. Evidence for a thiol group involved in the nucleotide-sugar binding site of the NeuAc a2-3 Gal B l-3 GalNAc a(2-6) sialyltransferase. Glycoconjugate J. 6, 1155127. Baubichon-Cortay H., Broquet P., George G. and Louisot P. (1989b) Evidence for an 0-glycan sialylation system in brain, Characterization of a p-galactoside a2,3-sialyltransferase from rat brain regulating the expression of an a-N-acetylgalactosaminide a2,6_sialyltransferase activity. Eur. J. Biochem. 182, 257-265. Broquet P., Morelis R. and Louisot P. (1975) The biosynthesis of cerebral glycoproteins: studies on mitochondrial mannosyltransferase. J. Neurochem. 24, 989-995. Deutsch V. and Lotan R. (1983) Stimulation of sialyltransferase activity of melanoma cells by retinoic acid. Expl Cell Res. 149, 2377245. Dion L. D., Blalock J. E. and Gifford G. E. (1978) Retinoic acid and the restoration of anchorage dependent growth to transformed mammalian cells. Expl Cell Res. 117, 15-22. Fischer I., Nolan C. E. and Shea T. B. (1987) Effects of retinoic acid on expression of the transformed phenotype in C6 glioma cells. Life Sci. 41, 463470. Hill D. L. and Grubbs C. J. (1982) Retinoids as chemopreventive and anticancer agents in intact animals (Review). Anticancer Res. 2, 11 l-124. Lotan R. (1980) Effect of vitamin A and its analogs (retinoids) on normal and neoplasmic cells. Biochim. biophys. Acta 605, 33-9 1. Lotan R., Kramer R. H., Neumann G., Lotan D. and
Nicholson G. L. (1980) Retinoic acid-induced modifications in the growth and cell surface components of a human carcinoma (HeLa) cell line. Expl Cell Res. 130, 401414. Lotan R., Neumann G. and Deutsch V. (1983) Identification and characterization of specific changes induced by retinoic acid in cell surface glycoconjugates of S91 murine melanoma cells. Cancer Res. 43, 303-3 12. Lotan R., Lotan D. and Amos B. (1988) Enhancement of sialyltransferase in two melanoma cell lines that are growth-inhibited by retinoic acid results in increased sialylation of different cell-surface glycoproteins. Expl Cell Res. 177, 2844294. Mookerjea S. and Yung J. W. M. (1975) Studies on uridine diphosphate-galactose pyrophosphatase and uridine diphosphate-galactose: glycoprotein galactosyltransferase activities in microsomal membranes. Archs Biochem. Biophys. 166, 223-226. Santala R. and Glaser L. (1977) The effect of cell density on the expression of cell adhesive properties in a cloned rat astrocytoma (C-6) Bichem. biophys. Res. Commun. 79, 285-29 1. Santala R., Gottlieb D. I., Littman D. and Glaser L. (1977) Selective cell adhesion of neuronal cell lines. J. biol. Chem. 252, 7625-7634. Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimato E. K., Goeke N. M., Olson B. J. and Klenk D. C. (1985) Measurement of protein using bicinchoninic acid. Analyf. Biochem. 150, 76685. Sporn M. B. (1977) Retinoids and carcinogenesis. Nurr. Ret>. 35, 65569. Takahashi N. and Breitman T. R. (1989) Retinoic acid acylation (retinoylation) of a nuclear protein in the human acute myeloid leukemia cell line HL60. J. biol. Chem. 246, 5159-5163.