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Specific modulation by ethanol of the protein synthesis pattern in the C2 rat hepatoma cell line
Journal of Hepatology. 1988; 6:85-93
85
Elsevier HEP 00379
Specific modulation by ethanol of the protein synthesis pattern in the C2 rat hepatoma cell line
M.N.
C h o b e r t 1, P. Vincens 2, G. GuellaEn 1, R. Barouki 1, Y. Laperche 1 M. A g g e r b e c k 1 T. Aissani l, A. Pawlak 1, P. Tarroux 2 and J. H a n o u n e 1 I Unit~ de Recherches INSERM U-99, H6pital Henri Mondor, Cr&eil and 2Untt~ Associ~e CNRS UA 686, Ecole Normale Sup&ieure, Parts (France)
(Received 30 June 1987) (Accepted 12 October 1987)
Summary The effect of ethanol on protein synthesis in the C 2 rat hepatoma cell line was analyzed by two-dimensional gel electrophoresis after the labeling with [35S]methionine of cells that were untreated or had been treated with 180 mM ethanol. In this cell line, this concentration of ethanol is known to induce gamma-glutamyl transpeptidase, a marker of alcoholism in man (Barouki et al., Hepatology 1983; 3: 323-329). In the present work we demonstrate that ethanol, besides causing a slight decrease in overall protein synthesis (less than 25%), primarily regulates the expression of two unique proteins among 1500 labeled products that were analyzed: one of these was induced and did not correspond to gamma-glutamyl transpeptidase, and one was repressed after 20 h of ethanol treatment. We conclude that the set of hepatic proteins altered by ethanol is likely to be very limited in number, which reflects the specificity of alcohol action on protein synthesis in the C 2 cell line.
Introduction Ethanol is known to exert multiple effects on liver cells at the nucleus [1,2], cytoplasm, and plasma membrane level [3]. The mechanisms involved in the alteration of protein metabolism or the activities of various enzymes have been widely discussed. Some
authors have proposed that ethanol acts by modifying the lipid environment of the cellular membrane [4]. This mechanism was particularly suggested to explain the modulation of membrane-bound enzyme activities [5,6] and the impairment of plasma membrane glycoprotein assembly [7]. Inhibition of protein and glycoprotein secretion has also been attrib-
This work was supported by the Institut National de la Sant6 et de la Recherche M6dicale, the D616gation G6n6rale h la Recherche Scientlfique et Technique, the Universit6 Paris-Val-de-Marne, and the Haut Comlt6 de l'Etude et d'Information sur l'alcoolisme. Correspondence: Marie-NoEle Chobert, Umt6 de Recherches INSERM U-99, H6pital Henri Mondor, 94010 Cr6teil, France. Tel: (1) 42075141, ext. 4945. 0168-8278/88/$03.50 (~ 1988 Elsevier Science Publishers B.V. (Biomedical Division)
86 uted to the metabolism of ethanol and acetaldehyde production, which is known to produce microtubular alteration [3,8,9]. The effect of ethanol on total protein synthesis remains very controversial [10-13]. Several studies indicate that, in vivo, protein synthesis is not affected by ethanol [8-10]. However, ethanol has been shown to alter protein synthesis in some in vitro systems, mainly when changes in redox potential are involved [10]. For example, in a few cases, the mechanism by which ethanol inhibits protein synthesis was further analyzed. In the C H O cell-free synthesis system, a decrease in translation was accounted for by an inhibition of Leu-tRNA synthetase by ethanol [11]. Another example is the inhibition of neutral amino acid transport by alcohol in certain cell lines [ 13]. The induction of some liver proteins by ethanol, such as cytochrome P-450 [14], glucose-6-phosphate dehydrogenase [15] and gamma-glutamyl transpeptidase (GGT) [16-19] appears to be more specific. We have shown that ethanol induces G G T in a rat hepatoma cell line (C2) without modifying other membrane or cytoplasmic enzyme activities [16]. In order to further determine the potency and the specificity of ethanol action on protein synthesis, we used twodimensional polyacrylamide gel electrophoresis to analyze the protein pattern of the C2 hepatoma cell line, especially the appearance or disappearance of individual proteins under conditions where gammaglutamyl transpeptidase activity is increased. In the present study, we demonstrate that ethanol produces a slight decrease in total protein synthesis. Surprisingly, we observed only a very limited, highly specific response to ethanol after 20 h of treatment.
Methods
L-y-Glutamyl-p-nitroanilide and glycylglycine were purchased from Sigma Chemical Co. L-[35S]Methionine (800 Ci/mmol) and 14C-methylated protein mixtures used as molecular weight standards were purchased from Amersham Radiochemical Centre, U.K. Ampholines (pH 5-7, and pH 3.5-10) were from LKB, Sweden.
M.N. CHOBERT et al.
Cell culture and ethanol treatment The C 2 cells derived from line H4IIEC 3 of the Reuber H35 hepatoma [20] were grown in a modified [21] Hams's Fl2 medium (Gibco) supplemented with 5% fetal calf serum (Flow Lab.), 200 units/ml of penicillin, 50 ~g/ml of streptomycin (Diamant) and 0.5 /~g/ml of fungizone (Squibb). Cells were routinely cultured as previously described [16], in a 7% CO~ humidified atmosphere at 37 °C, on Falcon plastic petri dishes; 2.5 x 104-105 cells and 105-106 cells were seeded per 6 cm and 10 cm diameter petri dishes, respectively. Experiments were performed during the exponential phase of growth. For ethanol treatment, culture medium was removed and fresh medium with or without 1% ethanol was added for 6 48 h. We have previously demonstrated [16] that G G T induction was not modified whether or not the plates were wrapped in parafilm to avoid ethanol evaporation. Gamma-glutamyl transpeptidase assays At each time point, control and ethanol-treated cells were washed with phosphate-buffered saline (PBS) at 4 °C, scraped and centrifuged at 300 x g and resuspended in PBS. G G T activity was determined according to the method of Orlowski and Meister [22] as previously described, using e-~,-glutamyl-p-ni'troanilide and glycylglycine as substrates [23]. Proteins were estimated by the method of Lowry et al. [24]. Measurement of [~SS]methionine incorporation in the total cell proteins as a function of time during ethanol treatment Cells were labeled with 20 lICi of [35S]methionine/ml of medium at the beginning of the experiment. At each time point, [~-~S]methionine incorporation was stopped by washing the cells three times with PBS at 4 °C. One volume of cells, resuspended in PBS as described for G G T assay, was mixed with 1 vol. of 4.2% sodium dodecyl sulfate (SDS), 0.125 M Tris HCI pH 6.8, 10% fl-mercaptoethanol, 13.2% sucrose and boiled for 3 min. Aliquots (2~d) were used for trichloroacetic acid (TCA)-precipitability measurement.
ETHANOL AND PROTEIN SYNTHESIS
Two-dimensional gel electrophoresis Cells were pulse-labeled with 75/iCi of [35S]methionine/ml of medium for 2 h before the end of ethanol treatment. The pulse was terminated by washing the cells as described above, and cell pellets were dissolved in 200-600/ll of O'Farrell lysis buffer [25]. Samples were centrifuged for 30 min at 10 000 × g and supernatants were used for TCA-precipitability measurement and isoelectric focusing. Samples were loaded onto 3 mm i.d. gel essentially according to the procedure of O'Farrell [25], except that the sample ( 10-30/xl containing 5-15/~g of proteins and 200 000 cpm) was applied to the acidic side of the gels, which were directly run at 500 V for 16 h. Gels were equilibrated for 40 min in 2.1% SDS, 5% fl-mercaptoethanol, 6.6% sucrose, 0.062 M Tris-HCl pH 6.8, and applied onto a stacking gel in 1% agarose prepared in equilibration buffer. The second-dimension electrophoresis was carried out in a 10% polyacrylamide gel slab as described previously [26] except that glycerol was replaced by 66% sucrose. Standard molecular weight markers (14C-labeled protein mixture, Amersham) were applied at one end of the gel tube, included in a 5 mm (length) by 3 mm (diameter) gel. En3Hance (New England Nuclear) was used for fluorography with Kodak X A R 5 films. Computer analysis Autoradiograms were analyzed using the HERMES computer system, a detailed description of which is found in Refs. 27-31. Briefly, the gels were scanned using a video camera [27]. These images are cleaned and filtered in order to reduce background and high frequency noise and to improve spot detection by mathematical morphology methods. Spots are detected by a computerized processing method [28] based on the use of a Gaussian model. The gels corresponding to different conditions are then matched to determine homologous spots [29]. A data array including the intensity of each spot in the different conditions tested in the experiment is computed by the system. Multivariate analysis [32] was performed with the data array, spots being considered as observations and gels as variables characterizing each spot throughout the series. In simple
87 language, each spot is described by its intensity in the different gels of the series. A full representation of spots requires an n-dimension space, n being the number of gels. The purpose of such a study is to analyze the relationships between gel patterns and different experimental biological conditions. The reduction of the number of axes by the transfer of the significant information to the first axes is useful for improving the interpretation of the experiments. This is done by computing a new axis system having the following properties: (i) the new axes are linear combinations of the old ones; (ii) the significant information carried along each axis decreases with the axis number; (iii) the new axes are independent from each other. The projections of the old axes (i.e., gels) can be mapped into the new coordinate system. Usually, the graph named factorial plane consists of two axes. The relative position of the gels over this plane indicates the relationships between these gels according to the considered axes. However, this approach is only a convenient representation for the overall information carried by the intensities of the spots in the series of gels, and allows for an easier study of the relationships between different biological conditions. Furthermore, variance analysis enables the detection of spot intensity variations under different conditions. As the migration of high molecular weight proteins in the upper part of the gel was not reproducible, the extreme areas of the gels were not taken into consideration for spot analysis.
Results
Time course of stimulation of GGT by ethanol in C2 cells The maximal increase of G G T activity was obtained with 180 mM ethanol [16]. Using this concentration, we observed stimulation of the enzyme as a function of time during ethanol treatment (see Fig. 1). In the control cells, G G T activity was relatively stable with a slight increase (40%) after 48 h of culture. The increase of G G T activity by ethanol was a slow process and the maximal increase (2.5-fold) was obtained within 48 h. A similar increase was obtained
88
M.N. CHOBERT et al
"2. o o.
whether G G T activity was expressed on the basis of protein (Fig. 1) or D N A concentration [16].
7
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Total protein synthesis during ethanol treatment in C2 cells
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U n d e r the same conditions in which a maximal in.=
crease in G G T activity was produced in C 2 cells by ethanol treatment, ethanol had only a minimal effect
== .= c m
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Fig. 1. Time course of stimulation of GGT by ethanol. C2 cells in the exponential phase of growth were exposed to 1% ethanol (180 mM) (C)) or untreated (•). At various time periods of treatment, control and treated cells were harvested, and enzyme activity was assayed as described in the Methods section. Results are expressed as mU/mg protein (1 mU of enzyme activity = 1 nmol product formed per min). Each point represents the mean value from two to five experiments, each assay being performed in duplicate.
mensional gel processing was performed as described in the Methods section. Since equal amounts of ra"dioactive material were loaded on each gel, any change in the intensity of an individual spot can be assumed to represent a change in the relative transla-
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C2 cell proteins during ethanol treatment analyzed by two-dimensional gel electrophoresis Ethanol-treated and control C2 cells were incubated for 2 h with [35S]methionine and then collected and dissolved in the O'Farrell lysis buffer. Two-di-
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on total protein synthesis. The control C2 cells incorporated [35S]methionine in TCA-precipitable material at a constant rate during the 48 h of incubation with the amino acid (Fig. 2). There was a slight and transient decrease in the incorporation of [35S]methionine in the presence of 180 mM ethanol, inhibition being maximal (25%) at 30 h. This slight inhibition was detected whether the labeled methionine was added at the beginning of the ethanol treatment (Fig. 2) or 2 h before each m e a s u r e m e n t of incorporation in T C A precipitable material (data not shown).
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Fig. 2. Effect of ethanol on the time course of incorporation of L-[35S]methionine in total proteins of C, cells. At zero time, cells in the exponential phase of growth were incubated in fresh medium in the presence or absence of 1% ethanol with 20 !tCi of [35S]methionine/ml.At the different time periods indicated, incorporation was stopped by harvesting the cells and dissolving them as described in Methods for TCA-precipitability measurement Each point is the mean value of two experiments. • = control cells; C) = 1% ethanol-treated cells.
tion of the protein in that spot. Each gel corresponding to control or ethanol-treated cells at 0, 6, 20 and 48 h was processed and a data base including all the data obtained was then constituted [27-31]. Representative fluorograms are shown in Fig. 3. The analysis of 1500 spots detected on the different gels showed that ethanol produced very small effects on the total protein pattern of the C~ cells. To fully interpret the information contained in the initial data, multivariate estimations were used. This factorial analysis is shown in Fig. 4. We can thus obtain an objective measurement of the global effects of ethanol on the protein pattern of the C 2 rat hepatoma cell line. The time-dependent effect of ethanol during 48 h of treatment is clearly demonstrated in Fig. 4A. In-
ETHANOL AND PROTEIN SYNTHESIS
89
deed, along axis 3, the patterns of control cells as well as cells treated for 6 h by ethanol are clearly separated from those of cells t r e a t e d for 20 and 48 h. ConA
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versely, using axis 5 (Fig. 4B), we can easily separate protein patterns as a function of time in control cells but not in ethanol-treated cells. As shown by inspection of the eigenvector constituents taken into account in axis 5, only control gels a p p e a r at different positions along this axis, which reflects their own variability. The i n d e p e n d e n t nature of spot variation between control cells during growth and ethanoltreated cells is thus demonstrated. A f t e r extraction of the significant data from the data base a variance analysis indicated that very few spots were specifically altered during time of ethanol treatment. O n e spot (a: M r = 37 000 + 1000; p l = 5.2 + 0.1) was present at zero time and d i s a p p e a r e d with time in control cells (Fig. 3 A and B). One spot was found to be induced by ethanol t r e a t m e n t (b: M r = 36 000 + 1000; p I = 6 + 0.1). A histogram of its intensity is presented in Fig. 5A. It was clearly absent from control cells at all time points and a p p e a r e d after 20 h of ethanol t r e a t m e n t (Fig. 3C). A unique spot (c: Mr = 24 000 + 1000; p I = 6.6 + 0.1) increased in control cells as a function of time but totally d i s a p p e a r e d after 20 h of t r e a t m e n t with ethanol (see Figs. 3 and 5B). The regions corresponding approximately to M r = 54 000, p I 6.8 and M r 63 000, p I 5.9 (boxed in Fig. 3) were modified to a limited extent. A few spots changed in shape and position when different control cells or when control and treated cells were compared at different times.
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Fig. 3. Two-dimensional gel autoradiograms from ethanoltreated or untreated C2 cells. Cells were plated and grown as described in Methods. Control and treated cells (180 mM ethanol) were labeled for 2 h with [35S]methionine at each time point. Two-dimensional gels were prepared as described in Methods, samples (200 000 cpm) being applied on the acidic side of the focusing gel. The vertical axis separates proteins as a function of molecular weight. The horizontal axis separates proteins as a function of pI. (A) Control cells, 0 h; (B) control cells, 20 h; (C) ethanol-treated cells, 20 h. (a) Spot present in A (control cells at 0 h) and absent in any other case. (b) Induced spot after 20 h of ethanol treatment (absent in A and B, present in C). (c) Repressed spot after 20 h of ethanol treatment (present in A and B, absent in C). The enclosed areas in A, B, C are regions where the shape and position of several spots are modified when the different gels are compared. The extreme upper and lower parts of the gels were not taken into consideration.
90
M.N. C H O B E R T et al.
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Fig. 4. Global effects of ethanol (lgt) mM) studied by factor analysis of the protein pattern of C_, cells. The gels correspondmg to control and treated cells are plotted onto two planes of a principal component analysis (respectively A (axes 1-3) and B (axes 3-5}). Time-effects during ethanol treatment are mainly c a m e d by axis 3 (A) Axis 5 only carries variations occurring in the different control gels, while treated gels are not separated on this axis ( B ) . / x / = 0 hour, [] = control, 6 h: • = ethanol, 6 h" z~ = control, 20 h, • = ethanol, 20 h, © = control, 48 h : • = ethanol, 48 h
In summary, C2 cell protein synthesis is not globally modified by ethanol treatment. We found two unique proteins regulated by ethanol. Spot b corresponds to a newly expressed protein in these cells following ethanol treatment. Spot c corresponds to a protein that is repressed by ethanol. Whether or not these events take place at the transcriptional, translational or different post-translational levels cannot be determined by the present approach.
48 -
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Fig. 5. Histograms of spot intensity for an induced (A: spot b) or a repressed protein (B: spot c)'. Control or ethanol-treated states at 0, 6, 20 or 48 h are represented. The values are selected from the data base.
Discussion
The effect of alcohol on protein synthesis has been studied previously using either total incorporation into TCA-precipitable material [10-13] or the increase in specific enzyme activity [14-16]. In the first case, the various results were sometimes contradictory and the importance of the inhibition of protein synthesis by ethanol in vivo remains questionable. In the latter case, the change in the activity of specific enzymes by ethanol was attributed either to a direct effect of ethanol on the plasma membrane [4-6] or to a stimulation of the synthesis of the protein [15,16]. None of these studies has given a clear view of the overall importance of ethanol effects on the synthesis of specific proteins.
ETHANOL AND PROTEIN SYNTHESIS The C 2 hepatoma cells have retained some of the characteristics of hepatocytes, such as tyrosine aminotransferase induction by glucocorticoids [20] and a low G G T activity [16], but lack other markers of liver differentiation [20]. In those cells, the inducuon of G G T [16], an enzyme which is a marker of alcoholism in man, occurs at ethanol concentrations that are readily found in the serum of alcoholics following an acute ingestion of alcohol [19]. Among many hepatoma-derived cells which we tested, C 2 was the only one that revealed the induction of G G T by ethanol; it thus constitutes a reasonable model of the in vivo effect of ethanol. We therefore decided to assess the effect of alcohol on protein synthesis in this particular cell line under conditions in which a specific increase in G G T activity is observed. Obviously, this might only represent a fraction of what is actually modified in the hepatocyte in vivo. The concentration of ethanol (180 mM) was chosen for the following reasons: (i) this corresponds to the optimal concentration that we have determined previously for G G T induction [16]; (ii) it is not toxic for the cells [16]; (iii) this concentration produces a mild effect on protein synthesis, and we expect smaller and more 'physiological' concentrations to produce even milder and probably not significant effects. Besides, the C, cell line is lacking in two of the main enzymes revolved in ethanol degradation, namely alcohol dehydrogenase and cytochrome P-450j (data not shown). In these cells, ethanol is probably not degraded to a very large extent. A single dose of ethanol was added to the cells and its acute effects (up to 48 h) on protein synthesis were determined using two-dimensional gel analysis. Our results show that ethanol modifies protein synthesis quantitatively and qualitatively after treatment of relatively short duration. Firstly, the incorporation of [3SS]methionine in total proteins is found to be slightly decreased by ethanol treatment in our conditions. This is probably correlated with the slight decrease in cell growth that we have observed. Recently, other authors have described an alteration of growth rate correlated with an accumulation of cells in G I phase and a low RNA content in rat liver tumor cells treated by ethanol [2]. Secondly, among all the pro-
91 tein spots analyzed, very few proteins are increased or decreased by ethanol treatment. One is clearly induced, and one is totally repressed after 20 h of treatment. These are rather minor proteins. The protein induced by ethanol cannot correspond to cellular GGT, which represents less than 0.0005% of total proteins in the C, cells. The sensitivity of the technique does not allow the detection of such rare proteins. Indeed, out of 200 000 cpm corresponding to total loaded proteins, only 1 cpm would correspond to GGT. In HTC cells [33], tyrosine aminotransferase represents 0.02-0.05% of total proteins and the easily detectable corresponding spot represents at least 100 cpm in these experiments with a 10 day exposure to X-ray films. In other experiments related to the "growth hormone domain' of rat liver [34], for a total load of 200 000 cpm, spots equivalent to 30 cpm correspond to the lower limit of detection. The total number of cellular mRNA products largely exceeds the 1500 spots detected in the present study, and 5000-11 000 different m R N A species can be estimated in hepatic cells [35,36]. The global analysis of gels reflects only a part of ethanol effects on protein expression. In other works on hepatic proteins, the set of spots that were analyzed never exceeded 1500 [33,36]. In the case of in vitro translation of isolated liver mRNAs, not more than 200 translation products were analyzed [34,35]. In HTC cells, glucocorticoids modify specifically 13 proteins out of a total number of 1000 spots [33]; 19 out of 231 hepatic proteins visualized are modulated by T.~ hormone [35]; 8 proteins among 250 are altered in the liver by growth hormone [34]. In a recent study of liver regeneration, 1600 spots were analyzed [36]; in this case, only a few qualitative changes were observed, but the intensity of many spots changed considerably. Other stress situations such as heat shock [37] and azotemia [38] modify a larger set of proteins. Compared to these results, ethanol in the C2 cell line modifies markedly two specific spots and its effects appear therefore to be much more limited than those of other effectors which also alter hepatic protein expression. Nevertheless, the two-dimensional gel analysis represents the most powerful tool to date for evaluating overall changes in the synthesis of pro-
92
M.N. CHOBERT et al.
teins, although the n u m b e r of changes is p r o b a b l y un-
cellular m e m b r a n e s , s o m e of t h e s e effects c o u l d also
derestimated. In a different system ( h a m s t e r fbroblasts, H A - l ) ,
be due to the specific m o d i f i c a t i o n of g e n e e x p r e s -
e t h a n o l has b e e n s h o w n to induce specifically two
m o d i f i e s g e n e e x p r e s s i o n was u n k n o w n . It has b e e n
sion. U n t i l recently, the m e c h a n i s m by which e t h a n o l
heat shock proteins, Hsp 70 and Hsp 87 [39]. T h e s e
suggested lately that e t h a n o l acts on the p r o m o t e r re-
do not c o r r e s p o n d to the m o d i f i e d spots o b s e r v e d
gion of the a p o l i p o p r o t e i n A - I g e n e [40], and stabi-
here. M o r e o v e r , in the studies with h a m s t e r fibro-
lizes the c y t o c h r o m e P-450 f o r m 3a [41].
blasts, e t h a n o l was used in v e r y acute stress conditions (6% for 1 h) w h e r e a s in o u r case it was used at 1% for 48 h. T h e s e latter e x p e r i m e n t a l c o n d i t i o n s were found to be o p t i m a l for the i n d u c t i o n of G G T in
Acknowledgements
C 2 cells [16]. T h e m a j o r conclusion of this study is that e t h a n o l
W e wish to t h a n k Drs. P. B e a u n e and N. P e r r o t for
can affect specifically the e x p r e s s i o n of a limited
c y t o c h r o m e P-450j d e t e r m i n a t i o n , Dr. P. Insel, Dr.
n u m b e r of p r o t e i n s in the C 2 h e p a t o m a cell line. A l -
F. P e c k e r , Dr. P. B e r t h e l o t and Dr. B. N a l p a s for
t h o u g h most of the cellular effects of e t h a n o l can be
their critical r e a d i n g of the m a n u s c r i p t , and Ms. L.
a c c o u n t e d for by its m e t a b o l i s m or its action on the
R o s a r i o for e x c e l l e n t secretarial assistance.
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ETHANOL AND PROTEIN SYNTHESIS
22 23
24
25 26
27
28
29
30
31
mation of spontaneous and virus-produced viable hybrids Proc Natl Acad Sci USA 1969; 62: 852-859. Orlowsk~ M, Meister A. Isolation of GGT from hog kidney. J Biol Chem 1965; 240: 338-347. Barouki R, Chobert MN, Billon MC, et al. Glucocorticoid hormones increase the activity of y-glutamyl transferase in a highly differentiated hepatoma cell line. Biochim Biophys Acta 1982; 721: 11-21. Lowry OH, Roberts NR, Kapphahn JH. The fluorometric measurement of pyndine nucleotides. J Blol Chem 1957; 224: 1047-1064. O'Farrell PH. High resolution two dimensional electrophoresis of proteins. J Biol Chem 1975; 250: 4007-4021. Barouki R, Finidori J, Chobert MN, Aggerbeck M, Laperche Y, Hanoune J. Biosynthesis and processing of GGT m hepatoma tissue culture cells. J Blol Chem 1984; 259: 7970-7974. Vincens P, Paris N, Pujol JL, et al. HERMES: a second generation approach to the automatic analysis of two dimensional electrophoresis gels. Part I: Data acquisition. Electrophoresis 1986; 7: 347-356. Vmcens P. HERMES: a second generation approach to the automatic analysis of two dimensional electrophoresis gels. Part II. Spot detection and integration. Electrophoresis 1986; 7: 357-367. Vincens P, Tarroux P. HERMES: a second generation approach to the automatic analysis of two d~mensional electrophoresis gels. part III: Spot list matching. Electrophoresis 1987; 8: 100-107. Vincens P, Tarroux P. HERMES: a second generation approach to the automatic analys~s of two dimensional electrophoresis gels. Part IV: Data base organization and management. Electrophoresis 1987; 8: 173-186. Tarroux P, Vincens P. HERMES: a second generation approach to the automatic analys~s of two dimensional electrophoresis gels. Part V: Data analysis. Electrophoresis
1987; 8: 187-199. 32 Kendall, M., Stuart H. The Advanced Theory of Statistics. London: Griffin, 1973; 243. 33 Ivarie RD, O'Farrell PH. The glucocorticoid domain changes in the rate of synthesis of rat hepatoma proteins. Cell 1978; 13: 41-55. 34 Liaw C, Seelig S, Manash CN, Oppenheimer JH, Towle HC. Interactions of thyroid hormone, growth hormone, and high carbohydrate, fat-free diet in regulating several rat liver messenger ribonucleic acid species Biochemistry 1983; 22: 213-221. 35 Seelig S, Liaw C, Towle HC, Oppenheimer JH. Thyroid hormone attenuates and augments hepatic gene expression at a pretranslational level. Proc Natl Acad Sci USA 1981; 78: 4733-4737. 36 Huber BE, Heilman CA, Wlrth PJ, Miller MJ, Thorgeirsson SS. Studies of gene transcription and translation in regenerating rat liver. Hepatology 1986; 6: 209-219. 37 Subjeck JR, Shyy qT. Stress protein systems of mammalian cells. Am J Physlol 1986; 250, 19: C1-C17. 38 Klnlaw WB, Schwartz HL, Mariash, CN, Bingham C, Carr FE, Oppenheimer JH. Hepatic messenger ribonucleic acid activity profiles in experimental azotemia in the rat. J Chn Invest 1984; 74: 1934-1941. 39 LI GC. Induction of thermotolerance and enhanced heat shock protein synthesis in chinese hamster fibroblasts by sodium arsenite and by ethanol. J Cell Physiol 1983; 115: 116-122.
40 Chao YS, Pan TC, Wu TJ, Hao QL, Yamin TF, Kroon PA Regulation of the promoter of rat apohpoprotein A-I gene in cultured cells. Fed Proc 1987; 46: 2157a. 41 Porter TD, Fujita VS, Coon MJ. Quantitation of cytochrome P 450 form 3a mRNA in tissues from untreated and ethanol-, acetone-, and imldazole-treated rabbits. Fed Proc 1987; 46: 2140a.
85
Elsevier HEP 00379
Specific modulation by ethanol of the protein synthesis pattern in the C2 rat hepatoma cell line
M.N.
C h o b e r t 1, P. Vincens 2, G. GuellaEn 1, R. Barouki 1, Y. Laperche 1 M. A g g e r b e c k 1 T. Aissani l, A. Pawlak 1, P. Tarroux 2 and J. H a n o u n e 1 I Unit~ de Recherches INSERM U-99, H6pital Henri Mondor, Cr&eil and 2Untt~ Associ~e CNRS UA 686, Ecole Normale Sup&ieure, Parts (France)
(Received 30 June 1987) (Accepted 12 October 1987)
Summary The effect of ethanol on protein synthesis in the C 2 rat hepatoma cell line was analyzed by two-dimensional gel electrophoresis after the labeling with [35S]methionine of cells that were untreated or had been treated with 180 mM ethanol. In this cell line, this concentration of ethanol is known to induce gamma-glutamyl transpeptidase, a marker of alcoholism in man (Barouki et al., Hepatology 1983; 3: 323-329). In the present work we demonstrate that ethanol, besides causing a slight decrease in overall protein synthesis (less than 25%), primarily regulates the expression of two unique proteins among 1500 labeled products that were analyzed: one of these was induced and did not correspond to gamma-glutamyl transpeptidase, and one was repressed after 20 h of ethanol treatment. We conclude that the set of hepatic proteins altered by ethanol is likely to be very limited in number, which reflects the specificity of alcohol action on protein synthesis in the C 2 cell line.
Introduction Ethanol is known to exert multiple effects on liver cells at the nucleus [1,2], cytoplasm, and plasma membrane level [3]. The mechanisms involved in the alteration of protein metabolism or the activities of various enzymes have been widely discussed. Some
authors have proposed that ethanol acts by modifying the lipid environment of the cellular membrane [4]. This mechanism was particularly suggested to explain the modulation of membrane-bound enzyme activities [5,6] and the impairment of plasma membrane glycoprotein assembly [7]. Inhibition of protein and glycoprotein secretion has also been attrib-
This work was supported by the Institut National de la Sant6 et de la Recherche M6dicale, the D616gation G6n6rale h la Recherche Scientlfique et Technique, the Universit6 Paris-Val-de-Marne, and the Haut Comlt6 de l'Etude et d'Information sur l'alcoolisme. Correspondence: Marie-NoEle Chobert, Umt6 de Recherches INSERM U-99, H6pital Henri Mondor, 94010 Cr6teil, France. Tel: (1) 42075141, ext. 4945. 0168-8278/88/$03.50 (~ 1988 Elsevier Science Publishers B.V. (Biomedical Division)
86 uted to the metabolism of ethanol and acetaldehyde production, which is known to produce microtubular alteration [3,8,9]. The effect of ethanol on total protein synthesis remains very controversial [10-13]. Several studies indicate that, in vivo, protein synthesis is not affected by ethanol [8-10]. However, ethanol has been shown to alter protein synthesis in some in vitro systems, mainly when changes in redox potential are involved [10]. For example, in a few cases, the mechanism by which ethanol inhibits protein synthesis was further analyzed. In the C H O cell-free synthesis system, a decrease in translation was accounted for by an inhibition of Leu-tRNA synthetase by ethanol [11]. Another example is the inhibition of neutral amino acid transport by alcohol in certain cell lines [ 13]. The induction of some liver proteins by ethanol, such as cytochrome P-450 [14], glucose-6-phosphate dehydrogenase [15] and gamma-glutamyl transpeptidase (GGT) [16-19] appears to be more specific. We have shown that ethanol induces G G T in a rat hepatoma cell line (C2) without modifying other membrane or cytoplasmic enzyme activities [16]. In order to further determine the potency and the specificity of ethanol action on protein synthesis, we used twodimensional polyacrylamide gel electrophoresis to analyze the protein pattern of the C2 hepatoma cell line, especially the appearance or disappearance of individual proteins under conditions where gammaglutamyl transpeptidase activity is increased. In the present study, we demonstrate that ethanol produces a slight decrease in total protein synthesis. Surprisingly, we observed only a very limited, highly specific response to ethanol after 20 h of treatment.
Methods
L-y-Glutamyl-p-nitroanilide and glycylglycine were purchased from Sigma Chemical Co. L-[35S]Methionine (800 Ci/mmol) and 14C-methylated protein mixtures used as molecular weight standards were purchased from Amersham Radiochemical Centre, U.K. Ampholines (pH 5-7, and pH 3.5-10) were from LKB, Sweden.
M.N. CHOBERT et al.
Cell culture and ethanol treatment The C 2 cells derived from line H4IIEC 3 of the Reuber H35 hepatoma [20] were grown in a modified [21] Hams's Fl2 medium (Gibco) supplemented with 5% fetal calf serum (Flow Lab.), 200 units/ml of penicillin, 50 ~g/ml of streptomycin (Diamant) and 0.5 /~g/ml of fungizone (Squibb). Cells were routinely cultured as previously described [16], in a 7% CO~ humidified atmosphere at 37 °C, on Falcon plastic petri dishes; 2.5 x 104-105 cells and 105-106 cells were seeded per 6 cm and 10 cm diameter petri dishes, respectively. Experiments were performed during the exponential phase of growth. For ethanol treatment, culture medium was removed and fresh medium with or without 1% ethanol was added for 6 48 h. We have previously demonstrated [16] that G G T induction was not modified whether or not the plates were wrapped in parafilm to avoid ethanol evaporation. Gamma-glutamyl transpeptidase assays At each time point, control and ethanol-treated cells were washed with phosphate-buffered saline (PBS) at 4 °C, scraped and centrifuged at 300 x g and resuspended in PBS. G G T activity was determined according to the method of Orlowski and Meister [22] as previously described, using e-~,-glutamyl-p-ni'troanilide and glycylglycine as substrates [23]. Proteins were estimated by the method of Lowry et al. [24]. Measurement of [~SS]methionine incorporation in the total cell proteins as a function of time during ethanol treatment Cells were labeled with 20 lICi of [35S]methionine/ml of medium at the beginning of the experiment. At each time point, [~-~S]methionine incorporation was stopped by washing the cells three times with PBS at 4 °C. One volume of cells, resuspended in PBS as described for G G T assay, was mixed with 1 vol. of 4.2% sodium dodecyl sulfate (SDS), 0.125 M Tris HCI pH 6.8, 10% fl-mercaptoethanol, 13.2% sucrose and boiled for 3 min. Aliquots (2~d) were used for trichloroacetic acid (TCA)-precipitability measurement.
ETHANOL AND PROTEIN SYNTHESIS
Two-dimensional gel electrophoresis Cells were pulse-labeled with 75/iCi of [35S]methionine/ml of medium for 2 h before the end of ethanol treatment. The pulse was terminated by washing the cells as described above, and cell pellets were dissolved in 200-600/ll of O'Farrell lysis buffer [25]. Samples were centrifuged for 30 min at 10 000 × g and supernatants were used for TCA-precipitability measurement and isoelectric focusing. Samples were loaded onto 3 mm i.d. gel essentially according to the procedure of O'Farrell [25], except that the sample ( 10-30/xl containing 5-15/~g of proteins and 200 000 cpm) was applied to the acidic side of the gels, which were directly run at 500 V for 16 h. Gels were equilibrated for 40 min in 2.1% SDS, 5% fl-mercaptoethanol, 6.6% sucrose, 0.062 M Tris-HCl pH 6.8, and applied onto a stacking gel in 1% agarose prepared in equilibration buffer. The second-dimension electrophoresis was carried out in a 10% polyacrylamide gel slab as described previously [26] except that glycerol was replaced by 66% sucrose. Standard molecular weight markers (14C-labeled protein mixture, Amersham) were applied at one end of the gel tube, included in a 5 mm (length) by 3 mm (diameter) gel. En3Hance (New England Nuclear) was used for fluorography with Kodak X A R 5 films. Computer analysis Autoradiograms were analyzed using the HERMES computer system, a detailed description of which is found in Refs. 27-31. Briefly, the gels were scanned using a video camera [27]. These images are cleaned and filtered in order to reduce background and high frequency noise and to improve spot detection by mathematical morphology methods. Spots are detected by a computerized processing method [28] based on the use of a Gaussian model. The gels corresponding to different conditions are then matched to determine homologous spots [29]. A data array including the intensity of each spot in the different conditions tested in the experiment is computed by the system. Multivariate analysis [32] was performed with the data array, spots being considered as observations and gels as variables characterizing each spot throughout the series. In simple
87 language, each spot is described by its intensity in the different gels of the series. A full representation of spots requires an n-dimension space, n being the number of gels. The purpose of such a study is to analyze the relationships between gel patterns and different experimental biological conditions. The reduction of the number of axes by the transfer of the significant information to the first axes is useful for improving the interpretation of the experiments. This is done by computing a new axis system having the following properties: (i) the new axes are linear combinations of the old ones; (ii) the significant information carried along each axis decreases with the axis number; (iii) the new axes are independent from each other. The projections of the old axes (i.e., gels) can be mapped into the new coordinate system. Usually, the graph named factorial plane consists of two axes. The relative position of the gels over this plane indicates the relationships between these gels according to the considered axes. However, this approach is only a convenient representation for the overall information carried by the intensities of the spots in the series of gels, and allows for an easier study of the relationships between different biological conditions. Furthermore, variance analysis enables the detection of spot intensity variations under different conditions. As the migration of high molecular weight proteins in the upper part of the gel was not reproducible, the extreme areas of the gels were not taken into consideration for spot analysis.
Results
Time course of stimulation of GGT by ethanol in C2 cells The maximal increase of G G T activity was obtained with 180 mM ethanol [16]. Using this concentration, we observed stimulation of the enzyme as a function of time during ethanol treatment (see Fig. 1). In the control cells, G G T activity was relatively stable with a slight increase (40%) after 48 h of culture. The increase of G G T activity by ethanol was a slow process and the maximal increase (2.5-fold) was obtained within 48 h. A similar increase was obtained
88
M.N. CHOBERT et al
"2. o o.
whether G G T activity was expressed on the basis of protein (Fig. 1) or D N A concentration [16].
7
E~ 6
Total protein synthesis during ethanol treatment in C2 cells
3 >~
5
U n d e r the same conditions in which a maximal in.=
crease in G G T activity was produced in C 2 cells by ethanol treatment, ethanol had only a minimal effect
== .= c m
m
E
o
6
'
'
24 TIME (
'
'
'
4'8
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Hours
Fig. 1. Time course of stimulation of GGT by ethanol. C2 cells in the exponential phase of growth were exposed to 1% ethanol (180 mM) (C)) or untreated (•). At various time periods of treatment, control and treated cells were harvested, and enzyme activity was assayed as described in the Methods section. Results are expressed as mU/mg protein (1 mU of enzyme activity = 1 nmol product formed per min). Each point represents the mean value from two to five experiments, each assay being performed in duplicate.
mensional gel processing was performed as described in the Methods section. Since equal amounts of ra"dioactive material were loaded on each gel, any change in the intensity of an individual spot can be assumed to represent a change in the relative transla-
70
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C2 cell proteins during ethanol treatment analyzed by two-dimensional gel electrophoresis Ethanol-treated and control C2 cells were incubated for 2 h with [35S]methionine and then collected and dissolved in the O'Farrell lysis buffer. Two-di-
"2. o.
on total protein synthesis. The control C2 cells incorporated [35S]methionine in TCA-precipitable material at a constant rate during the 48 h of incubation with the amino acid (Fig. 2). There was a slight and transient decrease in the incorporation of [35S]methionine in the presence of 180 mM ethanol, inhibition being maximal (25%) at 30 h. This slight inhibition was detected whether the labeled methionine was added at the beginning of the ethanol treatment (Fig. 2) or 2 h before each m e a s u r e m e n t of incorporation in T C A precipitable material (data not shown).
0
o
~
'
I
~4
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I (
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i
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Fig. 2. Effect of ethanol on the time course of incorporation of L-[35S]methionine in total proteins of C, cells. At zero time, cells in the exponential phase of growth were incubated in fresh medium in the presence or absence of 1% ethanol with 20 !tCi of [35S]methionine/ml.At the different time periods indicated, incorporation was stopped by harvesting the cells and dissolving them as described in Methods for TCA-precipitability measurement Each point is the mean value of two experiments. • = control cells; C) = 1% ethanol-treated cells.
tion of the protein in that spot. Each gel corresponding to control or ethanol-treated cells at 0, 6, 20 and 48 h was processed and a data base including all the data obtained was then constituted [27-31]. Representative fluorograms are shown in Fig. 3. The analysis of 1500 spots detected on the different gels showed that ethanol produced very small effects on the total protein pattern of the C~ cells. To fully interpret the information contained in the initial data, multivariate estimations were used. This factorial analysis is shown in Fig. 4. We can thus obtain an objective measurement of the global effects of ethanol on the protein pattern of the C 2 rat hepatoma cell line. The time-dependent effect of ethanol during 48 h of treatment is clearly demonstrated in Fig. 4A. In-
ETHANOL AND PROTEIN SYNTHESIS
89
deed, along axis 3, the patterns of control cells as well as cells treated for 6 h by ethanol are clearly separated from those of cells t r e a t e d for 20 and 48 h. ConA
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versely, using axis 5 (Fig. 4B), we can easily separate protein patterns as a function of time in control cells but not in ethanol-treated cells. As shown by inspection of the eigenvector constituents taken into account in axis 5, only control gels a p p e a r at different positions along this axis, which reflects their own variability. The i n d e p e n d e n t nature of spot variation between control cells during growth and ethanoltreated cells is thus demonstrated. A f t e r extraction of the significant data from the data base a variance analysis indicated that very few spots were specifically altered during time of ethanol treatment. O n e spot (a: M r = 37 000 + 1000; p l = 5.2 + 0.1) was present at zero time and d i s a p p e a r e d with time in control cells (Fig. 3 A and B). One spot was found to be induced by ethanol t r e a t m e n t (b: M r = 36 000 + 1000; p I = 6 + 0.1). A histogram of its intensity is presented in Fig. 5A. It was clearly absent from control cells at all time points and a p p e a r e d after 20 h of ethanol t r e a t m e n t (Fig. 3C). A unique spot (c: Mr = 24 000 + 1000; p I = 6.6 + 0.1) increased in control cells as a function of time but totally d i s a p p e a r e d after 20 h of t r e a t m e n t with ethanol (see Figs. 3 and 5B). The regions corresponding approximately to M r = 54 000, p I 6.8 and M r 63 000, p I 5.9 (boxed in Fig. 3) were modified to a limited extent. A few spots changed in shape and position when different control cells or when control and treated cells were compared at different times.
it-.q.
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Fig. 3. Two-dimensional gel autoradiograms from ethanoltreated or untreated C2 cells. Cells were plated and grown as described in Methods. Control and treated cells (180 mM ethanol) were labeled for 2 h with [35S]methionine at each time point. Two-dimensional gels were prepared as described in Methods, samples (200 000 cpm) being applied on the acidic side of the focusing gel. The vertical axis separates proteins as a function of molecular weight. The horizontal axis separates proteins as a function of pI. (A) Control cells, 0 h; (B) control cells, 20 h; (C) ethanol-treated cells, 20 h. (a) Spot present in A (control cells at 0 h) and absent in any other case. (b) Induced spot after 20 h of ethanol treatment (absent in A and B, present in C). (c) Repressed spot after 20 h of ethanol treatment (present in A and B, absent in C). The enclosed areas in A, B, C are regions where the shape and position of several spots are modified when the different gels are compared. The extreme upper and lower parts of the gels were not taken into consideration.
90
M.N. C H O B E R T et al.
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Fig. 4. Global effects of ethanol (lgt) mM) studied by factor analysis of the protein pattern of C_, cells. The gels correspondmg to control and treated cells are plotted onto two planes of a principal component analysis (respectively A (axes 1-3) and B (axes 3-5}). Time-effects during ethanol treatment are mainly c a m e d by axis 3 (A) Axis 5 only carries variations occurring in the different control gels, while treated gels are not separated on this axis ( B ) . / x / = 0 hour, [] = control, 6 h: • = ethanol, 6 h" z~ = control, 20 h, • = ethanol, 20 h, © = control, 48 h : • = ethanol, 48 h
In summary, C2 cell protein synthesis is not globally modified by ethanol treatment. We found two unique proteins regulated by ethanol. Spot b corresponds to a newly expressed protein in these cells following ethanol treatment. Spot c corresponds to a protein that is repressed by ethanol. Whether or not these events take place at the transcriptional, translational or different post-translational levels cannot be determined by the present approach.
48 -
L_ 6
20
48
4-
,I-
4-
C
Fig. 5. Histograms of spot intensity for an induced (A: spot b) or a repressed protein (B: spot c)'. Control or ethanol-treated states at 0, 6, 20 or 48 h are represented. The values are selected from the data base.
Discussion
The effect of alcohol on protein synthesis has been studied previously using either total incorporation into TCA-precipitable material [10-13] or the increase in specific enzyme activity [14-16]. In the first case, the various results were sometimes contradictory and the importance of the inhibition of protein synthesis by ethanol in vivo remains questionable. In the latter case, the change in the activity of specific enzymes by ethanol was attributed either to a direct effect of ethanol on the plasma membrane [4-6] or to a stimulation of the synthesis of the protein [15,16]. None of these studies has given a clear view of the overall importance of ethanol effects on the synthesis of specific proteins.
ETHANOL AND PROTEIN SYNTHESIS The C 2 hepatoma cells have retained some of the characteristics of hepatocytes, such as tyrosine aminotransferase induction by glucocorticoids [20] and a low G G T activity [16], but lack other markers of liver differentiation [20]. In those cells, the inducuon of G G T [16], an enzyme which is a marker of alcoholism in man, occurs at ethanol concentrations that are readily found in the serum of alcoholics following an acute ingestion of alcohol [19]. Among many hepatoma-derived cells which we tested, C 2 was the only one that revealed the induction of G G T by ethanol; it thus constitutes a reasonable model of the in vivo effect of ethanol. We therefore decided to assess the effect of alcohol on protein synthesis in this particular cell line under conditions in which a specific increase in G G T activity is observed. Obviously, this might only represent a fraction of what is actually modified in the hepatocyte in vivo. The concentration of ethanol (180 mM) was chosen for the following reasons: (i) this corresponds to the optimal concentration that we have determined previously for G G T induction [16]; (ii) it is not toxic for the cells [16]; (iii) this concentration produces a mild effect on protein synthesis, and we expect smaller and more 'physiological' concentrations to produce even milder and probably not significant effects. Besides, the C, cell line is lacking in two of the main enzymes revolved in ethanol degradation, namely alcohol dehydrogenase and cytochrome P-450j (data not shown). In these cells, ethanol is probably not degraded to a very large extent. A single dose of ethanol was added to the cells and its acute effects (up to 48 h) on protein synthesis were determined using two-dimensional gel analysis. Our results show that ethanol modifies protein synthesis quantitatively and qualitatively after treatment of relatively short duration. Firstly, the incorporation of [3SS]methionine in total proteins is found to be slightly decreased by ethanol treatment in our conditions. This is probably correlated with the slight decrease in cell growth that we have observed. Recently, other authors have described an alteration of growth rate correlated with an accumulation of cells in G I phase and a low RNA content in rat liver tumor cells treated by ethanol [2]. Secondly, among all the pro-
91 tein spots analyzed, very few proteins are increased or decreased by ethanol treatment. One is clearly induced, and one is totally repressed after 20 h of treatment. These are rather minor proteins. The protein induced by ethanol cannot correspond to cellular GGT, which represents less than 0.0005% of total proteins in the C, cells. The sensitivity of the technique does not allow the detection of such rare proteins. Indeed, out of 200 000 cpm corresponding to total loaded proteins, only 1 cpm would correspond to GGT. In HTC cells [33], tyrosine aminotransferase represents 0.02-0.05% of total proteins and the easily detectable corresponding spot represents at least 100 cpm in these experiments with a 10 day exposure to X-ray films. In other experiments related to the "growth hormone domain' of rat liver [34], for a total load of 200 000 cpm, spots equivalent to 30 cpm correspond to the lower limit of detection. The total number of cellular mRNA products largely exceeds the 1500 spots detected in the present study, and 5000-11 000 different m R N A species can be estimated in hepatic cells [35,36]. The global analysis of gels reflects only a part of ethanol effects on protein expression. In other works on hepatic proteins, the set of spots that were analyzed never exceeded 1500 [33,36]. In the case of in vitro translation of isolated liver mRNAs, not more than 200 translation products were analyzed [34,35]. In HTC cells, glucocorticoids modify specifically 13 proteins out of a total number of 1000 spots [33]; 19 out of 231 hepatic proteins visualized are modulated by T.~ hormone [35]; 8 proteins among 250 are altered in the liver by growth hormone [34]. In a recent study of liver regeneration, 1600 spots were analyzed [36]; in this case, only a few qualitative changes were observed, but the intensity of many spots changed considerably. Other stress situations such as heat shock [37] and azotemia [38] modify a larger set of proteins. Compared to these results, ethanol in the C2 cell line modifies markedly two specific spots and its effects appear therefore to be much more limited than those of other effectors which also alter hepatic protein expression. Nevertheless, the two-dimensional gel analysis represents the most powerful tool to date for evaluating overall changes in the synthesis of pro-
92
M.N. CHOBERT et al.
teins, although the n u m b e r of changes is p r o b a b l y un-
cellular m e m b r a n e s , s o m e of t h e s e effects c o u l d also
derestimated. In a different system ( h a m s t e r fbroblasts, H A - l ) ,
be due to the specific m o d i f i c a t i o n of g e n e e x p r e s -
e t h a n o l has b e e n s h o w n to induce specifically two
m o d i f i e s g e n e e x p r e s s i o n was u n k n o w n . It has b e e n
sion. U n t i l recently, the m e c h a n i s m by which e t h a n o l
heat shock proteins, Hsp 70 and Hsp 87 [39]. T h e s e
suggested lately that e t h a n o l acts on the p r o m o t e r re-
do not c o r r e s p o n d to the m o d i f i e d spots o b s e r v e d
gion of the a p o l i p o p r o t e i n A - I g e n e [40], and stabi-
here. M o r e o v e r , in the studies with h a m s t e r fibro-
lizes the c y t o c h r o m e P-450 f o r m 3a [41].
blasts, e t h a n o l was used in v e r y acute stress conditions (6% for 1 h) w h e r e a s in o u r case it was used at 1% for 48 h. T h e s e latter e x p e r i m e n t a l c o n d i t i o n s were found to be o p t i m a l for the i n d u c t i o n of G G T in
Acknowledgements
C 2 cells [16]. T h e m a j o r conclusion of this study is that e t h a n o l
W e wish to t h a n k Drs. P. B e a u n e and N. P e r r o t for
can affect specifically the e x p r e s s i o n of a limited
c y t o c h r o m e P-450j d e t e r m i n a t i o n , Dr. P. Insel, Dr.
n u m b e r of p r o t e i n s in the C 2 h e p a t o m a cell line. A l -
F. P e c k e r , Dr. P. B e r t h e l o t and Dr. B. N a l p a s for
t h o u g h most of the cellular effects of e t h a n o l can be
their critical r e a d i n g of the m a n u s c r i p t , and Ms. L.
a c c o u n t e d for by its m e t a b o l i s m or its action on the
R o s a r i o for e x c e l l e n t secretarial assistance.
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