Eur. J. Biochem. 195,723-729 (1991) 0FEBS 1991

0014295691000877

Identification of a novel nuclear protein synthesized in growth-arrested human hepatoblastoma HepG2 cells Giulia DONADEL ’, Carlo GARZELLI ’, Rainer FRANK and Franco GABRIELLI Institute of Biological Chemistry and ’ Department of Biomedicine, University of Pisa, Italy, European Molecular Biology Laboratory, Heidelberg, Federal Republic of Germany

(Received June 13/August 30, 1990) - EJB 90 0672

DNA synthesis of human hepatoblastoma HepG2 cells is reversibly inhibited by butyrate. When butyrate is removed from the culture medium, cells re-enter the cell cycle, synthesizing DNA with a time lag of about 12 h. HepG2 cells, growth-inhibited for 30 h with butyrate, synthesize and accumulate a nuclear protein, called D. Protein D synthesis is inhibited in cells which, released from the butyrate block, have resumed DNA synthesis as well as in growing cells never exposed to butyrate. Protein D has been purified from growth-arrested cells and partially sequenced. The amino acid sequences of five internal trypsin peptides indicate that protein D is a novel nuclear protein.

Cultured mammalian cells can be arrested in the G1 phase by serum deprivation [l - 31, cell, confluence [4] or treatment with chemical agents [5-71. GI-arrested cells are stated to be in the Go phase, i.e. quiescent. Qualitative differences between the G I and the Go phases are not well defined [l, 31. However, experimentally induced Go cells, when stimulated to proliferate, require a longer period than GI for reinitiating the DNA synthesis (the S phase)[l]. Besides, certain biochemical characteristics of experimentally induced Go cells are similar to those of quiescent normal cells, such as senescent cells [2] or terminally differentiated cells of adult tissues [5 - 71. The induced Go phase is characterized by repression as well as expression of specific genes [l - 4,6 - 91. A set of genes, called growth-arrested-specific genes, has been identified as being active in cultured mouse fibroblasts, made quiescent by serum deprivation. These genes are repressed with different kinetics when fibroblasts are induced to proliferate by addition of serum [3]. Two proteins, specifically synthesized in non-proliferating cells, have been identified [2, 8, 91. Statin [2] is a nuclear protein, synthesized in non-proliferating human fibroblasts, including senescent fibroblasts, serum-starved and confluent young fibroblasts. When serum-starved young fibroblasts are induced to proliferate by addition of serum, statin synthesis is greatly decreased . Quiescent chicken-heart mesenchymal cells, which do not proliferate in normal media containing plasma, synthesize and secrete a 20-kDa protein that is repressed when the cells are induced to proliferate by mitogens or are transformed by Rous sarcoma virus [8, 91. The function of growth-arrested-specific gene products, statin and the chicken heart mesenchymal cell 20-kDa protein is not known; however, the incompatibility between their expression and cell proliferation suggests that they may play a role in substaining the G o phase [3, 4, 81. Moreover, two Correspondence to F. Gabrielli, Institute of Biological Chemistry, via Roma 55,I-56126 Pisa, Italy Abbreviations. NaC1/Pi,phosphate-buffered saline.

proteins (RB protein and p53 protein) which negatively regulate proliferation have recently been detected in several different types of cancer cells [lo] (and references quoted therein). We have investigated the synthesis of nuclear proteins in human hepatoblastoma cells (HepG2 line) [I11 treated with butyrate. Butyrate inhibits cellular proliferation and its major effect on cell progression is G1/Go block [6] (see also references quoted therein). Moreover, butyrate can induce morphological and biochemical changes which mimic cellular differentiation [5-7, 12, 131. In this report we describe the identification of a nuclear protein (called protein D), which is synthesized and accumulates in HepG2 cells growth-inhibited by butyrate. The expression of this protein is repressed in proliferating cells. Protein D has been purified and partially sequenced. The analysis of the amino-acid sequences indicates that protein D is a novel nuclear protein. MATERIALS AND METHODS Materials

Cell-culture materials (Gruppo Flow, Italy); sodium butyrate (Merck, FRG); Triton X-100 (Sigma, USA); urea (Bio-Rad, USA); trypsin, sequencing grade (Boehringer, FRG); electrophoresis chemicals (Serva, FRG); L - [ ~ ~ S ] methionine and [3H]thymidine (Amersham, England) ;X-AR X-ray film (3 M, Italy). Cell culture HepG2 cells [ll] were grown in RPMI-1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U penicillin/ml, 100 pg streptomycin/ml, 2.5 pg amphotericin B/ml. Additions of 1 M sodium butyrate pH 7.4 (10 mM final concentration) were made directly to the flasks as indicated in the legends of the figures.

724 Determination of D N A .qvnthesis

lo5-

Cultures (lo3 cells in 0.250 ml complete medium) were set up in 96-well microtiter plates and pulsed for the final 6 h with 0.5 pCi [nzethyl- 3H]thymidine (25 Ci/mmol). The cells were washed twice with NaCl/Pi (1.85 mM NaH2P04, 0.84 mM NaHP04, 0.15 M NaC1, pH 7.3), collected by means of a semiautomatic cell harvester and the radioactivity measured in a liquid scintillation counter.

8 6 4 -

.E

Preparation of nuclear proteins

-

.1.

10"8

-

6 -

.HepG2 cells were washed twice with NaC1/Pi, centrifuged P 0 4 at 600 x g and the cell pellet stored at - 8 0 "C. Nuclei and a total nuclear proteins were prepared as previously described [14]. All the procedures were carried out at 0-4"C, and phenylmethylsulphonyl fluoride (1 mM final concentration) was added to all solutions. Cells were suspended in hypotonic 1o3 solution (10 mM Tris/Cl pH 7.5,4 mM MgC12, 1 mM CaC12) I and lysed by addition of Triton X- 100 (0.4% final concen0 6 12 18 24 30 tration). An equal volume of isotonic solution (50 mM Tris/ Cl pH 7.5, 300 mM sucrose, 25 mM KC1, 4 mM MgC12, Time ( h ) 1 mM CaC12) was added and the nuclei were collected at 1000 x g and washed twice in the isotonic solution. The nuclei Fig. 1. DNA synthesis in HepG2 cells. At the times indicated, DNA were lysed by suspension in 0.2 M EDTA and the nuclear synthesis was assayed in ( 0 )cells never exposed to butyrate; (A)cells treated with 10 mM butyrate for 30 h and at zero time transferred proteins extracted by adding an equal volume of 0.4 M into fresh culture medium; (m) cells continuously treated with 10 mM H2S04. The proteins extracted were precipitated (12 h at butyrate from 30 h before to 30 h after zero time. Arrows indicate - 2 0 T ) by addition of 10 vol. of acetone, collected by times at which cells were collected in a parallel experiment, shown in centrifugation (10000 x g for 15 mim), dried, dissolved in 9 M Fig. 2 urea/lO% 2-mercaptoethanol and stored at - 80°C. Proteins were labelled by growing cells for 8 h with ~ - [ ~ ~ S ] m e t h i o n i n e (100 pCi/ml). q

Electrophoresis Two-dimensional electrophoresis was performed as described [15]. The first-dimension gel contained 15% acrylamide, 0.08% bisacrylamide, 7.5 M urea, 0.36% Triton X-100. Each sample track to be andlysed in the second dimension was cut out and soaked in the SDS gel electrophoresis buffer [I61 containing 2% 2-mercaptoethanol. The seconddimension gel contained 14% acrylamide, 0.375 bisacrylamide and 0.1 OO/ SDS. One-dimensional SDS polyacrylamide gels were run in the same way as those for two-dimensional electrophoresis. Gels were stained with Serva blue R. Detection of radioactively labeled proteins was carried out by autoradiography of dried gels on X-AR X-ray films.

eluted from the gel by shaking the pieces twice for 3 h with an equal volume of 0.1% trifluoroacetic acid in water. Residual water was extracted from the gel matrix by treatment with acetonitrile. The concentrated eluates were extracted twice with isoamyl alcohol/heptane (1 :4) to remove traces of SDS [17]. Separation of the tryptic peptides was performed by reversed-phase high-performance liquid chromatography on a Vydac 218TP5 column (1.6 x 250 mm). Nine peaks were selected from the elution diagram and the material was subjected to gas-phase sequence analysis according to Gausepohl et al. [18]. Five peaks contained peptides more than 85% pure. The initial sequencing yields were between 45 pmol (peptide 1) and 25 pmol (peptide 5 , see Fig. 5) indicating that all peptides were derived from the same protein and not from a minor contaminant.

Purification and amino-acid sequence determination ofprotein D

RESULTS

Total nuclear proteins from about 2-4 x l o 7 cells were fractionated by preparative two-dimensional polyacrylamide gel electrophoresis. Gel pieces containing stained protein D were cut from several gels and loaded together on an SDS/ polyacrylamide gel. Then, ordinary SDS/polyacrylamide gel electrophoresis [16] was performed (Fig. 4). At this step, 412 pg protein D was concentrated in a single band. A band corresponding to about 4 pg protein D was precisely excised from the gel, cut in pieces (about 1 x 1 mm) and incubated in water with frequent changes for 16 h. The washed gel pieces were then immersed in 200 p1 of 100 mM ammonium hydrogen carbonate pH 8.5 containing trypsin at an enzyme/ protein mass ratio of approximately 1:5. Following incubation at 37 ^C for 8 h, the protein fragments produced were

DNA synthesis of human hepatoblastoma HepG2 cells is strongly inhibited by 10 mM sodium butyrate. The inhibition of DNA synthesis is reversible. When sodium butyrate is removed from the medium, HepG2 cells begin to synthesize DNA again with a time lag of about 12 h. (Fig. 1). We have examined the patterns of nuclear protein synthesis in growing and growth-inhibited HepG2 cells, as well as in cells recovering from growth inhibition, at 9,17 and 25 h after the removal of the butyrate from the culture medium (Fig. 2). The cell cultures were metabolically labelled for 8 h with L[35S]methionine in RPMI- 1640 medium containing 20% fetal calf serum and the normal amount of methionine. Unlabelled methionine was not subtracted from the culture medium in order to avoid the inhibitory effect on cell growth

726

Fig. 2. Two-dimensionul gel unalysis of total acid-soluble nuclear proteins synthesized b,v HepG2 cells in dijferent growth conditions. The protcins were metabolically labelled with ~-[~'S]methionine for 8 h and analyzed by two-dimensional electrophoresis and autoradiography. Electrophoresis in a first-dimension Trilon/urca/polyacrylamide gel (left to right) was followed by electrophoresis in a second-dimension SDS/ polyacrylamide gel (top to bottom). (A) Pattern for growing cells; (B) pattern for cells growth-inhibited for 30 h with 10 mM butyrate; pattern for 26 cells (C) 9 h, (D) 17 h and (E) 25 h after the removal of butyrate from the culture medium. Arrows, without identification symbols, point to polypeptides which are more intensely labelled in pattern (B) for growth-inhibited cells as compared to pattern (A) for growing cells. The dotted areas show the migration areas of HZA, H I B and H2A-1 histones which, since they do not contain methionine [13, 241, do not show up i n autoradiograms. The H2B spot includes H2B- 1 and H2B -2 variants [13,14,21]. H2A-U, H2A histones combined with ubiquitin. Histones were identified by their mobility in relation to H4 in Triton/urea and SDS/polyacrylamide gel electrophoresis [14, 151

due to shortage of an essential amino acid in the culture medium [l]. Comparative analysis of the autoradiograms showed that a protein (called D) was synthesized in growth-inhibited HepG2 cells treated for 30 h with butyrate (Fig. 2B). Synthesis of protein D was also detectable in HepG2 cells that, after being treated for 30 h with butyrate and then transferred into a normal culture medium, had not yet resumed DNA synthesis (Fig. 2C). Protein D synthesis was inhibited in growing cells never treated with butyrate (Fig. 2A), as well as in cells which, treated for 30 h with butyrate and then grown for 17 h (Fig. 2D) or for 25 h (Fig. 2E) in normal culture medium, had resumed DNA synthesis. In autoradiograms with exposure

times longer than those shown in Fig. 2A, D and E, the spot for protein D becomes slightly visible, indicating that a minimal rate of protein D synthesis may take place in growing cells (data not shown). In HepG2 cells, butyrate stimulates synthesis of other nuclear proteins. These proteins include HI" histone and at least four other nuclear proteins (indicated by arrows, without identification symbols, in Fig. 2B). These four proteins and histone HI" behave like protein D. They are more actively synthesized in non-growing butyrate-treated HepGZ cells (Fig. 2B) than in growing cells (Fig. 2A) and their synthesis is reduced with different kinetics in cells released from the butyrate block (Fig. 2C, D and E). HI" histone is a replication-

727

Fig. 3. Two-dimensional gel analysis of total acid-soluble nuclear proteins of HepG2 cells. Electrophoresis was the same as in Fig. 2. Proteins were stained by Serva blue R. Total acid-soluble nuclear proteins of (A) growing cells and (B) cells growth-inhibited for 30 h with 10 mM butyrate. For other details see Fig.2

independent variant, which accumulates in normal quiescent cells, such as adult tissues [12, 14, 191, as well as in experimentally growth-inhibited cultured cells [6, 7, 12, 19, 201. The positive effect of butyrate on H1" histone synthesis has been shown in several types of cells [6,7,20], (see also references quoted therein). It has been suggested that H1" is an inhibitor of cell proliferation and/or of transcription ( for a review see [13]). Our experiments indicate that butyrate also stimulates H1 synthesis in HepG2 cells. Unlike replicating human normal liver cells and other human hepatoma cells, replicating HepG2 cells have a high content of H l 0 [14] (Fig. 3A), which is further increased by butyrate treatment (Fig. 3B). Densitometer scanning of autoradiograms obtained from one-dimensional SDS gels indicates that, in butyratetreated cells, H1" synthesis is about twice as large as in the control cells (data not shown). Butyrate inhibits the synthesis of several nuclear proteins, nucleosomal histones included (Fig. 2B). The synthesis of most of the histones is cycle-regulated [19]; therefore the inO

hibitory effect of butyrate on nucleosomal histone synthesis appears to be a consequence of the inhibitory effect of butyrate on cell-cycle progression. Synthesis of H2B histone is the least inhibited. H2B histone includes H2B - 1 and H2B - 2 variants which are not separated in the electrophoretic conditions of our experiments (Figs 2 and 3) [13, 14,211. Similarly, different inhibitory effects on different nucleosomal histones have been observed in butyrate-treated murine neuroblastoma cells [20]. The nucleosomal histone synthesis is resumed 25 h after removal of butyrate block (Fig. 2E). HepG2 cells, growth-inhibited for 30 h with 10 mM butyrate, accumulate a large amount of protein D (Fig. 3B). This becomes clearly visible by staining with Serva blue R when a large amount of total nuclear protein is loaded on the twodimensional gel (Fig. 3B). A minimal amount of protein D is present in growing cells (Fig. 3A). We assume that protein D is localized in nuclei since we obtained it by acidic extraction of purified nuclei. The identical extraction procedure, when used for HepG2 cell cytoplasm and for the culture medium of

728 1.

-Ser-Pro-Ala-Leu-Leu-Leu-Ser-Gln-Leu-Leu-Pro-Tyr-

Met-Glu-Asn-

Fig. 4. SDS/pol-vucrylaniide gel electrophoresis of purified protein D. The molecular mass markers are (from the top): bovine serum albumin, egg albumin, glyceraldehyde-3-phosphatedehydrogenase, carbonic anhydrase. trypsinogen, soybean trypsin inhibitors, cc-lactoalbumin. Histone molecular masses were calculated from the aminoacid sequence of unmodified polypeptides. Lane 1, histones of bovine lung; lane 2, purified preparation of protein D; lane 3, total acidsoluble nuclear proteins of HepG2 cells growth-inhibited for 30h with 10mM butyrate. For other details see Fig. 2

2.

-Leu-Gin-Gly-Glu-Leu-Leu-Val-Ala-Ala-Ile-Val-

3.

-Val-Gln-Gly-Glu-Gly-Gly-Ser-Val-Ala-

4.

-Thr-Leu-Ala-Leu-Glu-Leu-Ala-Pro-

5.

-1le-Leu-Ser-Val-Asn-Val-

Fig. 5. Amino-acid .sequences of the five internal trypsin peptides qf protein D

quences in the EMBL Data Library Or the cal Research Fundation Data Bank.

Biomedi-

DISCUSSION butyrate-treated HepG2 cells, gave negative results. Histones constitute the largest fraction of nuclear proteins both in butyrate-treated and control cells. As compared to the control cells, butyrate-treated cells contain an increased amount of H1' and a reduced amount of HIA histone. The same cells do not show major changes in the relative amounts of nucleosomal histones (Fig. 3B), even though, in butyratetreated cells, the synthesis of several nucleosomal histone fractions is greatly reduced (Fig. 2B). This may be explained by the different turnover rates of different histones. H1B has a rapid turnover [22], while that of H1A and nucleosomal histones is very slow [19,22]. Inhibition of synthesis for 30 h does not appear to be sufficient to cause detectable reduction in the amount of histones which turnover slowly. In butyratetreated cells, the nucleosomal histone spots are wider than those of the control cells because of nucleosomal histone hyperacetylation caused by butyrate inhibition of histone deacetylase [5].In direct relation to their acetyl group content, the migration of histone hyperacetylated polypeptides is slower in Tritonlurea gel electrophoresis [19, 211 and it causes the widening of nucleosomal histone spots in second-dimension electrophoresis. The protein D of growth-arrested HepG2 cells was purified (Fig. 4). Its molecular mass was around 20- 26 kDa: the value was about 26 kDa when the calibration curve was drawn using non-nuclear proteins commonly used for determining molecular masses by means of SDS/polyacrylamide gel electrophoresis but only about 20 kDa when histones were used as molecular mass markers. Histones are very basic proteins and in SDS gel electrophoresis they have a slower migration rate than that of routinely used molecular mass markers. Protein D was partially digested by trypsin and five internal peptides were sequenced (Fig. 5). The amino-acid sequences of these peptides show no similarities with the se-

The results presented indicate that HepG2 cells, growthinhibited by butyrate, synthesize and accumulate an acidsoluble nuclear protein (called protein D) of 20 - 26 kDa. The synthesis of protein D is inhibited in growing HepG2 cells and in butyrate-treated HepG2 cells which resume DNA synthesis after removal of the butyrate from the culture media. Primary structure analysis indicates that protein D is a novel nuclear protein. The biological function of protein D remains to be determined. Accumulation of protein D in growth-inhibited HepG2 cells might point to the need to sustain growth inhibition or might be related to differentiation induced by butyrate in these cancer cells and not to growth control. Synthesis of protein D is cell-cycle regulated as is the synthesis of quiescence-specific protein [2, 4, 81, as well as being an expression of growth-arrested-specific genes [3]. The discovery of quiescence-specific proteins and of these genes has led to the hypothesis that certain of these proteins and genes might act as negative regulators of cell proliferation. Recently, two proteins that act as inhibitors of cell proliferation have been identified. It has been assumed that in normal cells the inhibitory activity of these proteins balances the effects of other proteins that are activators of cell proliferation and are expressed by proto-oncogenes [lo]. Therefore, abnormal cell proliferation may result from an increased activity of activators and/or from the reduced activity of cell proliferation inhibitors. p53 protein, a negative regulator of cell proliferation, forms stable complexes with the transforming proteins of SV40 virus and type-5 adenovirus [lo, 231. In cells containing these complexes, the levels of p53 protein are 100-fold higher than in nontransformed cells. Moreover, mutant p53, able to form complexes with wild-type p53 protein, are oncogenic presumably because the mixed oligomers have no anti-proliferative activity [lo, 231.

729 We may hypothesize that protein D plays a role in the negative control of cell proliferation, assuming that in HepG2 cells the butyrate-induced accumulation of protein D is necessary for counteracting abnormally high activities of endogenous factors which sustain HepG2 cell proliferation in butyrate-free media. Therefore, it will be interesting to discover the biological function of protein D. Our current research aims at synthesizing and sequencing its cDNA in order to deduce the complete amino-acid sequence of protein D and to carry out a functional investigation. The work at the Institute of Biological Chemistry of the University of Pisa was supported by grants (40% and 60%) to F. Gabrielli from the Italian Ministry for the University and Scientific and Technological Research. We wish to thank Dr L. Bartalena for the gift of HepG2 cells and Mr P. Bertelli for technical help.

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Identification of a novel nuclear protein synthesized in growth-arrested human hepatoblastoma HepG2 cells.

DNA synthesis of human hepatoblastoma HepG2 cells is reversibly inhibited by butyrate. When butyrate is removed from the culture medium, cells re-ente...
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