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Disruption of the coordinate expression of muscle genes in a transfected BC3H1 myoblast cell line producing a low level of the adenovirus E1A transforming protein JOES. MYMRYK' Department of Biochemistry, McMaster University, Hamilton, Ont., Canada L8S 4KI AND

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JANICE D. OAKES,SENTHIL K. MUTHUSWAMY, PIETRO D'AMICO, STANLEYT. BAYLEY,' AND RAYMOND W. H . LEE^ Department of Biology, McMaster University, Hamilton, Ont., Canada L8S 4KI Received March 30, 1992 MYMRYK, J. S., OAKES,J. D., MUTHUSWAMY, S. K., D'AMICO, P., BAYLEY,S. T., and LEE, R. W. H. 1992. Disruption of the coordinate expression of muscle genes in a transfected BC3H1 myoblast cell line producing a low level of the adenovirus E1A transforming protein. Biochem. Cell Biol. 70: 1268-1276. Mouse BC3H1 myoblasts were stably transfected with the adenovirus 5 EIA gene. One clonal line, BC3E7, was found to differ in some important respects from those previously reported for E1A-transformed myoblasts. In contrast to BC3Hl cells which differentiate when confluent in medium containing 0.5% fetal calf serum (FCS), BC3E7 cells failed to elongate and align, to express acetylcholine receptor and creatine kinase, and to down-regulate expression of Pand Y-actins and tropomyosin isoform (TM) 1. However, increased synthesis of TMs 2, 3, and 4, and myosin light chain 1 associated with differentiation in BC3Hl still occurred in BC3E7 cells, and most surprisingly, a-actin was produced at a significant level in both proliferating and confluent BC3E7 cells. Interestingly, myogenin was expressed in confluent BC3E7 cells in 0.5% FCS, but not in 20%. The level of EIA expression in BC3E7 cells was found to be very low by analysis of mRNA, by immunoprecipitation of E l A protein, and by the ability of BC3E7 cells to complement the EIA-deficient adenovirus mutant d1312. These results suggest that different levels of E1A may be needed to repress different promoters and that E1A does not block myogenic differentiation by repressing myogenin expression, but represses each muscle gene independently. Key words: actin, adenovirus 5 E l A , BC3H1 myoblasts, myogenin. MYMRYK,J. S., OAKES,J. D., MUTHUSWAMY, S. K., D'AMICO, P., BAYLEY,S. T., et LEE, R. W. H. 1992. Disruption of the coordinate expression of muscle genes in a transfected BC,Hl myoblast cell line producing a low level of the adenovirus E l A transforming protein. Biochem. Cell Biol. 70 : 1268-1276. Les myoblastes BC3H1 de souris sont transfectes de facon stable avec le gkne ElA de I'adCnovirus 5. Une lignCe clonale, BC3E7, diffkre sous plusieurs aspects importants de celles dkja rapportkes pour les myoblastes transformks par EIA. Au contraire des cellules BC3H1 qui se differencient lorsque confluentes dans un milieu contenant 0,5% de serum de veau foetal, les cellules BC3E7 sont incapables de s'allonger et de s'aligner, d'exprimer le recepteur de l'acktylcholine et de la creatine kinase et de contr6ler nkgativement l'expression de la P- et de la Y-actine et de l'isoforme 1 de la tropomyosine (TM). Cependant, la synthbe accrue des TM 2,3, et 4 et de la chaine 1Cgkre 1 de la myosine, associee a la differenciation en BC3H1, s'effectue encore dans les cellules BC3E7 et, fort ktonnamment, l'a-actine est produite en quantitks importantes dans les cellules BC3E7 proliferantes et confluentes. De facon intbessante, la myogknine est exprimke dans les cellules BC3E7 confluentes dans le milieu contenant 0,5% de serum de veau foetal, mais non dans le milieu avec 20% de ce serum. L'analyse du mRNA, I'immunoprCcipitation de la protCine E1A et le pouvoir des cellules BC3E7 de complkmenter I'adCnovirus mutant dl312 deficient en E1A permettent de montrer que le taux d'expression du gene ElA dans les cellules BC3E7 est trts bas. Ces rksultats suggtrent que diffkrents taux de E l A seraient nkcessaires pour reprimer divers promoteurs et que le gkne EIA ne bloque pas la differenciation myogknique en reprimant l'expression de la myogenine, mais reprime chaque gkne des muscles de facon independante. Mots clis : actine, EIA de l'adenovirus 5, myoblastes BC3H1, myogknine. [Traduit par la redaction]

Introduction Cellular differentiation involves changes in the pattern of gene expression which give t h e differentiated cell its characteristic phenotype. BC3H1 myoblasts (Schubert et al. ABBREVIATIONS: FCS, fetal calf serum; TM, tropomyosin (isoform); CK, creatine kinase; AChR, acetylcholine receptor; MHC, myosin heavy chain; MLC, myosin light chain; Ads, human adenovirus serotype 5; DMEM, Dulbecco's modified Eagle's medium; PFU, plaque forming units; G418, Geneticin; SDS, sodium dodecyl sulfate; dNTP, deoxynucleoside triphosphate; kDa, kilodalton(s); NP-40, Nonidet P-40; BSA, bovine serum albumin; 2D PAGE, two-dimensional gel electrophoresis; IEF, isoelectric focusing. '~esearchstudent of the National Cancer Institute of Canada. ' ~ u t h o rto whom all correspondence should be addressed. 3 ~ r e s e n taddress: Microbix Biosystems Inc., 341 Bering Avenue, Toronto, Ont., Canada M8Z 3A8. Printed in Canada / lmprime au Canada

1974), when induced to differentiate by either reducing serum level in t h e growth medium o r allowing cell-cell contact upon confluency, activate muscle-specific gene transcription a n d accumulate muscle-specific proteins such as muscle CK a n d myokinase (Schubert et al. 1974), the nicotinic AChR (Patrick et al. 1977), the insulin receptor (Standaert et al. 1984), a-actin (Strauch a n d Rubenstein 1984; Strauch et al. 1986; Strauch a n d Reeser 1989; T a u b m a n et al. 1989), troponin T (Taubman et al. 1989), a n d the sarcomeric isoforms of M H C , M L C 2 a n d 3, a n d a-tropomyosin (Taubman et al. 1989). Concomitantly, expression of the nonmuscle p- a n d Y-actins (Strauch a n d Rubenstein 1984; Strauch et al. 1986; Taubman et al. 1989), t h e nonsarcomeric isoforms of a-tropomyosin, M H C , a n d myosin regulatory light chain are reduced (Taubman et al. 1989).

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MYMRYK ET AL.

This developmentally regulated expression of such a large array of unlinked genes suggests the existence of a complex regulatory mechanism signalling the precise temporal activation and inactivation of genes to achieve the differentiated state. There is increasing evidence that members of the myoD gene family, encoding proteins containing a domain homologous to the basic helix-loop-helix structure of the myc product, are involved in dictating the expression of the muscle-specific genes (Olson 1990; Weintraub et al. 1991). BC3H1 cells d o not express myoD (Davis et al. 1987), but when these myoblasts are induced to differentiate, expression of myogenin, another of these regulatory genes, precedes by several hours the expression of muscle-specific genes (Edmondson and Olson 1989; Brunetti and Goldfine 1990). It has been shown that transfection of myogenin into C3H10T1/2 and 3T3 fibroblasts induced the appearance of MHC immunofluorescence-positive cells (Wright et al. 1989; Edmondson and Olson 1989), while exposure of BC3Hl and L6A1 myoblasts t o antisense oligonucleotides of myogenin inhibited AChR and CK protein expression, respectively (Brunetti and Goldfine 1990; Florini and Ewton 1990). From these observations, it has been suggested that, like all other members of the myoD gene family of master regulatory genes, myogenin alone is sufficient to activate muscle differentiation. The antithesis of cellular differentiation is oncogenic cellular transformation. Two of the effects transformation has on cells are to prevent them from escaping from the cell cycle to enter Go and to prevent them from expressing differentiation-specific genes. Nuclear oncogenes such as adenovirus EIA are able to produce both of these effects in cells and so are useful tools for analyzing the molecular mechanisms underlying differentiation. The main products of the Ad5 E1A gene are two related proteins of 289 and 243 residues. In addition to immortalizing cells as part of the transformation process, the larger of these proteins is a potent activator of transcription by virtue of the internal 46. residue sequence unique to this protein (Berk et al. 1979; Jones and Shenk 1979), while both proteins have been reported to be able to repress transcriptional enhancers (Borrelli et al. 1984; Velcich and Ziff 1985; Lillie et al. 1986; Jelsma et al. 1989). Webster et al. (1988) showed that each of these proteins was able to repress transcription of musclespecific genes such as a-actin, MHC, and CK, in rat L8 and mouse C2 myoblasts transfected with the appropriate ElA sequences. More recently, Enkemann et al. (1990) found that transfection of mouse 23A2 myoblasts with EIA inhibited expression of genes for MyoD and myogenin, as well as troponin I, and concluded that ElA blocks differentiation by repressing the expression of myogenic regulatory genes. As part of a study on the effect of Ad5 E1A on the differentiation of BC3H1 cells, we have isolated a line of these cells stably transformed with EIA that expresses E l A at a very low level. In this BC3E7 line, myogenin expression depends on the mode of induction of muscle differentiation. However, regardless of the level at which the myogenin gene is expressed, BC3E7 cells fail to differentiate morphologically and to express several muscle-specific genes. In addition, changes in the expression of some other genes normally associated with differentiation still occur, and in particular, the a-actin gene is expressed constitutively. These results raise some interesting questions about the effect of E l A on the control of gene expression during myogenesis.

Materials and methods Cell lines and viruses

BC3H1 cells were obtained from the American Type Culture Collection (Rockville, Md.) and maintained in DMEM supplemented with 20% FCS (Gibco), penicillin (100 U/mL), and streptomycin (100 pg/mL) (Sigma) at 37°C in a 5% C0,-enriched atmosphere. BC3E7 cells were maintained under the same conditions. Adenoviruses d1520, d1309, and dl312 were grown and titrated on human 293 cells. dl309 is wild type for EIA (Jones and Shenk 1979). Both dl520 and dl312 were constructed from d1309; dl520 contains a deletion that removes the 5 '-splice site for the 13s EIA mRNA (Haley et al. 1984) and dl312 lacks most of the ElA gene (Jones and Shenk 1979). Cells were infected 3 days after plating at a multiplicity of infection of 10 PFU/cell. Transfection of B C f l cells with plasmids

Exponentially growing BC3H1 cells in 100-mm dishes were transfected by the calcium phosphate protocol of Chen and Okayama (1989), using for each dish 15 pg of either pElAneo3, a plasmid containing both the EIA and neomycin resistance genes. or pPBdx4, containing the neornycin-resistance gene alone (generously provided by P. Brinkley and F. L. Graham, McMaster University). Transfected cell lines were selected and maintained for 13 passages in medium containing 400 pg G418/mL (Gibco), after which the G418 concentration was reduced to 200 pg/mL. RNA isolation and analysis

Total cellular RNA was extracted as described by Chomczynski and Sacchi (1987). For Northern hybridization analysis, 6 pg of RNA was electrophoresed on formaIdehyde-agarose gels (Selden 1991) and blotted onto Gene Screen membrane (DuPont) using 10 x SSC as transfer buffer (1 x SSC is 0.15 M NaCl - 0.15 M sodium citrate, pH 7.0). After either baking or UV cross-linking, filters were prehybridized in 1% SDS - 1 M NaOH - 10% dextran sulfate at 65°C for 6 h and hybridized with [32~]phosphatelabelled DNA probes (1-2 x lo6 cpm/mL) in the same mixture at 60°C for 16-20 h. Probes were labelled by primer extension (Pharmacia) and separated from unincorporated dNTP by chromatography on Sephadex G-50 (Pharmacia) following established protocols (Struhl 1991). After hybridization, the membranes were washed to a final stringency of 0.5 x SSC - 0.1% SDS at 65°C and exposed to Kodak XAR-5 film for 36-72 h at -70°C with intensification. The probes used were pMHaA-1, which contains a full length human skeletal a-actin cDNA (Gunning et al. 1983; obtained from L. Kedes, University of Southern California, Los Angeles); pLE2, which carries the Ad5 EIA region (Jelsma et al. 1988); and BSM13-MGN#l l (a generous gift from Dr. W.E. Wright, University of Texas Southwestern Medical Center, Dallas), which contains the myogenin cDNA (Wright et al. 1989). For primer extension analysis of EIA transcripts, RNA, isolated as above, was further purified by another round of phenolchloroform treatment. Primer extension was performed essentially as described by Jones et al. (1985), except that annealing of the primer to RNA was performed at 60°C and the concentration of dNTPs added after the annealing step was increased from 0.33 pM to 0.33 mM. The synthetic 25 nucleotide primer AB485 (a generous gift from P.E. Branton, McGill University) hybridizes to the sequence from + 56 to + 81 relative to the ElA mRNA cap site. Zmmunoprecipitation of Ad5 EIA and E2A 72-kDa proteins

Cells were labelled with [35~]methionine(100 pCi/mL, ICN Biomedicals; 1 Ci = 37 GBq) in methionine-free medium at 22-25 h postinfection. The cells were lysed in 100 pL of buffer X (50 mM Tris-HCI (pH 8.5) - 250 mM NaCl - 1% NP-40 - 2 mg BSA/mL). A 15 000 x g supernatant was prepared and immunoprecipitated using either 5 pL of a mouse monoclonal antibody H2-19 directed against the Ad5 72-kDa DNA-binding protein (Rowe et al. 1984) or 5 pL of a mouse monoclonal antibody M73 directed against the Ad2 ElA proteins (Oncogene Science

BfOCHEM. CELL B l o t . VOL. 70, 1992

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days after plating

days after plating

FIG. 1. Comparison of BC3E7 and BC3H1 cells. Phase-contrast micrographs of (A) BC3E7 cells at 8 days and (B) BC H1 cells at 6 days after plating, and maintained in medium supplemented with 20% FCS. Analyses of (C) cell-surface AChR using '25~-labelled a-bungarotoxin (BTX) binding and (D) CK activity in BC3Hl and BC3E7 cells at intervals after plating. In C and D, the medium supplemented with 20% FCS was replaced with medium supplemented with 0.5% FCS at 3 days after plating for BC3H1 cells and at 4 days for BC3E7 cells.

Inc.), and 70 pL of 3% protein A - Sepharose (Pharmacia Ltd.) in buffer X. The resin was washed six times in 0.75 mL of the same buffer with centrifugation, and the immunoprecipitate was analyzed by SDS-polyacrylamide gel electrophoresis and fluorography.

Two-dimensional polyacrylamide gel electrophoresis Cells were labelled with [35~]methionine(50 pCi/mL, ICN Biomedicals) in methionine-free medium for 6 h at 37"C, collected in 200 pL lysis buffer (9.5 M urea - 2% (w/v) NP-40 - 2% ampholines - 5% /3-mercaptoethanol) (OIFarrell 1975), and analyzed by 2D PAGE essentially as described by O'Farre11(1975), using a pH range of 5-7 and an 11% separating gel. After electrophoresis, the gels were stained and fixed with 0.1% Coomassie blue in 50% methanol - 10% acetic acid, destained in 5% methanol - 7% acetic acid, dried, and exposed to Kodak XAR-5 film. The positions of contractile proteins in the gels were determined by using purified rabbit muscle actin, bovine muscle TM, and bovine muscle MLC (all from Sigma) as markers. The identities of the TM isoforms were determined by Western immunoblotting (Towbin et al. 1979) using a rabbit anti-mouse TM antibody (ICN Biomedicals). Assays for acetylcholine receptor and creatine kinase Creatine kinase was assayed as described by Simard and Connolly (1987). Quantitation of cell surface AChR was performed essentially as described by Olson et al. (1983).

Results Expression of muscle-specific genes in BC3E7 cells pElAneo3, a plasmid carrying genes for both Ad5 E1A and neomycin resistance, was transfected into BC3Hl cells. Upon confluency, a large proportion of these cells were round, flattened, and contact inhibited, in contrast to untransfected BC3H1 cells which under the same conditions become elongated and aligned to form streams (Schubert et al. 1974; Fig. 1B). From the transfected cells, we established 13 lines, one of which, BC3E7 (Fig. lA), we have examined in detail. This line has retained G418 resistance and the morphologically undifferentiated phenotype for over 50 passages. Its growth rate is slower than that of parental BC3H1 cells and the relative times for the analyses described below were chosen to compensate for this difference. BC3H1 cells were also transfected with pPBdx4, a plasmid containing only the neomycin resistance gene; all lines established from these transfected cells were morphologically indistinguishable from BC3Hl cells. One of these lines, BC3P7, was used as a control in some of our studies on BC3E7. A number of muscle-specific markers were studied to determine the extent of inhibition of myogenic differentiation in BC3E7 cells. BC3Hl and BC3E7 cells were plated at

MYMRYK ET AL. -- IEF

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FIG. 2. Autoradiographs of two-dimensional gels of proteins from [35~]methionine-labelled BC3H1and BC,E7 cells at different times after plating. A representative autoradiograph of proteins from BC3E7cells 11 days after plating is shown at the upper right. The positions of the molecular mass markers are indicated to the left. Regions A, B, and C of the gel are shown for proliferating cells (at 3 and 4 days after plating for BC3H1and BC3E7cells, respectively), and for serum starved cells (at 10 and 11 days after plating, respectively). Medium was replaced at 3 days after plating for BC,H1 cells and at 4 days for BC3E7 cells with medium supplemented with 0.5% FCS. (A) a, /3, and Y isoforms of actin; (B) TM isoforms numbered 1-5; (C) MLC 1 indicated by the arrowhead; an unidentified protein to the right, whose expression was not affected by ElA, was used as a marker in the autoradiographs.

10' cells/60-mm dish or 5 x lo4 cells/35-mm dish in medium containing 20% FCS, and at 3 days after plating for BC3Hl and at 4 days for BC3E7, while the cells were still proliferating, the medium was replaced with one containing 0.5% FCS. Cells can still undergo an additional round of division (Patrick et al. 1977) and appear confluent a day after serum starvation. Levels of cell-surface AChR and CK activities were determined in the two cell lines growing under these conditions using [125~]a-bungarotoxin binding and CK enzyme assays, respectively. Figures 1C and 1D show clearly that both AChR level and CK activity remained low in BC3E7 cells compared with the large increases associated with differentiation in BC3H1 cells. The synthesis of contractile proteins was monitored by labelling cells with [35~]methionineat various stages of culture and analyzing the labelled proteins by 2D PAGE (Fig. 2). In these gels, the positions of actin, TM, and MLC 1 were determined using purified standards. The migration pattern of these proteins was similar to that reported by

Garrels (1979). The identities of the TM isoforms were confirmed as described in Materials and methods. In BC3H1 cells, a decrease in the levels of nonmuscle p- and Y-actins and a concomitant increase in muscle-specific a-actin were observed during differentiation (Fig. 2A). In BC3E7 cells, a-actin was found in proliferating cells even before they reached confluency; in addition, 0- and Y-actins were not down-regulated under differentiation conditions. Five TM isoforms could be resolved: TM 1, TMs 2 and 3 making up P-tropomyosin, and TMs 4 and 5 making up a-tropomyosin (Fig. 2B). During differentiation of BC3H1 cells, the expression of TM 5 was unchanged, TM 1 was downregulated, and TMs 2, 3, and 4 were induced. Under differentiation conditions, these same changes occurred in BC3E7 cells, except that the down-regulation of TM 1 was partially inhibited (Fig. 2B). MLC 1 induction observed in BC3H1 cells was also found in BC3E7 cells upon confluency (Fig. 2C). The unexpected finding that muscle a-actin was synthesized in both proliferating and confluent

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FIG. 3. Actin mRNA levels in BC3Hl and BC3E7cells. RNA was extracted from (A) BC3H1 and (B) BC,E7 cells on the indicated days postplating and analyzed by Northern hybridization using actin cDNA as probe. The positions of the nonmuscle P- and Y-actin mRNAs, which run as a single band (2100 bp), and the muscle-specific a-actin mRNA (1500 bp) are indicated. BC3E7 cells led us to look at actin mRNA levels. Northern analysis of total RNA was carried out using as probe, pMHaA-1, which contains a full-length a-actin cDNA and therefore hybridizes to all three actin transcripts (Gunning et al. 1983). In BC3Hl cells, following the trigger to differentiate, the level of 0- and Y-actin mRNAs fell and this was followed by a large increase in the level of a-actin (Fig. 3A). In contrast, at all stages of growth in BC3E7 cells, the levels of all three actins mRNAs remained significant and unchanged (Fig. 3B). As not all aspects of myogenesis were blocked in BC3E7 cells, we examined the effect of EIA on myogenin expression. A plasmid, BSM13-MGN#11, containing myogenin cDNA (Wright et al. 1989) was used as a probe for Northern analysis. Untransfected BC3Hl cells expressed maximal levels of myogenin mRNA by 5 days after plating (Fig. 4E), as observed previously (Brunetti and Goldfine 1990). A similar but perhaps slightly reduced level of myogenin expression was seen in BC3E7 cells by day 5 after plating (Fig. 4A). In the above experiments cells were exposed to conditions of both serum starvation and cell-cell contact. Since cellcell contact alone is sufficient to induce differentiation of BC3H1 myoblasts (Schubert et al. 1974; Patrick et al. 1977), we examined the expression of several muscle-specific genes in cells which were maintained through confluency in 20% FCS. CK activity, AChR levels, and actin mRNA levels in BC3E7 cells treated in this way behaved similarly to those found for BC3E7 cells maintained in 0.5% FCS (data not shown), although AChR levels were more depressed. Surprisingly, using similar amounts of total cellular RNA as before (cf. Figs. 4B and 4D), we were unable to detect the presence of myogenin transcripts in BC3E7 cells grown in 20% FCS at up to 9 days after plating (Fig. 4C). In BC3Hl cells however, the pattern of myogenin expression was the same under both growth conditions (cf., Figs. 4E and 4F). E1A expression in BC3E7 cells In contrast to previous studies on ElA-transfected myoblasts, which indicated that all muscle-specific genes examined were repressed by ElA (Webster et al. 1988; Enkemann et al. 1990), the results just described showed that in BC3E7 cells, E1A blocks only some of these genes. In an attempt to understand this difference, we examined

1992

FIG. 4. Myogenin mRNA levels in BC3E7 and BC3H1 cells. RNA was isolated from cells on the indicated days postplating, and 10 pg of each sample was electrophoresed and (A,C,E,F) analyzed by Northern analysis or (B,D) stained with ethidium bromide. RNA from BC3E7cells was plated in medium with 20% FCS and (A,B) transferred 4 days later to medium with 0.5% FCS or (C, D) maintained in medium with 20% FCS. RNA from BC3H1cells was plated in medium with 20% FCS and (E) transferred 3 days later to medium with 0.5% FCS or (F) maintained in medium with 20% FCS. ElA expression in BC3E7 cells. We were unable to detect E l A mRNAs in BC3E7 cells by Northern analysis, as other authors have done with their transfected myoblasts (Webster et al. 1988; Enkemann et al. 1990). However, primer extension analysis of BC3E7 RNA indicated that the transfected E1A gene was transcriptionally active in the BC3E7 cell line (Fig. 5A, lanes 5 and 6), although the level of expression was low compared with that in 293 cells, an Ad5 DNAtransformed human embryonal kidney cell line (Graham et al. 1977) (lanes 2 and 3), and in BC3Hl cells infected with the phenotypically wt Ad5 dl309 (lane 4). No band was detected in uninfected BC3H1 cells (lane 7). Production of E1A proteins in BC3E7 cells was confirmed by electrophoretic analysis of labelled cell extracts immunoprecipitated with an E l A-specific monoclonal antibody (Fig. 5B, lane 4). Compared with an immunoprecipitate from 293 cells (lane l), the amount of E1A protein in BC3E7 cells was very much reduced and most of it was in the more slowly migrating form, indicating that the EIA gene product in these cells was predominantly in the phosphorylated form (Richter et al. 1988; Dumont et al. 1989). No E1A protein was detected in either BC3H1 or neomycin-resistant BC3P7 cells (lanes 2 and 3). Other proteins appeared to coprecipitate with E1A from the BC3E7 cells, as they were not present in the E l A precipitates from either BC3H1 or BC3P7 cells. More recent studies (Mymryk et al. 1992) suggest that these proteins are the mouse equivalents of the human cellular factors of 300, 107, and 105 kDa (p300, p107, and pRb) with which E1A is known to associate (cf., Fig. 5B, lanes 1 and 4) and through which E l A is thought to act (Yee and Branton 1985; Harlow et al. 1986). These results show that not only are these factors present in the BC3E7 cell line, but that the E1A protein is capable of specifically associating with them. One of the major functions of E l A is to activate other viral genes such as E2A during the early phase of adenovirus infection. To determine whether the E l A proteins in BC3E7 were biologically active, we tested the ability of BC3E7 cells to complement the ElA-deficient Ad5 mutant dl312 in activating transcription of the E2A gene. BC3Hl, BC3P7, and BC3E7 cells were infected with Ad5 dl312 and labelled with [35~]methionine,and cell extracts were

MYMRYK ET AL.

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FIG.5. Estimates of the level of EIA expression in BC3E7 cells. (A) Analysis of RNA by primer extension. The products of extending the primer AB485 hybridized to RNA from 293 cells (lanes 2 and 3), dl309-infected BC3H1cells (lane 4), BC3E7cells 4 days (lane 5) and 7 days (lane 6) after plating, and uninfected BC,H1 cells (lane 7). For lane 2, the reaction contained 10 pg RNA; all other reactions contained 20 pg. The arrowhead indicates the position of the 81-nucleotide product. The bulk of the labelled primer can be seen at the bottom of the autoradiograph. Size markers shown in lane 1 correspond to (from top) 109, 100, 90, 76, 71, 67. and 57 nucleotides. (B) Immunoprecipitation of E1A protein. Equal numbers of counts of [35~]methionine-labelled extracts from 293 cells (lane l), BC3H1 cells (lane 2), BC3P7 cells (lane 3), and BC3E7 cells (lane 4) were immunoprecipitated with an E1A-specific monoclonal antibody, and the precipitates were analyzed by gel electrophoresis and fluorography. Bands corresponding to ElA and to cellular proteins p105/p107 and p300 from human 293 cells are indicated to the right. The positions of molecular mass markers in kDa are shown on the left. (C) Complementation of the EIA-deficient mutant Ad5 dl312 to produce the E2A 72-kDa protein. BC3H1cells (lanes 1 and 2), BC3P7cells (lanes 3 and 4), and BC3E7 cells (lanes 5 and 6) were infected with either Ad5 dl312 (lanes 2, 4, and 6) or Ad5 dl309 (lanes 1, 3, and 5) and were labelled with [35S]methioninefor 2 h at 24 h postinfection. Cell extracts were immunoprecipitated with a monoclonal antibody against the adenovirus E2A 72-kDa protein and the immunoprecipitates were analyzed by gel electrophoresis and fluorography. The arrowhead indicates the position of the 72-kDa protein.

immunoprecipitated with a monoclonal antibody against the E2A-encoded 72-kDa protein and analyzed on denaturing gels (Fig. 5C, lanes 2, 4, and 6). The 72-kDa protein was detected only in extracts of infected BC3E7 cells (lane 6), indicating that the E1A product produced in BC3E7 cells was able to activate the viral E2A gene. The amount of 72-kDa protein produced was significantly less than that obtained in BC3Hl cells (lane I), BC3P7 cells (lane 3), or BC3E7 cells (lane 5) infected with d1309, indicating that constitutive expression of E I A was limiting. T o confirm that the differences we observed between BC3E7 cells and the previously reported EIA-transfected myoblasts were due to differences in level of E A expression and not to the different cell type used, we produced a high level of EIA expression in BC3H1 cells by infecting them with Ad5 dl520 on day 3 after plating. This virus was used as it does not synthesize the larger E l A protein; as a consequence, it replicates poorly, thereby allowing the cells to

FIG. 6. Expression of actin mRNAs in Ads-infected BC3Hl cells. BC3Hl cells were infected 3 days postplating with either dl312 or d1520. RNA was extracted at the indicated hours after infection and analyzed by Northern blotting. The filters were probed with a plasmid containing sequences for either (A) actin or (B) ElA. The positions of mRNAs for P- and Y-actin, a-actin, and EIA are indicated to the left.

survive longer than if wt virus had been used (Zerler et al. 1987; Moran and Zerler 1988; Howe et al. 1990). Northern analyses showed high levels of EIA transcription within 48 h of infection (Fig. 6B) and the infected cells had the characteristics reported for other EIA-transfected myoblasts (Webster et al. 1988; Enkemann et al. 1990), such as undifferentiated morphology upon confluency and lowered levels of CK activity and surface AChR (Mymryk et al. 1992). In addition, a-actin transcription was greatly repressed by 24 h and was completely blocked by 48 h after infection, while nonmuscle 0- and Y-actin mRNAs persisted up to 72 h after infection (Fig. 6A). That these changes were due specifically to E I A expression was shown by infecting BC3H1 cells with Ad5 dl312 which produces no E1A (Fig. 6B). This virus had no obvious effect on the changes in morphology, CK activity, AChR levels (Mymryk et al. 1992), or transcription of actin genes (Fig. 6A) that accompany differentiation. Discussion Our results show that the BC3E7 line of EIA-transfected BC3H1 cells expresses the E I A gene at a low level compared with the levels reported in the E1A-transfected myoblast lines L8, C2, and 23A2 (Webster et al. 1988; Enkemann et al. 1990). Although Webster et al. and Enkemann et al. were able to detect EIA mRNA in L8, C2, and 23A2 cells by Northern analysis, the amount of E I A RNA produced in BC3E7 cells was too low for us t o detect by this method. Only by using the more sensitive primer extension technique were we able to show the presence of E I A transcripts. Immunoprecipitation of cell extracts with a n E1A-specific monoclonal antibody, and complementation of the EIA-deficient Ad5 d1312, confirmed that a low level of E I A protein was produced in BC3E7 cells. In fact, none of our clonal lines of EIA-transfected BC3H1 cells produced high levels of EIA mRNA, suggesting that E I A may have detrimental effects on the viability of BC3H1 cells. The low level of E I A expression in BC3E7 cells was sufficient to prevent these cells from differentiating morphologically, and to block increases in CK activity and surface AChR and decreases in the synthesis of 0- and Y-actins and TM 1. On the other hand, unlike EIA-transfected L8, C2, and 23A2 myoblasts where E I A completely

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inhibited transcription of all muscle-specific genes examined (Webster et al. 1988; Enkemann et al. 1990), in BC3E7 cells a number of changes normally associated with differentiation still occurred, such as increased synthesis of TM 2, 3, and 4 isoforms and of MLC 1. Furthermore, the musclespecific a-actin gene, which is expressed in BC3H1cells and other myoblasts only when differentiated, was expressed in BC3E7 at all stages of culture, even during proliferation. We demonstrated that these differences were probably due to a lower level of EIA expression in BC3E7 and not to a difference in cell line by infecting BC3H1 cells with an adenovirus producing only the smaller E l A protein. In these infected cells, the smaller E l A protein was expressed at a significantly higher level, and the repression of morphological differentiation and the changes in gene expression associated with it were similar to those in the L8, C2, and 23A2 lines (Mymryk et al. 1992; Webster et al. 1988; Enkemann et al. 1990). In preliminary experiments (J.S. Mymryk, R. W.H. Lee, K. Shire, and S. T. Bayley, unpublished) using Western blots with a monoclonal mouse antibody specific for muscle actins (clone no. HUC1-1, ICN Biomedicals Inc., Costa Mesa, Calif.), we have found that, besides BC3E7, cells of other ElA-transformed BC3H1 lines that failed to differentiate morphologically also expressed a-actin during proliferation. These lines included one transformed by wt EIA, as well as a line transformed by an E1A mutant dl1 151 that produces a truncated product consisting essentially of only exon 1 of the smaller E1A protein (Mymryk et al. 1992). With the same technique, we have also found that a-actin production was reduced in BC3E7 cells when the level of E l A proteins was raised by infecting at 10 PFU/cell with either Ad5 dl520 or Ad5 d1309, a virus producing both E1A proteins. These results suggest that the disruption of coordinate expression of muscle-specific genes we observed in BC3E7 cells is not peculiar to that line and does not depend on the transactivation function provided by the unique region of the 289 residue E1A protein. Furthermore, expression of muscle-specific genes appears to be completely repressed by high levels of either the smaller E l A protein alone or both E1A proteins. Both Webster et al. (1988) and Enkemann et al. (1990) have shown by transient expression assays that E1A is capable of repressing transcription from the promoters of the muscle-specific genes for a-actin, troponin I, and CK. The failure of the low level of E1A protein produced in BC3E7 cells to repress genes for MLC 1 and several TM isoforms could be explained if these genes required more E1A protein for repression than others. This would be consistent with past observations that transcriptional transactivation and efficiency of transformation can be affected by varying E1A concentrations (Brunet and Berk 1988; Adami and Babiss 1990). However, this would not account for the surprising observation that BC3E7 cells express a-actin constitutively. Presumably this expression is also brought about in some way by the low level of E l A protein, and the explanation may lie in the ability of E1A proteins to transactivate as well as repress transcription. Our preliminary observation, mentioned above, that transformation with E l A mutant dl1 151 also leads to constitutive expression of a-actin, suggests that transactivation by the 289 residue protein may not be involved. However, it is clear

that the smaller, 243 residue E1A protein is capable of activating expression of some genes (Bagchi et al. 1990; J.S. Mymryk and S.T. Bayley, in preparation), and it is possible that the balance between activation and repression for a particular gene depends on a variety of factors, including the concentration of the smaller E1A protein. Thus a high concentration may account for the repression of a wide range of muscle-specific genes observed in L8, C2, and 23A2 lines and in Ad5-infected BC3H1 cells, whereas the low concentration in BC3E7 cells may repress some genes but activate others. Our results showed that only when differentiation in BC3E7 cells was induced by cell-cell contact in the presence of 20% FCS, was E1A able to repress myogenin expression; in 0.5% FCS, myogenin expression was only slightly less than in BC3H1 cells. This difference is most likely due to the presence of E1A because myogenin expression in untransfected BC3H1 cells is identical under both experimental conditions. While it is possible that high serum levels may alter the ability of E1A to repress transcription of certain genes, this seems unlikely as we observed no major effects of changing serum levels on the expression of other muscle-specific markers. In addition, we are not aware of any studies describing a serum sensitivity for any E l A function. Our results may indicate that there are two signalling pathways leading to the activation of the myogenin gene, one triggered by cell-cell contact and the other by serum starvation, and that the one triggered by cell-cell contact is more sensitive to down-regulation by E1A than the other. This would explain the absence of myogenin expression in BC3E7 cells under conditions which provide only the cellcell contact signal, and the slightly reduced level of myogenin expression in BC3E7 as compared with BC3H1 cells under more stringent conditions which provide both signals. The BC3E7 cell line may provide a model in which to study these two pathways further, as well as how the interplay between cellular proliferation and differentiation affects myogenin expression. It has been suggested that E l A blocks myogenic differentiation by repressing the expression of the myoD group of genes which are the putative master regulators of musclespecific gene expression (Enkemann et al. 1990). Our results show that E1A does not block myogenic differentiation by repressing myogenin expression, as serum starved BC3E7 cells express myogenin, yet remain morphologically undifferentiated and fail to express appreciable levels of AChR and CK activity. On the contrary, E l A appears to act directly and independently on each muscle gene to repress expression, overriding the effects of myogenin. The ability of E1A to repress a variety of muscle-specific promoters in transient expression assays (Webster et al. 1988; Enkemann et al. 1990) also provides further evidence for this argument. Because the relative degree of sensitivity of each individual gene to repression by E l A is likely to differ, this would explain the apparent disruption of the coordinate regulation of muscle-specific gene expression observed in BC3E7 cells, as the limiting level of E1A produced in these cells would not affect all available cellular targets of ElA. As E1A proteins appear to exert their effects by binding to cellular proteins, the next step is to identify the factors in myoblasts with which these oncogenic proteins interact. Using EIA deletion mutants, we have shown in a separate

MYMRYK ET AL.

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study (Mymryk et al. 1992) that repression of differentiation in BC3HI myoblasts by EIA correlates with the ability of E1A to bind to p300, but not to p107 or plO5. Acknowledgements We thank Natalie Veitch for cell culture, cloning, and expansion of cell lines and other technical assistance; Erika Hansel1 for the CK assays; Carole Evelegh for the initial transfections; and Barbara Panning for help with the primer extension analysis. This work was supported by grants from the National Cancer Institute of Canada, the Medical Research Council of Canada, and the Natural Sciences and Engineering Research Council of Canada. R.W.H.L. was a University Research Fellow of the Natural Sciences and Engineering Research Council of Canada. S.K.M. is the recipient of the Canadian International Development Agency/McMaster University Scholarship. Adami, G.R., and Babiss, L.E. 1990. The efficiency of adenovirus transformation of rodent cells is inversely related to the rate of viral E1A gene expression. J. Virol. 64: 3427-3436. Bagchi, S., Raychaudhuri, P., and Nevins, J.R. 1990. Adenovirus ElA proteins can dissociate heteromeric complexes involving the E2F transcription factor: a novel mechanism for E1A transactivation. Cell, 62: 659-669. Berk, A.J., Lee, F., Harrison, T., Williams, J., and Sharp, P.A. 1979. Pre-early adenovirus 5 gene product regulates synthesis of early viral messenger RNAs. Cell, 17: 935-944. Borrelli, E., Hen, R., and Chambon, P. 1984. Adenovirus-2 E1A products repress enhancer-induced stimulation of transcription. Nature (London), 312: 608-612. Brunet, L.J., and Berk, A.J. 1988. Concentration dependence of transcriptional transactivation in inducible E1A-containing human cells. Mol. Cell. Biol. 8: 4799-4807. Brunetti, A., and Goldfine, I.D. 1990. Role of myogenin in myoblast differentiation and its regulation by fibroblast growth factor. J. Biol. Chem. 265: 5960-5963. Chen, C., and Okayama, H. 1989. High-efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell. Biol. 7: 2745-2752. Chomczynski, P., and Sacchi, N. 1987. Single-stepmethod of RNA isolation by acid guanidinium thiocyanate - phenol - chloroform extraction. Anal. Biochem. 162: 156-159. Davis, R.L., Weintraub, H., and Lassar, A.B. 1987. Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell, 51: 987-1000. Dumont, D.J., Tremblay, M.L., and Branton, P.E. 1989. Phosphorylation at serine 89 induces a shift in gel mobility but has little effect on the function of adenovirus type 5 E1A proteins. J. Vjrol. 63: 987-991. Edmondson, D.G., and Olson, E.N. 1989. A gene with homology to the myc similarity region of MyoDl is expressed during myogenesis and is sufficient to activate the muscle differentiation program. Genes Dev. 3: 628-640. Enkemann, S.A., Konieczny, S.F., and Taparowsky, E. J. 1990. Adenovirus 5 E1A represses muscle-specific enhancers and inhibits expression of the myogenic regulatory factor genes, MyoDl and Myogenin. Cell Growth Differ. 1: 375-382. Florini, J.R., and Ewton, D.Z. 1990. Highly specific inhibition of IGF-I stimulated differentiation by an antisense oligodeoxyribonucleotide to myogenin mRNA. J. Biol. Chem. 265: 13 435 - 13 437. Carrels, J.I. 1979. Changes in protein synthesis during myogenesis in a clonal cell line. Dev. Biol. 73: 134-152. Graham, F.L., Smiley, J., Russell, W.C., and Nairn, R. 1977.

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