EXPERIMENTAL

CELL

RESEARCFi

z@&

105-112

(19%)

tor Responsiveness: Rol JOSEPH Molecular

and Cell Biology,

FL WOLF, T. El. Morgan

R. HIRSCHHORN,’

RICKY School

of Biological

Sciences,

A differentiation-defective variant (DD-1) of the MM14 myoblasts acquired the ability to synthesize DNA in response to treatment with epidermal growth W. Lim and S. D. Hauschka, 1984, Dev. Biol. 195, 48) and no longer expressed myogenic determinant genes (i.e., MyoD and myogenin) (P. R. Mueller3 and B. Weld, 19SgT Science 246, 780). To determine the effect of expression of MyoD on EGF responsiveness, LID-I. cells were cotransfected with a MyoD expression vector and with pRSVneo. A clone, MyoDDcells, which was G418 resistant, formed multinuclear syncitia, and also expressed MyoD and myogenin, was further characterized. EGF responsiveness, as assessed by DNA synthesis, was decreased 5- to lofold in the MyoDD-1 cells from that in G4lSresistant control DD-1 cells, despite similar EGF receptor numbers and binding affinities of the receptors. Responsiveness of MyoDDcells to fibroblast growth factor (FGF) was also diminished although to a lesser extent. To determine the effects of deer-eased myogenic determinant gene expression on mitogen responsiveness, MM14 myoblasts were grown in medium supplemented with 5 piIf5-bromo-2-deoxyuridine (BUdR-MM14). BUdRMM14 cells had decreased expression of MyoD and myogenin, did not fuse, and haa an altered morphology, from round to flat. The eflect on fusion and cell sbape was reversed by growtb in control medium. BUdR-MM14 cells were responsive to EGF and had enhanced responsiveness to FGF. The combined studies support the view that expression of MyoD and/or myogenin contributes to negative regulation of mitogen responsiveness@ 1992 Academic Press, Inc.

mJaR

IN’TRODUC-ITON

Terminal differentiation of many cell types is accompanied by a loss of the ability of the cells to replicate. In addition, earlier stages in the differentiation processes also may involve a decrease in proliferative capacity-

’ Current address: Biology MD 217’01. ‘To whom correspondence dressed.

Department, and

reprint

Hood requests

College, should

Fredrick, be ad-

an

AND SHELDON Uniuersity

of Kentucky,

ivL STEIPW~ Lexington,

Kentucky

4Q506-02256

For example, in a model system of adipocyte differentiation prelineage cells proliferate extensively in response to a. range of mitogens while cells committed to the adipocyte lineage, although not terminally differentiated, exhibit a marked reduction in proliferation in response to defmed mitogens or 5% fetal bovine serum [l]. An intimate association between lineage commitment and regulation of proliferative capacity is suggested by studies of the effects of the rrmscle determinant gene MyoD on cell proliferation [& 3]* Microinjection of MyoD into quiescent 3T3 fibroblasts results in inhibition of serum-stimulated transition from the GOto the S phase of the cell cycle [2]. Expression of myogenic determinant genes in myoblasts also appears to be associated with a reduced proliferation potential; e.g*?MMl4 myoblasts express the myogenic determinant genes, 0D and myogenin, and have restricted mitogen responsiveness [4, 51 as compared to that of DD-1 cell variants that do not express these genes. The MM14 myoblasts do not significantly proliferate in response to treatment with epidermal growth factor (EGF) and will proliferate in response to fibroblast growth factor GF) only when FGF is used in concert with serum. M 4 myoblasts have receptors for botb growth factors; hence> nonresponsiveness is not simply the result of a lack of appropriate receptors. In addition, there is a nonproliferative response to FGI?. In that regard, FGF inhibits the differentiation event of fusion of MM14 myoblasts to form myotubes [6]. An inverse correlation between expression of myogenic determinant genes and proliferation otential of cells is further supported by findings fr udies of the differentiation-defectiv 14 myoblasts, the DD-1 cells. The D their altered morphology, flat as compared to the rounded morphology of the M were found to be uriable to fuse defective? did not express the myogenic genes, MyoD or rnyogenin, and did replicate in response to treatment with either or FGF [49 ‘7]. Thus, studies with the -1 cells and MM14 myoblasts provide an opportunity for examination of the effect of manipulation of myogenic d~~e~rni~a~t gene expression on growth factor respons terminant gene related cells. In this study? myogenic

106

WOLF,

HIRSCHHORN,

expression has been modulated by stable transfection of the DD-1 cells with MyoD and by treatment of MM14 myoblasts with 5bromodeoxyuridine (BUdR), a treatment previously reported to inhibit the expression of the myoblast phenotype 15, 81. The cells have been characterized with respect to a functional attribute of skeletal muscle lineage, fusion of cells, and with respect to the relationship between expression of myogenic determinant genes and growth factor responsiveness. MATERIALS

AND

METHODS

Cell culture. Seed cultures of the mouse myoblasts (MMl4) and DD-1 cells were kindly supplied by Dr. S. Hauschka (Seattle, WA). The growth medium (GM) and low serum medium used for differentiation (DM) were as described previously [9]. Briefly, GM was Ham’s F-10 with the calcium concentration adjusted to 1.1 mMwith calcium chloride, supplemented with 3% chick embryo extract (v/v), 15% horse serum (v/v), and antibiotics (10,000 U/ml, penicillin G; 0.5 pg/ ml, streptomycin sulfate). MM14 myoblasts continue to proliferate if they are fed with fresh growth medium every 12 h. Commitment of MM14 myoblasts to differentiate and change of the other cells from growing to quiescent were accomplished by shifting growing cultures from GM to DM (Ham’s F-10 containing 1.1 rnM calcium chloride with 2% horse serum). Conditioned DM was prepared by culture of DD-1 cells in DM for 48 h. DNA synthesis was estimated either by autoradiography of [3H]thymidine-labeled cells [lOI or by measurement of [3H]thymidine incorporation into an acid-insoluble fraction Ill]. For both detection methods, cells were grown in GM to a relatively sparse density (3 to 4 X lO* cells per 35-mm tissue culture dish), washed twice with physiological saline, and shifted to DM for 24 or 48 h. Cells were then treated with EGF or FGF (10 and 20 rig/ml, respectively) (Collaborative Research, Bedford, MA) for 24 h in the presence of [3H]thymidine (20 Ci/mmol; New England Nuclear, Boston, MA) and analyzed by one of the methods above. 7Yansjection. The expression vector pEMSVscribe and its MyoDcontaining derivative pEMClls [12a] were most graciously supplied by Dr. Lassar (Seattle, WA). DD-1 cells were seeded at 2.5 X 10’ cells per lo-cm tissue culture dish in Dulbecco’s modified Eagle’s medium (DMEM) containing 15% fetal bovine serum (FBS). Transfection was carried out essentially as described by Wigler et ul. [13]. Ten micrograms of the pEMClls plasmid plus 1 pg pRSVNeo [14] plasmid plus 10 pg DD-1 genomic DNA were coprecipitated and added to each culture dish followed by the addition of 10 PM chloroquine (Sigma, St. Louis, MO). The culture dishes were incubated at 37’C in 5% CO2 for 6 h and then fresh DMEM with 15% FBS was exchanged for the transfection medium. The medium was changed with like medium every 12 h for 48 h and then DMEM with 15% FBS supplemented with 400 &g/ml Geneticin (G418) (GIBCO, Gaithersburg, MD) was added. After approximately 2 weeks, G418-resistant clones were selected and subcloned and frozen stocks were made for subsequent studies. Utilizing a strategy similar to that described above some DD-1 cells were transfected solely with pRSVneo. A G418-resistant clone was isolated and the cells were designated NeoDD-1 cells. Total cellular RNA was isoRNA isolation and blot hybridization. lated by the guanidinium thiocyanate-phenol-chloroform method described by Chomczynski and Sacchi [I5]. In some experiments, poly(A)+ RNA was isolated using the Micro-Fast track kit (Invitrogen, San Diego, CA). RNA was size-fractionated by 1% agarose/2.2 &f formaldehyde gel electrophoresis and then transferred overnight to reinforced nitrocellulose membranes (Micron Separations, Inc., Westborough, MA) in 10X SSC buffer essentially as described [16]. The gels were loaded with poly(A)* RNA (approximately 2 Kg/lane) or with total RNA (approximately 15 pg/lane), based upon spectro-

AND

STEINER

photometric estimation, to which 2 ~1 of ethidium bromide (40 ng/Fl) was added. Gels were illuminated with ultraviolet light to estimate sample equivalence. cDNA probes were prepared by nick translation (kit from GIBCO; Gaithersburg, MD) in the presence of [a-“P]dATP (3000 Ci/mmol; ICN Biomedicals, Irvine, CA). The following cDNA probes were utilized MyoD insert was obtained as a 1.8-kb fragment by EcoRI digestion of the pEMClls plasmid [6], myogenin was obtained as a 1.5.kb fragment by EcoRI digestion of pEMSV-MGN plasmid (generously supplied by Dr. W. Wright, Dallas, TX) [17], and pRSVNeo plasmid was as described by Gorman et aZ. [14]. Western immunoblots. A polyclonal antibody against MyoD (generously supplied by Dr. H. Weintraub, Seattle, WA), at a dilution of l/500, was utilized to examine MyoD protein levels by Western blot analysis. SDS-polyacrylamide gel electrophoresis (7.5% gel), transfer of proteins to a polyvinylidene difluoride membrane (Immobilon, Millipore Corp.), and detection were performed as previously described [16]. The procedure utilized for “‘I-labeled 1251-labeled EGF binding. EGF equilibrium binding was essentially that of Lim et al. [4] except that the binding was routinely performed at 1O’C rather than 37’C to minimize uptake of the labeled EGF. Growing cells were seeded at approximately 5 X 105 cells per 35mm tissue culture dish and allowed to attach in GM for 3 to 4 h. The cells were washed twice with F-10 medium plus 0.5% horse serum and incubated with F-10 medium plus 0.1% bovine serum albumin plus iz51-labeled EGF (106 &i/pg; ICN Biomedicals) at 1O’C for 3 h. The medium was then removed and the cells were washed eight times with cold phosphate-buffered saline containing 0.1% BSA. Bound radioactive material was solubilized by incubation of cells in 1 ml 0.5 N NaOH at 37°C for 1 h, and ‘? was estimated by gamma counting (Beckman Model 5500).

RESULTS

DD-1 cells were cotransfected with the MyoD expression plasmid pEMClls and pRSVNeo plasmid (utilized as a G418-selectable marker) or with pRSVNeo alone and a number of G418-resistant clones were obtained. One of the MyoD-transfected G418-resistant clones, termed MyoDDcells, fused into syncitia and was further characterized. A second G418-resistant clone, termed NeoDD-1 cells, was isolated from DD-1 cells transfected with pRSVNeo alone. When growing at low cell population density, MyoDDcells (Fig. 1A) have a round morphology that is indistinguishable from that of MM14 myoblasts (Fig. 1B) and quite different from the flattened morphology of the DD-1 cells (Fig. 1C). Moreover, MyoDDcells were capable of forming multinucleated syncitia (Fig. 1D). However, the MyoDDcells differed from MM14 myoblasts with respect to culture conditions which promote cell fusion. MM14 myoblasts fuse either following shift to DM or when cultured to high density in GM. Fusion of MyoDDcells was primarily observed when cells were grown to high density in GM although there was a low level of fusion following shift to DM. The characteristic of fusing at high cell population density in GM but not following shift to DM was observed with six additional G418 clones; however, these clones lost the ability to fuse upon subcloning (data not shown). The ability to fuse was a stable characteristic in the MyoDD-

FIGI. Effect of MyoD growing low-population-density and Methods.

transfection iVIM14

on morphological phenotype of DD-1 cells. (A) Growing ~ow-popu~~t~o~-de~s~~~ MyoDD-1; @3) myoblasts; (C) growing DD-1 cells; (II) fusing MyoDDcells. For conditions of’ growth see Materiak

1 cells and therefore these cells were utilized in subsequent studies. The morphological and fusion characteristics of the ~~oD~~~ cells in GM suggest that these cells are expressing a myoge~i~ determinant gene(s), while the &atus of expression of such genes in the serum-deprived yoDD-3. cdls is unclear. Therefore, the level of expression of &IyoL) and myogenin, the two myogenic determinant genes expressed in MM14 myoblasts, was measured by Northern blot analyses of RNA samples from growing versus serum-deprived cells. Moreover, since the mRNA from the transfected i%lyoB typically has a decreased e~e~trophoreti~ mobility as compared to

that of iWyoI3 m NA transcribed from the endogenous gene, the relativ expression of the transfected and en dogenous Lyons could be determines [Ek]O There was si~ni~~ant expression of ~~0~ in ing ~yo~~“~ cells (Fig. ZA, lane 7). In view of th ecreased electrophoretic mobility of the Lyon RNA from growing MyoDDcells as compared to that of ~~o~i~g M~~4 myoblasts (Fig. ZAY lane 7 versus lane I)> the My cells appear to express only the transfected There was a gro~h-de~e~d~~t alteratiQ~ in &.Fy& expression in MyoDDcells; i-e., these cells did not express significant levels of &Q when cultured in D medium (Fig- ZA lane 8). In r&cast* there was su

108

WOLF,

HIRSCHHORN,

AND

STEINER

12345676 FIG. 2. (A) Expression of MyoD mRNA in exponentially growing and serum-deprived MM14 myoblasts, DD-1 cells, MyoDDcells, and NeoDD-1 cells. Lane 1, total RNA from growing MM14 myoblasts; lane 2, total RNA from serum-deprived MM14 myoblasts; lanes 3 and 4, blank; lane 5, total RNA from growing DD-1 cells; lane 6, total RNA from serum-deprived DD-1 cells; lane 7, total RNA from growing MyoDDcells; lane 8, total RNA from serum-deprived MyoDDcells; lane 9, total RNA from growing NeoDD-1 cells; lane 10, total RNA from serum-deprived NeoDD-1 cells. The blot was probed with nick-translated MyoD cDNA. (B) Expression of myogenin mRNA in exponentially growing and serum-deprived MM14 myoblasts, DD-1 cells, MyoDDcells, and NeoDD-1 cells. Lane 1, total RNA from serum-deprived NeoDD-1 cells; lane 2, total RNA from growing NeoDD-1 cells; lane 3, total RNA from serum-deprived MyoDDcells; lane 4, total RNA from growing MyoDDcells; lane 5, total RNA from serum-deprived DD-1 cells; lane 6, total RNA from growing DD-1 cells; lane 7, total RNA from serum-deprived MM14 myoblasts; lane 8, total RNA from growing MM14 myoblasts. For experimental details see Materials and Methods.

stantive expression of this gene in the MM14 myoblasts cultured in DM as well as GM (Fig. 2A, lanes 2 and 1, respectively). Since MyoD can activate myogenin in other cell systems [12b] and the MM14 myoblasts express both MyoD and myogenin, the expression of myogenin in the MyoDDcells was determined. Myogenin was expressed in both growing and quiescent MyoDDcells (Fig. 2B, lanes 4 and 3, respectively). Hence, expression of all myogenic determinant genes was not inhibited by culture of MyoDDcells in DM. Controls exhibited the expected patterns of expression, namely, myogenin expression in both growing and DM-cultured MM14 myoblasts and absence of myogenic determinant gene expression in DD-1 cells and NeoDD-1 cells (Fig. 2B). MyoD mRNA expression in exponentially growing MyoDDcells was accompanied by synthesis of readily detectable levels of MyoD protein (Fig. 3, lane 6). A cross-reacting, slightly electrophoretically less mobile protein of approximately 50 kDa was observed in extracts from all cells. There was little MyoD protein de-

123456789 FIG. 3. Western blot of MyoDDprotein in exponentially growing and serum-deprived MM14 myoblasts, DD-1 cells, MyoDDcells, and NeoDD-1 cells. Lane 1, molecular weight marker of approximately 50 kDa; lane 2, total cellular protein growing MM14 myoblasts; lane 3, total cellular protein from serum-deprived MM14 myoblasts; lane 4, total cellular protein from growing DD-1 cells; lane 5, total cellular protein from serum-deprived DD-1 cells; lane 6, total cellular protein from growing MyoDDcells; lane 7, total cellular protein from serum-deprived MyoDDcells; lane 8, total cellular protein from growing NeoDD-1 cells; lane 9, total cellular protein from serum-deprived NeoDD-1 cells. A cross-reacting, slightly electrophoretically less mobile protein of approximately 50 kDa can be observed in extracts from all of the cells. For conditions of growth, serum deprivation, and Western blotting see Materials and Methods.

monstrable in MyoDDcells shifted to DM medium (Fig. 3, lane 7). In contrast, MyoD protein was detectable in growing and serum-deprived MM14 myoblasts (Fig. 3, lanes 2 and 3). The phenotype of the MyoDDcells with respect to responsiveness to EGF and FGF was determined since there is a distinctive difference between DD-1 cells and MM14 myoblasts with respect to responsiveness to these growth factors. The MyoDDcells were 10 to 20% as responsive to EGF as the NeoDD-1 cells, as determined by autoradiography of [3H]thymidine-labeled nuclei (Fig. 4A) or by incorporation of [3H]thymidine into an acid-precipitable fraction (Fig. 4B). There was also a difference in responsiveness of MyoDDcells and NeoDD-1 cells to FGF; however, the reduction in responsiveness of MyoDDcells to FGF was significantly less than the reduction in responsiveness to EGF (Fig. 4). The decrease in responsiveness of MyoDDcells to EGF and FGF does not represent a global reduction in mitogen responsiveness since stimulation of DNA synthesis by complete medium, GM, is comparable in MyoDDcells and NeoDD-1 cells (Fig. 4). One possible mechanism for the decreased responsiveness of the MyoDDcells to EGF is via alterations in the EGF receptor, either a decrease in receptor number or a change in binding capacity. Analysis of EGF binding revealed little difference between MyoDDcells and NeoDD-1 cells (Fig. 5). Based on the EGF binding saturation levels, the Kd was calculated to be about l-l.5 X 10PgM for both cell types and the receptor number was calculated at approximately 4000 for the MyoDDcells and approximately 5000 for the NeoDD1 cells. Thus, the decreased ability of the MyoDDcells to respond to EGF does not appear to reflect a substantive change in the number or binding affinities of the EGF receptors. These data suggest that expression of myogenic determinant genes results in a decreased responsiveness of cells to EGF and FGF. To further explore this relationship, the possibility of decreasing expression of these

&fyoD

AND

GROWTH

FACTOR

10

RE§PONSIVENESS

0 EGF

FGF

GM

EGF

MyoDD-

FGF NeoDD-

EGF

GM

FGF MyoDD-

1

GM 1

EGF

CGF NedID-

GM 1

FIG. 4. Effect of EGF, FGF, and GM on DNA synthesis of serum-deprived MyoDDcells and NeoDD-1 cells. (A) DNA synthesis as assessed by autoradiography of H-labeled nuclei. The above is representative of a single experiment done in triplicate. Error bars represent the standard deviation of triplicate plates. The nuclei of at least 10 fields of cells were counted, representing approximately 500 cells per plater (B) DNA synthesis as assessed by [‘Hjthymidine incorporation into an acid-precipitable fraction. The above is a single experiment done in triplicate that is representative of three separate experiments performed in triplicate. The error bars represent the standard deviation of the triplicate plates of a single experiment. For experimental details see Materials and Methods.

genes by treatment of MM14 myoblasts with BUdR and then analyzing mitogen responsiveness was examined. We determined that MM14 myoblasts, in medium supplemented with BUdR (BUdRMMl4) lost, in a dosedependent marmer, the ability to fuse (Fig. 6). When BUdR-MM14 myoblasts were shifted to GM without BUdR and then to DM, they reacquired the ability to fuse (data not &own). Northern blot analyses were performed to determine the extent of MyoD and myogenin expression in the BUdR-MM14 myoblasts. There was a marked decrease in the expression of MyoD (Fig. 7A) while myogenin was not detectable (Fig. 7B). The ability of BUdR-MM14 myoblasts to respond to EGF and FGF was determined. BUdR-MM14 cells were capable of initiating DNA synthesis in response to EGF treatment,

12C

0

1

2

3 nM free

4

5

6

?

EGF

FIG. 5. Epidermal growth factor binding capacity in MyoDDas compared to NeoDD-1 cells. Growing cultures were incubated with varying concentration of *“I-labeled EGF at 1O’C for 3 h to achieve binding equilibrium. For additional experimental details see Materials and Methods.

whereas there was no demonstrable EGF-mediated DNA synthesis in control MM14 rny~~~~st~ (Table l)Moreover, there was a 26-fold increase in responsiveness of BUdR-MM14 cells to FGF as corn of control MM14 myoblasts. Thus, there is an inverse relationship between mitogen responsiveness and expression of rnyo~e~i~ determinant genes when this gene expression is modulated by either transfection with h!yoL) or by treatment with BUdR.

Introduction of a MyoD expression vector into DD-1 cells (MyoDDcells) results in e ression of both MyoD and myogenin and in a change in the jmorphological phenotype from flat, stel shape to one that is round and more like that of 14 myoblasts. Moreover, the MyoDDcells have the a multinucleated syncitia. The ability myogerzin and to convert nonmuscl muscle cells has been de 12b]; bowever, an unan current study is that when DM, a marked decrease in exp%?ss10n occurs~ This decreased expression of &fy marked decrease in the ability of We interpret the decreased fusio in DM to indicate that expression o ed under low serum conditions virus promoter and, since the 1 cells do rmt @ nous lklyol3, fusion i creased* A r~k fo blast fusion has also been suggested BC3Hl cells [I$]. Additional G4X%-resistan isolated following t~a~sf~~t~~~ with

110

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HIRSCHHORN,

AND

STEINER

FIG. 6. Effect of 5-bromodeoxyuridine on morphological phenotype of MM14 myoblasts. (A) MM14 myoblasts in DM; (B) MM14 myoblasts after four passages in DM supplemented with 0.5 &’ BUdI% (C) same as in B, only with 1.0 ,LL&~BUdI$ (D) same as in B, only with 5.0 PM BUdR. For experimental details see Materials and Methods.

pRSVNeo. Like the MyoDDcells, cells from tbese clones also formed syncitia at high cell population densities and exhibited only very limited fusion when shifted to DM. Unlike the MyoDDcells, the fusion phenotype was not stable upon subculture of these other clones (data not shown). Introduction of MyoD into DDW1 cells results in marked inhibition in the ability of EGF to stimulate DNA synthesis and some inhibition in the ability of FGF to stimulate DNA synthesis. Conversely, inhibition of myogenic determinant gene expression by BUdR treatment of MM14 myoblasts results in marked increases in the mitogenic responsive-

ness of the cells to EGF and to FGF. The data suggest that &fyoD may not directly affect EGF and FGF responsiveness since the MyoDDcells have reduced responsiveness to these mitogens under culture conditions in which there is little Lyon expression. &fyoD expression is not demonstrably increased for at least 6 h following EGF stimulation of quiescent MyoDDcells (Wolf and Steiner, unpublished observations). Moreover, BUdRMM14 cells respond to EGF and FGF under conditions in which MyoD expression is ~minished but detectable. Since myogenin is expressed at relatively abundant levels in MyoDDcells under conditions in which respon-

&fyoD

AND

GROWTH

I FIG. 7. (A) Effect poly(A)+ RNA MM14 with 5 PM BUdR (data myoblasts in DM; lane lane 4, poly(A)+ RNA

FACTOR

of BUdR on MyoD mRNA expression of MM14 myoblasts. Lane 1, poly(A)+ RNA MM14 myoblasts in GM; lane 2 myoblasts in GM supplemented with 5 PM BUdR. DD-1 cells did not have detectable &ryoD in GM or G not shown). (B) Effect of BUdR on myogenm mRNA expression of MM14 myoblasts. lane 1, poly(A)+ RNA from MM14 2, poly(A)+ RNA from MM14 myoblasts in DM supplemented with 5 &f BUdR$ lane 3, poly(A)+ RNA from DD-1 cells; from DD-1 cells in DM supplemented with 5 PM BUdR. For experimental details see Materials and Methods”

TABLE

Ceils

of BUdR Treatment and FGF Stimulation Additions

111

2

siveness to EGl? and FGF is diminished and since exgene is not depression of this myogenic determinant tectable in BUdI&MM14 cells which do respond to the mitogens, it is possible that myogenin is more directly involved in decreased mitogen responsiveness. To test this hypothesis studies are in progress to determine if transfection of DD-1 cells with myogenin results in inhibition of EGF responsiveness. It is unlikely that endogenous MyoD will be expressed in myogenin-transfected DD-1 cells, since autoregulation of expression of the former gene does not occur in MyoDDcells, which have a relatively abundant level of expression of myogenin. There are a number of potential mechanisms for decreased growth factor responsiveness due to MyoD and1 or myogenin expression. Loss of surface growth factor receptors does not appear to be the basis for the decreased growth factor responsiveness since EGF receptor number and affinity are similar in MyoDDcells and in control cells. This finding directly parallels the findings with MM14 myoblasts, which are not responsive to EGF despite possessing EGF receptors, whose

Ef%ct

RESPONSIVENESS

1

of MM14 of DNA

[3H]Thymidinea

Myoblasts Synthesis cpm into

-BUdR +BUdR

+EGF +EGF

0 801 Z!I 4

-IXJciR +BUdR

+FGF fFGF

66 k 21 1693 31 22

on EGF

DNA/cell

X 103

IVote. MM14 myoblasts were grown for four passages in growth medium supplemented with 5 &!f BUdR and then shifted to DM as indicated, EGF (20 rig/ml) or FGF (25 rig/ml), and labeled thymidine. The above represents a single experiment performed in triplicate and is representative of three independent experiments done in triplicate. 2 represents the standard deviation of the counts of triplicate experiments. a Radioactivity incorporated into untreated (i.e., BUdR), unstimuiated (EGF or FGF), and treated, unstimulated cells was subtracted from all values.

number and binding affinity are comparable to those of DD-1 cells [4]. Possible postreceptor mechanisms include inhibition of the EGF signal transduction patbway or alteration of an additional signaling system which is needed for stimulation of replic&ion. Postreceptor alteration in signal transduction is an interpretation that is consistent with findings of Clegg and Hauschka [191 utilizing heterokaryons between MM14 myocytes and DD-1 cells to indicate that a diffusible factor was produced by myocytes that inhibited mitogen responsiveness of the DD-1 cells Cell-extracellular matrix interactions might be involved as an additional signaling system? since extracellular matrix has been demonstrated to have an important roIe [20] in the regulation of myoblast proliferation. n this regard, a consequence of AJyoD expression is an apparent alteration in celi-substratum atta y the change in morphology of the fiat to round. The results of these studies would support the hypothesis that expression of MyoD and/or myogenin contributes to negative regulation of mitogen responsiveness and might at least partially explain the basis for inhibition of proliferation by these genes We thank Drs. Marion Steiner and Lester Goldstein for their critical reading of the manuscript and Dr. Mike Clark for his heipful discussions. We thank Dr. Weintraub for providmg antibody against MyoD and Drs. Lassar and Wright for providmg MyoD and myogenin expression vectors, respectively. This work was supported (in part) by a Grant-in Aid from the American Heart Association, Kentucky Affihate (S.M.S.) and Grant CA 43798 from NIH (R.R.H.).

1. 2.

3. 4. 5. 6. 7.

Hoerl, B. J., and Scott, R. E. (1989) J. CeQ. I%ysiol. lsgY 6875. Crescenzi, M., Fleming, T. P., Lassar, A. B., Wemtraub, H., and Aaronson S. A. (1990) hoc. NatL Acad Sci. USA $7? 84428446. Sorrentino, B. R., Pepperkok, R. L.? Davis, R” L., Ansorge, W., and Philipson, L. (1990) iVature WEnP 813-815. Lim, R. W., and Hauschka, S. D. (1984) Kiev. &ol. 1 Lim, R. W., Linkhart, T., Merrill, Go, Clegg, C., and Hauscbka, S. D. (1980) J. Cell Viol. $7, 24~1. Linkhart, T. A., C!egg, C. H., and Hauschka, S. D. (1980) J. Supramol Struct. ld,483-498. Mueller, P. R.T and Weld, B. (1989) Sctince %4

112 8. 9.

WOLF,

HIRSCHHORN,

Merrill, G. F., Clegg, C. H., Linkhart, T. A., and Hauschka, S. D. (1980) Eur. J. Cell Biol. 22, 402. Steiner, S., Manley, G., and Adams, T. (1984) Exp. Cell Res.

AND 14. 15.

155,289-293. 10. 11. 12a.

Keutzer, J. C., and Hirschhorn, R. R. (1990) 153-15s. Handler, J. A., Danilowicz, R. M., and Eling, Chem. 265, 3669-3673. Davis,

R. L., Weintraub,

Exp.

Cell Res.

16.

J. Biol.

17.

51,

18.

W. E., Las-

19.

S., Axel, R., Urlaub, G., and Sci. USA 76, 1373-1376.

20.

H., and Lassar,

T. E. (1990)

188,

A. B. (1987)

Cell

987-1000. 12b. Thayer, M. J., Tapscott, sar, A. B., and Weintraub, 13.

Wigler, Chasin,

Received Revised

S. J., Davis, H. (1989)

M., Pellicer, A., Silverstein, L. (1979) Proc. Natl. Acad.

March version

4, 1992 received

April

28, 1992

R. L., Wright, Cell 58,241-248.

STEINER

German, C. R., Padmanabhan, Science 22 1,551-553. Chomczynski, P., and Sacchi, 156-159. Steiner, CellRes. Wright, 607-617.

S. M., Keutzer,

R., and Howard, N.

(1987)

Anal.

J. C., and Hirschhorn,

B. H. (1983) Biochem.

162,

R. R. (1991)

Exp.

192, 643-646. W. E., Sassoon,

Brennan, T. J., Edmondson, CellBiol. 110, 929-937. Clegg, C. H., and Hauschka, 947. Rapraeger, A. C., Krufka, 252, 1705-1708.

D. A., and Lin,

V. K. (1989)

D. G., and Olson, S. D. (1987) A., and

Olwin,

Cell 56,

E. N. (1990)

J, Cell Biol.

J.

105,937-

B. B. (1991)

Science