Oncogene (1997) 14, 63 ± 73  1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00

Transcriptional down-regulation of myogenin expression is associated with v-ras-induced block of di€erentiation in unestablished quail muscle cells

Simona Russo, Franco TatoÁ and Milena Grossi

Dipartimento di Biologia Cellulare e dello Sviluppo, UniversitaÁ di Roma `La Sapienza', Via degli Apuli 1, Roma, Italy

Unestablished quail myoblasts were infected with a (Weintraub et al., 1991; Li and Olson, 1992). An retroviral vector encoding the oncogenic form of H-Ras additional element of this regulatory circuit is in order to investigate the mechanism by which this represented by the Id protein, that might sequester oncoprotein interferes with terminal di€erentiation. E12/E47 into inactive dimers, thus preventing func- Primary quail myogenic cells exhibit the simultaneous tional complex formation between E12/E47 and MyoD expression of the muscle regulatory genes myf-5, MyoD family members (Benezra et al., 1990). Muscle-speci®c and myogenin in proliferative conditions. v-ras-trans- isoforms of the MEF-2 transcription factor have been formed myoblasts displayed an altered growth control isolated, and are thought important in transactivating and lost the competence for terminal di€erentiation. E-box-negative muscle genes as well as in cooperating When expression of myogenic regulatory genes was with MyoD family members (Molkentin et al., 1995). analysed, it was immediately apparent that the di€erence Another feature of myogenic di€erentiation is repre- between normal and v-ras-transformed cells was limited sented by the mutual exclusion between proliferation to a severely decreased level of myogenin expression. and expression of muscle-speci®c genes: permanent Forced expression of exogenous myogenin in v-ras- withdrawal from cell cycle is required for the activation transformed quail myoblasts led to a striking recovery of the myogenic di€erentiation program (Okazaki and of the competence for terminal di€erentiation. The Holtzer, 1966; Nadal-Ginard, 1978). present data show that: (i) repression of myogenin With the exception of the D7 mutant of c-myc, expression is linked to the di€erentiation defective lacking the last leucine of the Leucine Zipper, and of phenotype of quail myoblasts transformed by v-ras as a cytoplasmic mutant of SV40 Large T, that can well as other retroviral oncogenes; (ii) correction of the confer anchorage-independent proliferation, but fail to di€erentiation-defective phenotype of v-ras-transformed inhibit muscle cell di€erentiation (La Rocca et al., myoblasts by exogenous myogenin entailed reactivation 1994; Tedesco et al., 1995), transformation of muscle of endogenous myogenin and of the E-box-dependent cells by various oncogenes generally results in the transactivating function. These results strongly indicate inhibition of the di€erentiation potential (reviewed in that myogenin expression plays a central role in AlemaÁ and TatoÁ , 1994). From the analysis of regulating the transition into the terminally di€erentiated transformed myoblasts and rhabdomyosarcoma cell state and that its transcriptional down-regulation lines, several potential mechanisms have been pro- represents a nodal step in v-ras-induced block of posed, among which repression of MyoD expression di€erentiation. (Lassar et al., 1989; Bengal et al., 1992; Caruso et al., 1993) and interference with MRF transactivating Keywords: v-ras; oncogenes; transformation; myogen- function (Lassar et al., 1989; Falcone et al., 1991; esis; myogenin Miner and Wold, 1991; Arnold et al., 1992; Braun et al., 1992; Li et al., 1992). Thus, the molecular mechanism by which transformation interferes with muscle di€erentiation does not appear to be unique, Introduction but it can vary depending both on the oncogene under investigation and the muscle cell model used. For In vitro transformation of di€erentiating cells belonging instance, although terminal di€erentiation is almost to several lineages, including myogenic cells, is often completely blocked, MyoD expression can be retained associated with a reduction or loss of their differentia- in v-src-andv-jun-transformed quail myoblasts tive potential (Graf, 1992; Enrietto and Beug, 1994). It (Falcone et al., 1991; Grossi et al., 1991), whereas c- is well established that skeletal myogenic di€erentiation jun-induced block of di€erentiation in the C2C12 cell depends on the concerted action of muscle transcrip- line is associated with MyoD down-regulation (Bengal tion factors belonging to the MyoD and the MEF-2 et al., 1992). Similarly, expression of a transforming families. The four known Muscle Regulatory Factors ras gene has been shown to lead to transcriptional (MRF) of the MyoD family (MyoD, myogenin, myf-5 silencing of MyoD in murine myogenic cell lines and MRF4) are thought to activate muscle gene (Konieczny et al., 1989; Lassar et al., 1989). With transcription by binding to a speci®c DNA sequence, regard to the mechanism of action of oncogenic Ras, called E-box, after heterodimerization with ubiqui- there are con¯icting reports on the inhibition of tously expressed E2A gene products such as E12/E47 MyoD transactivating function: in one instance, activated Ras was shown to prevent MyoD from transactivating an E-box containing reporter gene Correspondence: M Grossi, Sezione di Science Microbiologiche, Via (Lassar et al., 1989), whereas in a more recent report delgi Apuli 1, 00185-Roma, Italy Received 24 June 1996; revised 6 September 1996; accepted 6 activated Ras apparently could not inhibit MyoD September 1996 transactivating function (Kong et al., 1995). v-ras oncogene and muscle differentiation S Russo et al

64 On these grounds, we have decided to re-investigate „—˜le I the mechanism of interference with muscle differentia-

tion in cells transformed by the v-ras oncogene. We †irus yn™ogene gell line

took advantage of the fact that unestablished avian ‚ous —sso™i—ted virus I ‚e†EI G w@‚e†IA

et —lFD IWVSA cells can be transformed by a single oncogene and have @p—l™one

used primary cultures of quail myogenic cells as a more

gII

rEr—s r—sA

physiological model to investigate the relationship w@ et —lFD IWVTA

between oncogenic ras-induced transformation, pertur- @u—hn

bation of the myogenic regulatory circuit and block of evi—n erythro˜l—stosis virus

@iƒRA

er˜E˜ @Cer˜EeA

terminal di€erentiation. Our results indicate that v-ras w@ei†AB ei†@‚e†EIA

et —lFD IWVSA alone is sucient to cause loss of growth control and @p—l™one to prevent terminal di€erentiation of primary quail

myoblasts. Interference with myogenic di€erentiation irythro˜l—stosis virus ƒIQ ƒIQ@‚e†EIA w@se—A se—

et —lFD IWVVA by v-ras resulted selectively associated with transcrip- @unight

tional repression of myogenin expression and lack of E-

‚ous s—r™om— virus @€r—gueA

box dependent transactivating function. Forced expres- sr™ ‚ƒ†E€‚Ee w@sr™A

et —lFD IWVSA sion of exogenous myogenin corrected to a large extent @p—l™one

the di€erentiation defective phenotype of v-ras-

pujin—mi s—r™om— virus

transformed quail myoblasts. We conclude that fps w@fpsA

expression of myogenin, rather than that of MyoD or pƒ†@‚e†EIA et —lFD IWVSA

myf-5, represents a major regulatory, rate-limiting step @p—l™one mil

w@milA

that is preferentially a€ected in v-ras-transformed, €ePHHEwrP@‚e†EIA et —lFD IWVTA

unestablished quail myoblasts. @qr—f

evi—n s—r™om— virus IU

jun

w@junA eƒ†EIU

@qrossi et —lFD IWWIA

— fos

fosA Results w@ ePEfos@‚e†IA

er˜f is the relev—nt on™ogeneD we

Growth behaviour and competence for terminal Belthough in the present ™ontext vE h—ve preferred to refer to these ™ells —s w@ei†A ˜e™—use of the

er˜eD in the str—in of ei† here di€erentiation of v-ras-transformed quail myoblasts presen™e of — se™ond on™ogeneD vE

A polyclonal population of v-ras-transformed quail usedF €revious studies showed th—t vEir˜e interferes spe™i®™—lly with erythroid gene tr—ns™ription @enke et —lFD IWVVAD ˜ut does not

et —lFD IWVWY

myoblasts, referred to as QM(ras) (see Table 1), was interfere with qu—il myo˜l—st di€erenti—tion @qrossi

—

ePEfos is — repli™—tion defe™tive —vi—n et —lFD IWWRAF

generated by infection at high multiplicity of early- g—ss—rEw—lek passage myogenic cells with the avian retroviral vector retrovir—l ve™tor en™oding — q—gEpos polyprotein from pf‚Ewƒ† @t

C11, encoding the oncogenic form of p21v-ras of Harvey qhysd—elD unpu˜lishedA murine sarcoma virus (Kahn et al., 1986). Rous associated virus 1 (RAV1) contains no oncogene and

served as a control. Infected cells were passaged two-

gompeten™e for termin—l differenti—tion —nd r—te of entry three times in Growth Medium (GM), to ensure virus „—˜le P

r—sEtr—nsformed qu—il myo˜l—sts

spread by reinfection, before analysing the e€ects of into ƒ ph—se of norm—l —nd vE

oncogene expression on growth control and differentia- gells qw hw

C C C C

7frd 7wrg 7frd tion potential. 7wrg

Loss of growth control in QM(ras) was evidenced by QFR VP THFH QV

increased proliferation rate, very high saturation w@‚e†IA r—sA RFH VP QFH TW

density and acquired anchorage-independent prolifera- w@ gells were seeded in dupli™—te QS mm dishesD —s spe™i®ed in

tion (20% soft agar cloning eciency), as previously w—teri—ls —nd methodsF PR h l—terD ™ells were fed with either qw or

reported for QM(src), QM(fps), QM(AEV), and hwF RV h l—ter ™ells in qw were either ®xed —nd st—ined for wrg

w frd Y ™ells in m

QM(jun) (Falcone et al., 1985; Grossi et al., 1991); expression or fed with fresh qw ™ont—ining PH

w frd F PR h m QM(RAV1) remained strictly anchorage-dependent for hw were fed with fresh hw with or without PH

proliferation (50.01% soft agar cloning eciency). l—terD frd El—˜elled ™ells in qw or hw were ®xed —nd st—ined to determine the per™ent—ge of ™ells th—t entered into ƒ ph—seY ™ells

QM(ras) showed a severely reduced competence for ™ultiv—ted in hw without frd were st—ined for wrg expressionF

morphological di€erentiation after cultivation in „he v—lues shown —re the —ver—ge of dupli™—te s—mplesD di€ering less

Di€erentiation Medium (DM). As shown in Table 2, th—n IH7Y the d—t— ™ome from — represent—tive experimentD whi™h immuno¯uorescence analysis of these cultures con- in™luded the xorthern ˜lot —n—lysis shown in pigures I —nd P ®rmed that the absence of fusion re¯ected inhibition of di€erentiation: while QM(RAV1) retained the ability to form multinucleated myotubes that expressed the muscle-speci®c isoform of Myosin Heavy Chain apparent that the di€erence in the rate of entry into (MHC), QM(ras) gave only rise to a small number of S phase (less than twofold) between normal and abortive, terminally di€erentiated myocytes and/or transformed myoblasts in DM did not account for oligonucleated myotubes. We also compared the the large di€erences in the competence for terminal ability of these cells to enter into S phase by BrdU di€erentiation (over 10 ± 20-fold). Accordingly, only a labelling in GM or DM (see Table 2). The labelling small percentage of QM(ras) that failed to enter into S indices in GM suggest that, in these conditions, normal phase in the 24 h labelling period was capable of and ras-transformed myoblasts could proliferate with a di€erentiating, while the vast majority of unlabelled comparable eciency. Furthermore, it was also QM(RAV1) had terminally di€erentiated. These data v-ras oncogene and muscle differentiation SRussoet al 65 do not suggest a simple and direct relationship between Expression of myogenic regulatory and coregulatory v-Ras-induced loss of growth control and block of genes in normal and v-ras-transformed quail myoblasts di€erentiation. The behaviour of QM(ras) was compared with that Experimental evidence indicates that proper functioning of quail myoblasts transformed by oncoproteins acting of MRFs such as MyoD, myf5, myogenin and MRF4 upstream or downstream of c-Ras in the signal and of their positive (E12/E47) and negative (Id) co- transduction cascade. Polyclonal populations of regulators is required for myogenic di€erentiation transformed quail myoblasts, referred to as QM(onc) (Benezra et al., 1990; Lassar et al., 1991; Lyons and (see Table 1), were generated by infection at high Buckingham, 1992). In order to understand why multiplicity with avian retroviruses encoding oncopro- di€erentiation was blocked in QM(ras), we have teins that can act as constitutively activated versions of analysed the pattern of expression of these genes in growth factor receptors (ErbB, Sea), non-receptorial cells grown in GM or DM, with the exception of the tyrosine kinases (Src, Fps), serine-threonine kinases MRF4 homologue, that is not expressed in cultured quail (Mil, the avian equivalent of mammalian Raf1) and muscle cells, and of E2A, whose expression showed no transcription factors (Jun, Fos). Cells infected by substantial di€erences between normal and transformed transforming retroviruses displayed a variable degree quail myoblasts (unpublished observations). of morphological conversion and of loss of growth Most established myogenic cell lines express either control; cloning eciencies in soft agar ranged from MyoD or myf-5 in the proliferative phase and myogenin 15% in the case of QM(sea) to 2 ± 5% in the case of expression only begins at the onset of terminal QM(mil)andQM(fos). Upon passaging, QM(fos) di€erentiation (reviewed in Olson and Klein, 1994). progressively began to exhibit a reduced growth rate Normal quail myoblasts have been shown to express and an increased tendency to di€erentiate, that simultaneously MyoD, myf-5 and myogenin (Pownal suggested an instability of the transformed state in and Emerson, 1992). Accordingly, as shown in Figure these cells and prevented further investigations. 2a, phenotypically normal QM(RAV1) accumulated As above reported for QM(ras), myoblasts trans- transcripts from MyoD, myf-5 and myogenin both in formed by the other oncogenes showed a severely proliferative (GM) and di€erentiative (DM) conditions. reduced competence for morphological di€erentiation While expression of MyoD was similar in GM and DM, and expression of MHC after cultivation in DM. The myf-5 expression was down-regulated upon terminal observed inhibition of MHC protein expression in di€erentiation and myogenin was up-regulated in DM. transformed myoblasts was due to the virtually Three lines of evidence suggest that the expression of complete absence of MHC mRNA accumulation (see myogenin in proliferating QM(RAV1) cultivated in GM Figure 1). Strongly reduced accumulation of muscle- cannot be quantitatively accounted for by contaminat- speci®c Myosin Light Chain (MLC) mRNA was also ing di€erentiated cells: (i) the virtual absence of MHC apparent, although some residual RNA could be and MLC transcripts characteristic of the di€erentiated observed. Thus, expression of v-ras, as well as of state in QM(RAV1) maintained in GM (see Figure 1); other single, retroviral oncogenes, is sucient to (ii) when RNA was extracted from QM(RAV1), the disrupt growth control and to cause a severe inhibition incidence of di€erentiated cells in GM did not exceed of the di€erentiative potential of primary quail 3 ± 4% (see Table 2); (iii) at least 82% of QM(RAV1) in myoblasts. GM, from which RNA was extracted and analysed in Figures 1 and 2, were still competent to enter the S phase in the subsequent 24 h (see Table 2). These data strongly suggest that myogenin transcription is a characteristic of replicating quail myoblasts and does not require permanent cell cycle withdrawal or irreversible commitment to terminal di€erentiation. Id transcripts in QM(RAV1) were present at low level in GM and were further down-regulated in DM. src ras sea mil fps AEV jun RAV1-GM RAV1-DM Northern blot analysis of the expression of myogenic regulatory and co-regulatory genes in QM(ras) clearly GAPDH indicated that v-ras-induced block of di€erentiation was associated to a severe reduction of myogenin transcript accumulation both in GM and in DM (see MLC Figure 2b and c). Surprisingly, the di€erentiation defective phenotype of QM(ras) did not appear to involve repression of MyoD transcription, as reported MHC for oncogenic ras-transformed murine myogenic cell lines (Lassar et al., 1989; Kosniezcky et al., 1989): MyoD transcript levels were comparable with those of Figure 1 Northern blot analysis of muscle-speci®c structural normal QM(RAV1) both in GM (Figure 2b) and in transcripts in normal and transformed quail myoblasts. Total RNA was extracted from transformed quail myoblasts cultivated DM (Figure 2c). Furthermore, maintenance of MyoD in DM for 72 h and from QM(RAV1) cultivated either in GM for transcription corresponded to high levels of the protein 48 h or in DM for 72 h. The same ®lter was sequentially product, that was correctly localized in the nuclei of hybridized with the indicated probes, as described in Materials QM(ras) (Figure 3). On the contrary, expression levels and methods, as well as with the other probes shown in Figure 2a of myf-5 and Id in QM(ras) did not noticeably vary and c. Parallel cultures of QM(RAV1) and QM(ras)were analysed by immuno¯uorescence for MHC expression and DNA from those observed in QM(RAV1), at least in GM synthesis as described in Table 2 (Figure 2b), thus indicating that expression of neither v-ras oncogene and muscle differentiation S Russo et al 66 RAV1-GM RAV1-DM src ras sea mil fps AEV jun RAV1 src ras sea mil fps AEV jun RAV1 a b c GAPDH

Myf5

MyoD

Myogenin

Id

Figure 2 Northern blot analysis of muscle regulatory and coregulatory gene transcripts in normal and transformed quail myoblasts. All the cells were cultivated for either 48 h in GM or 72 h in DM before RNA extraction. (a) Shows normal QM(RAV1), cultivated in GM or DM; (b) shows transformed quail myoblasts cultivated in GM; (c) shows transformed quail myoblasts cultivated in DM. Blots in b and c contain also RNA from QM(RAV1), cultivated in the corresponding medium, as an internal reference. The same ®lters were sequentially hybridized with the indicated probes as described in Materials and methods. The presence of high levels of myogenin transcripts in QM(RAV1) cultivated in GM cannot be accounted for by the low level of contaminating di€erentiated cells. Furthermore, the majority of these cells cultivated in GM was capable of entering into S phase in the 24 h period after the time point at which the RNA was extracted (see Table 2)

of this oncogene, we extended the analysis of myogenic regulatory and co-regulatory gene expression to quail muscle cells transformed by other retroviral oncogenes. From the Northern blot analysis shown in Figure 2, it is immediately apparent that the expression of myogenin was found to represent a preferential target not only for the v-ras oncogene, but also for the other oncogenes here analysed, and its levels were generally very low as compared to normal, proliferating cells. QM(AEV) apparently represented a notable exception to this rule and maintained a high level of these transcripts in GM (B), but such a feature tended to disappear upon cultivation in DM (C). MyoD expression, though showing some ¯uctuation among transformed cells cultivated in GM (B), remained substantially high, particularly in DM (C). Even in the case of QM(jun), Figure 3 Expression of endogenous MyoD in QM(ras). QM(ras), displaying the lowest level of residual MyoD expression, cultivated in GM, were stained by immuno¯uorescence with a previous clonal analysis has shown that MyoD down- MyoD-speci®c rabbit polyclonal antiserum (R4B4), as described in Materials and methods. MyoD is expressed and correctly regulation is not required to prevent di€erentiation localized in QM(ras) nuclei. Bar, 50 mm (Grossi et al., 1991). Unexpectedly, MyoD transcripts were undetectable by Northern blot analysis in QM(mil) under all experimental conditions. This surprising result contrasts with maintenance of MyoD expression in gene represented a crucial target in v-ras-induced block myoblasts expressing v-Ras, acting just upstream of of terminal di€erentiation in quail myoblasts. Alto- Mil/Raf in the mitogenic pathway and might re¯ect gether these data suggest that the observed repression di€erences between v-Mil and v-Ras in activating the of myogenin expression in QM(ras) might represent a MAP kinase cascade in quail myoblasts; further work is nodal point in the pathway through which the v-ras required to clarify this point. Furthermore, it is oncogene prevents terminal di€erentiation of unestab- interesting to note that MyoD down-regulation in lished quail myoblasts. QM(mil) did not result in the up-regulation of myf-5, as previously observed in vitro (Peterson et al., 1990) and in vivo (Rudnicki et al., 1992). Figure 2 also shows Control of myogenin expression is a preferential target that none of the analysed oncogenes were capable of for oncogene-induced block of di€erentiation inhibiting myf-5 expression and, on the contrary, in In an e€ort to understand whether the repressed most cases myf-5 transcripts were more abundant than myogenin phenotype of QM(ras) was strictly peculiar in normal cells, both in GM (B) and DM (C). Id levels v-ras oncogene and muscle differentiation SRussoet al 67 were strongly up-regulated in most transformed length rat myogenin cDNA insert (see Materials and myoblast populations when cultivated in GM (B), methods). Polyclonal populations of stably transfected possibly a re¯ection of a speci®c synergistic e€ect cells were selected for G418 resistance and are referred between oncogene action and serum growth factors to as QM(ras)/neo or QM(ras)/myogenin. that, however, was not operating in QM(ras)and As compared to parental QM(ras) both QM(ras)/ QM(jun). Accordingly, in DM (C), where serum neo and QM(ras)/myogenin displayed a 4 ± 5-fold growth factors are reduced, Id transcript levels in reduction in the cloning eciency both in liquid and transformed cells were substantially reduced, but still semi-solid medium. When the di€erentiation potential higher than in normal di€erentiating myoblasts. was analysed, QM(ras)/neo behaved like the parental QM(jun) appeared to express the comparatively highest level of Id in DM (C); it is not clear whether this ®nding re¯ects a myoblast-speci®c e€ect of v-Jun. Altogether, the data in Figure 2 suggest that a myoblasts transformed by functionally distinct onco- genes may express altered, distinguishable repertoires of myogenic regulatory and co-regulatory factors. However, reduced levels of myogenin expression emerged as a major feature common to the pheno- types elicited by v-ras and other oncogenes, thus suggesting that this alteration might play a central role in the transformation-induced block of differentia- tion. The data also suggest that the negative modulatory function of Id on myogenic differentiation may contribute to prevent terminal di€erentiation in those transformed, unestablished quail myoblasts where Id expression is up-regulated. b Correction of QM(ras) di€erentiation defective phenotype by forced expression of myogenin To further dissect the mechanism by which the v-ras oncogene can interfere with myogenic di€erentiation, we next asked whether we could correct the differentia- tion-defective phenotype of QM(ras)byforced expression of exogenous myogenin, as these cells ostensibly di€ered from normal cells only for a strongly reduced level of myogenin transcripts accumu- lation in GM. Accordingly, QM(ras) were transfected with the expression vector MDR1, encoding G418 resistance, or MDR1-myogenin, containing also a full

c

„—˜le Q ixpression of exogenous myogenin ™orre™ts the differE

enti—tion defe™tive phenotype of vEr—sEtr—nsformed qu—il myo˜l—sts

C

@7A gells wrg

w@r—sAEneoGe SFH

w@r—sAEneoGf IFP

w@r—sAEwyohGe IHFS

w@r—sAEwyohGf QFW

w@r—sAEmyogeninGe PSFH

w@r—sAEmyogeninGf IWFH

w@r—sAEneoGe ™lFQ SFH

w@r—sAEneoGe ™lFS IFS

w@r—sAEmyogeninGe ™lFP PQFH

w@r—sAEmyogeninGe ™lFS QSFS

w@r—sAEmyogeninGe ™lFII IQFH

w@r—sAEmyogeninGe ™lFIQ RTFT

w@r—sA were st—˜ly tr—nsfe™ted with either wh‚ID wh‚IEwyoh

or wh‚IEmyogenin expression ve™torsD —s des™ri˜ed in w—teri—ls Figure 4 Terminal di€erentiation occurs in QM(ras) after

—nd methodsF €oly™lon—l popul—tionsD sele™ted for qRIV resist—n™eD transfection of exogenous myogenin expression vector. Polyclo-

derived from di€erent experiments —re denoted ˜y Ge or GfF glon—l nal populations of QM(ras) expressing either the MDR1

str—ins were derived ˜y pi™king well isol—ted ™olonies growing in backbone vector, referred to as QM(ras)/neo, or the MDR1-

softE—g—r —nd ™ells were exp—nded in liquid mediumF wrg myogenin vector, referred to as QM(ras)/myogenin, were analysed

expression w—s —ssessed —fter ™ultiv—tion in hw for P ± Q d—ys —s for the competence to undergo terminal di€erentiation. Immuno-

des™ri˜ed in w—teri—ls —nd methodsF „he d—t— from two ¯uorescence with MHC-speci®c antibodies was performed on cells

represent—tive poly™lon—l popul—tions —nd — few —g—rEderived ™lon—l cultivated in DM for 2 days: (a) QM(RAV1); (b) QM(ras)/neo; (c)

C

str—ins —re shown —nd refer to the proportion of nu™lei in wrg QM(ras)/myogenin. Nuclei were counterstained with ¯uorescent

r—sAEwyoh did myotu˜es —nd myo™ytesF „he poor growth of w@ Hoechst dye and simultaneously visualized with MHC staining. not —llow su˜™loning of these ™ells @see text for det—ilsA Bar, 100 mm v-ras oncogene and muscle differentiation S Russo et al 68

cells and only in DM gave rise to a small number of n MHC+, terminally di€erentiated cells (see Figure 4b and Table 3); QM(ras)/myogenin, on the contrary,

could di€erentiate 4 ± 20-fold more eciently into neo myogeni RAV1 + MHC , multinucleated myotubes (see Figure 4c and GM DM GM DM GM DM Table 3) in DM. Clonal cell strains were derived from soft agar colonies of both cell types; most of the clones of QM(ras)/myogenin displayed a differentia- GAPDH tion potential comparable to, or even higher than, that of parental polyclonal population. Table 3 illustrates the percentage of cells capable of under- going terminal di€erentiation in representative exam- rat myogenin ples of polyclonal QM(ras)/neo and QM(ras)/ myogenin as well as of soft-agar derived sub-clones. This set of data strongly suggests that myogenin- induced correction of QM(ras) di€erentiation defective phenotype did not interfere with maintenance of the quail myogenin transformed state. Even though endogenous MyoD was synthesized and correctly localized in the nuclei of QM(ras) (see Figure 3), we investigated the possibility that exogenous MyoD might also function like exogenous MyoD myogenin in restoring the competence for terminal di€erentiation, as previously observed in a MyoD- expressing rat rhabdomyosarcoma cell line (Arnold et al., 1992). However, stable MDR1-MyoD (see desmin Materials and methods) transfectants, referred to as QM(ras)/MyoD, were very dicult to generate and were characterized by poor growth and early senescence that allowed only a limited characteriza- tion by immuno¯uorescence. In most of the experi- α-actin ments we had no evidence for any correction of the QM(ras) di€erentiation defective phenotype and only in two out of six independent transfections, polyclonal populations of QM(ras)/MyoD exhibited a modest MLC increase in the percentage of MHC expressing, terminally di€erentiated cells upon cultivation in DM, as compared to their matched QM(ras)/neo Figure 5 Induction of endogenous myogenin and muscle-speci®c control transfectants (see Table 3). The poor growth mRNAs by exogenous myogenin in QM(ras). Total RNA was properties of QM(ras)/MyoD contrasted with the extractedfromQM(ras)/neo, QM(ras)/myogenin and QM(RAV1), respectively referred to as neo, myogenin and excellent growth properties of QM(ras)/myogenin RAV1, cultivated in GM or DM for 2 days. The same ®lter and this discrepancy prevented a meaningful compar- was sequentially hybridized with the indicated probes as described ison of the two populations. Given the modest e€ects in Materials and methods exerted by exogenous MyoD in QM(ras)/MyoD, we can conclude that, unlike exogenous myogenin, forced expression of additional MyoD is insucient to override v-ras-induced block of di€erentiation in been corrected to a large extent in QM(ras)/myogenin. unestablished myoblasts. This phenotypic correction, however, was not com- Upon further analysis, it was immediately apparent plete, and although the `corrected' myotubes accumu- that expression of exogenous rat myogenin in QM(ras) lated muscle-speci®c gene products, sarcomeric reactivated the expression of endogenous quail structures could never be observed. These observa- myogenin and activated transcription of muscle- tions are in keeping with the fact that QM(ras)/ speci®c genes such as desmin, a-cardiac actin and myogenin maintained a fully transformed phenotype MLC (Figure 5). Unlike parental QM(ras), QM(ras)/ that, as in the case of oncogenic ras-transformed neo expressed a low level of endogenous myogenin in ®broblasts (Lloyd et al., 1989), does not depend on DM, that might be sucient to account for the inhibition of myogenin expression and, thus cannot be presence of low levels of desmin and a-cardiac actin neutralized by forced expression of exogenous transcripts (Figure 5). myogenin. These data strongly indicate that myogenin expres- sion in QM(ras) is necessary and sucient to rescue Exogenous myogenin restores muscle-speci®c promoter the competence for terminal di€erentiation, without activity and E-box-dependent transactivating function in interfering with the maintenance of the transformed QM(ras) state. Although we cannot distinguish the relative contributions by the endogenous and the exogenous Finally, to further elucidate the mechanism by which v- proteins, these results show that the di€erentiation ras interferes with myogenic di€erentiation and how defective phenotype of v-ras-transformed cells had exogenous myogenin overrides v-ras e€ects, we have v-ras oncogene and muscle differentiation SRussoet al 69 Olson, 1992). These data suggest that interference of v-ras with myogenin expression is mostly exerted at the transcriptional level. Furthermore, expression of exogenous myogenin in QM(ras) restores the transcrip- tion of endogenous myogenin (see Figure 5), presumably by indirectly restoring the functionality of the myogenin autoregulatory loop (Braun et al., 1989; Dechesne et al., 1994). Since QM(ras) maintained expression of MyoD and myf-5 (see Figure 2), and at least MyoD was correctly localized in the nucleus (see Figure 3), we asked whether these cells were competent to express a simple, E-box dependent reporter construct, despite the evident loss of competence for di€erentiation. Figure 6b shows that the 4R-CAT construct, containing four copies of the right E-box of the MCK enhancer, was barely expressed in QM(ras)/neo either in GM or in DM. This ®nding suggests that, as previously observed in mammalian cells (Lassar et al., 1989), oncogenic Ras may interfere with the transactivating function or the DNA-binding activity of MyoD and/or Myf5 also in quail cells. On the contrary, QM(ras)/myogenin expressed this construct with high eciency in GM, with a further increase upon cultivation in DM. Figure 6c shows that QM(ras)/myogenin also recovered the ability to express alpha-CAT and TNI-CAT reporter constructs, where the CAT gene is driven by more complex regulatory regions from a-cardiac actin and Troponin I genes respectively. Both constructs were expressed in a di€erentiation-dependent manner. Figure 6 Transactivation of muscle-speci®c reporter constructs is We could not investigate the MEF2box-dependent recovered in QM(ras)/myogenin. Polyclonal QM(ras)/neo and transactivating function because QM(ras) were found QM(ras)/myogenin were transiently cotransfected in GM with to express high levels of a MEF2-like activity, that RSV-lacZ and either Myo1565-CAT (a), 4R-CAT (b), alpha-CAT caused high expression of CAT vectors contain- (c) or TNI-CAT (c); 15 h later, fresh GM or DM was added and ing either the correct (MHCemb/MEF262) or the CAT protein expression was determined after a further 48 h, as described in Materials and methods. Myo84(-/-)CAT in a and mutated (MHCemb/MEF2mtx2) MEF2-speci®c con- TK-CAT in b served as negative control. The b-actin promoter sensus sequence (unpublished observations). As ex- construct (beta-CAT) served as a positive, di€erentiation- pected, both QM(ras)/neo and QM(ras)/myogenin were independent control. Data from several experiments were pooled capable of expressing the di€erentiation-independent and are expressed as pg of CAT protein after normalization to the b-galactosidase enzymatic activity. Transfection experiments were construct beta-CAT. These results strongly indicate performed several times with at least two independent prepara- that the close link between myogenin expression, E-box tions of each plasmid with comparable results dependent transactivating function and competence for terminal di€erentiation in quail myoblasts accounts for the correction of the di€erentiation defective phenotype of QM(ras) by exogenous myogenin. studied the functionality of the myogenin 5' regulatory region and the competence to express E-box or MEF2- box driven reporter genes as well as complex, muscle- Discussion speci®c constructs in QM(ras)/neo and QM(ras)/ myogenin. Transformation of myogenic cells and cell lines usually Both cell types were transiently transfected with interferes with their competence for terminal differ- CAT constructs as described in Materials and entiation into post-mitotic myotubes, although the methods. As shown in Figure 6a, the pMyo-1565- mechanisms involved may vary depending on the cell CAT, containing the whole 5' regulatory region of type and the oncogene analysed (reviewed in AlemaÁ mouse myogenin (see Materials and methods), showed and TatoÁ , 1994). Several studies from di€erent a low, but signi®cant, level of expression in QM(ras)/ laboratories have analysed the mechanism by which neo, both in GM and in DM. The stable expression of the ras oncogene interferes with terminal di€erentiation exogenous myogenin in QM(ras) resulted in a 4 ± 6- in mammalian myogenic cell lines (Olson et al., 1987; fold increased level of expression of the pMyo-1565 Konieczny et al., 1989; Lassar et al., 1989). In a more construct (Figure 6a), reaching a level comparable to recent report, it has been suggested that oncogenic Ras that observed in normal cells (data not shown). could not interfere with E-box-dependent transactiva- Control experiments with normal quail myoblasts tion by MyoD or MRF4, while it interfered with the and normal or v-ras-transformed quail ®broblasts transactivation of more complex muscle-speci®c con®rmed that expression of this CAT-construct is reporter genes (Kong et al., 1995), but these results strictly muscle-speci®c also in quail cells, as previously contrast with previous data showing that activated Ras shown in mouse and chicken cells (Edmondson and inhibited MyoD transactivating function (Lassar et al., v-ras oncogene and muscle differentiation S Russo et al 70 1989). Here we have attempted to re-investigate the patterns of myogenic regulatory and coregulatory mechanism(s) by which transformation interferes with gene expression in normal and transformed quail muscle di€erentiation by analysing primary quail myoblasts (see Figure 2), overexpression of Id and myoblasts transformed by the v-ras oncogene and repression of myogenin expression emerged as the most comparing their properties with those of untrans- evident and frequent alterations of the myogenic formed cells as well as of myoblasts expressing several regulatory circuit, though not always coordinate. di€erent retroviral oncogenes. While up-regulation of Id expression had not been In this paper we focussed our attention on the previously observed in oncogene transformed cell lines consequences of v-ras-induced transformation on the (Miner and Wold, 1991; Kong et al., 1995), our data expression of MRF belonging to the MyoD family, of clearly implicate that many oncogenes can elevate Id E12, the heterodimeric MRF partner encoded by the expression levels in quail myoblasts, although this is E2A gene, and of Id, a putative negative regulator of not strictly required to observe inhibition of differ- MRF/E12 heterodimer formation. Since our goal was entiation. Forced expression of Id in murine cell lines to identify which components of the myogenic has been shown to interfere with myogenic differentia- regulatory circuit were perturbed by the v-ras tion (Jen et al., 1992) and increased Id expression levels oncogene, we ®rst analysed the pattern of expression might contribute to block terminal di€erentiation in of muscle regulatory and co-regulatory genes in transformed quail myoblasts. untransformed QM(RAV1). These cells, that do not Previous studies on oncogene-transformed myogenic express exogenous oncogenes, displayed a normal cell lines suggested MyoD expression and/or function- growth control (i.e. anchorage-dependent prolifera- ing as the regulatory step a€ected by the transformed tion) and retained the competence for full terminal state (Konieczny et al., 1989; Lassar et al., 1989; Miner di€erentiation upon serial subcultivation. Our results and Wold, 1991; Bengal et al., 1992). However, the show that QM(RAV1) appeared to transcribe the avian comparative analysis of unestablished myoblasts homologues of the mammalian myf-5, MyoD, myogen- strongly indicates that oncogene-induced repression of in, E2A and, to a lower extent, Id genes before terminal MyoD expression seems to be an exception rather than di€erentiation and in the presence of high levels of a rule, as it has been observed only in the case of the v- growth factors; MRF4 expression, on the contrary, was mil oncogene. On the contrary, a major target for never observed, as also reported for chick embryo oncogene action appeared to be myogenin expression, muscle cells in vitro (Yoon and Boettiger, 1994). that was severely a€ected in most transformed Expression of myogenin prior to terminal differentia- myoblasts. Such a reduced level of transcript tion has previously been reported for primary cultures accumulation represents the most clear and distinctive of quail embryo myoblasts (Pownal and Emerson, feature of transformed quail myoblasts, and even 1992). Thus, unestablished quail myoblasts display two QM(AEV), that retain abundant myogenin expression notable di€erences from established cell lines: (i) myf-5 in GM, tend to down-regulate this gene when and MyoD expression is not mutually exclusive as cultivated in DM (see Figure 2b and c). observed in C2C12 (Peterson et al., 1990) and L6 cells Although we cannot exclude that QM(ras) may (Braun et al., 1989); (ii) myogenin expression depends display other unidenti®ed defects in the control of neither on growth factor withdrawal nor on irreversible terminal di€erentiation, that might cooperate with growth arrest, in contrast with previous reports myogenin repression, we found that stable transfection (Edmondson and Olson, 1989; Wright et al., 1989; of QM(ras) with a myogenin expression vector led to reviewed in Olson and Klein, 1994). Recent data reactivation of endogenous myogenin and to a suggest that also in C2C12 cells myogenin can be considerable, yet partial, recovery of the differentiative expressed before permanent cell cycle withdrawal and potential, including muscle-speci®c gene expression as terminal di€erentiation, but this requires growth factor well as multinucleated myotube formation. In several withdrawal (Andre s and Walsh, 1996). studies it has been observed that loss of growth control As expected, v-ras-induced transformation was is compatible with maintenance of the di€erentiation accompanied by the loss of growth control (i.e. potential (Bignami et al., 1982; Lassar et al., 1989; La acquired anchorage-independent proliferation) and a Rocca et al., 1994; Tedesco et al., 1995), and we also severely reduced competence for terminal differentia- found that forced expression of myogenin did not cause tion. Surprisingly, QM(ras) di€erentiation defective phenotypic reversion from the transformed state of phenotype was ostensibly linked only to a drastically QM(ras), as suggested by maintenance of anchorage- reduced level of myogenin transcript accumulation, independent proliferation in QM(ras)/myogenin. while expression of the other regulatory genes, The possibility of correcting the di€erentiation including myf-5, MyoD, E2A and Id was not defective phenotype of QM(ras) through forced particularly a€ected. Since our data show that expression of myogenin allowed to directly investigate myogenin is expressed in proliferating QM(RAV1), the mechanism by which v-Ras inhibited terminal prior to terminal di€erentiation and in the presence of myogenic di€erentiation. The present results implicate high levels of growth factors (see Table 2 and Figure a reduced eciency of myogenin transcription and a 2a; see also Pownal and Emerson, 1992), down- severe de®ciency in the E-box-speci®c transactivating regulation of myogenin in QM(ras) seems to represent function in QM(ras), despite abundant expression of a cause rather than an e€ect of the loss of the myf-5 and MyoD in these cells. Both defects were competence for terminal di€erentiation. Furthermore, eciently corrected by the forced expression of repression of myogenin expression was not a unique exogenous myogenin, that led to reactivation of property of the v-ras oncogene and was observed also endogenous myogenin transcription, recovered compe- in quail muscle cells transformed by other retroviral tence for E-box dependent transactivation as well as to oncogenes. In spite of the complex and variable transactivation of complex muscle-speci®c CAT con- v-ras oncogene and muscle differentiation SRussoet al 71 structs. These data lend further support to the view Materials and methods that myogenin expression plays a central role in regulating the choice toward terminal di€erentiation Cell culture and viruses and that v-Ras-induced transcriptional repression of Quail embryo myoblasts were prepared from breast myogenin represents a rate limiting step in the muscles of 10-day-old Japanese quail embryos as pre- mechanism by which this oncogene inhibits terminal viously described (TatoÁ et al., 1983). Polyclonal popula- di€erentiation in unestablished quail myoblasts. Beside tions of transformed quail myoblasts were established from the E-box-dependent transactivating function, the primary/early-passage cultures infected at high multiplicity MEF2 function, recognized as strictly required for with the appropriate avian transforming retrovirus; origin activating the myogenin promoter (Edmondson and and oncogenes of the viral strains here used are listed in Olson, 1992), might also represent an important target detail in Table 1. Uninfected and infected quail myoblasts were routinely propagated on collagen-coated dishes in for v-Ras signalling. Elucidation of this important issue Dulbecco's modi®ed Eagle's medium supplemented with will require the development of avian-speci®c reagents 10% fetal calf serum, 10% tryptose phosphate broth and and assays. 1% chicken serum (referred to as growth medium, GM). Previous analysis of ras-transformed myogenic cell To assay myogenic di€erentiation, cells were plated on lines suggested a role for MyoD repression in the block collagen-coated dishes at 2 ± 2.56104 per cm2 in GM. 24 h of di€erentiation (Konieczny et al., 1989; Lassar et al., later, cultures were fed with di€erentiation medium, DM, 1989), but a general validity for such a mechanism is which consisted of F14 medium (Vogel et al., 1972) hard to reconcile with the present data as well as with supplemented with 2% foetal calf serum and 0.5 mg/ml of the near normal muscle formation in MyoD null mice bovine insulin. Cells were routinely fed every other day. (Rudnicki et al., 1992). On the contrary, it is Transformed cells were assayed for colony formation in soft-agar by suspending 103 and 104 cells in Dulbecco's interesting to note that ras-transformed unestablished modi®ed Eagle's medium containing 10% foetal calf serum, myoblasts in vitro and myogenic bHLH gene knock- 10% tryptose phosphate broth, 2% chicken serum, vitamins outs in vivo (Hasty et al., 1993; Nabeshima et al., 1993) (ICN, Costa Mesa, CA) diluted 1:100, folic acid (8 mg/ml) point to the same conclusion, i.e. that myogenin and 0.35% Bacto-Agar and layering the mixture in expression is essential in taking the decision to enter duplicate on a hard base of 0.7% agar with the same the terminally di€erentiated state. It may not be just a supplements. mere coincidence that in continuous myogenic cell lines The rate of entry into S phase of normal and transformed myogenin expression is repressed by growth factors and quail myoblasts was assayed by measuring the percentage of that in unestablished myoblasts oncogenes such as ras nuclei which incorporated bromo-deoxyuridine (BrdU) in 5 repress ongoing myogenin expression. It can be GM or DM. 1.5610 cells were plated in duplicate into 35 mm dishes in GM and 20 ± 24 h later the medium was envisaged that, as observed in unestablished myo- replaced with either GM or DM. After 48 h, cultures were blasts, constitutive myogenin expression represents a fed with fresh GM or DM containing 20 mM BrdU and strong bias toward terminal di€erentiation and that allowed to incorporate the analogue for 24 h. reversible repression of this regulatory gene is selected during the immortalization process in the development of continuous cell lines. Thus, restoration of myogenin Northern blot analysis transcription by growth factor withdrawal in myogenic cell lines (Andre s and Walsh, 1996) preceeds and, Total RNA was prepared by lysing the cells with a bu€er containing 1% SDS, 10 m Tris-HCl (pH 7.5), possibly, drives cells toward terminal di€erentiation. M 100 mM NaCl and 0.1 mM EDTA. Following centrifuga- Similarly, in the case of QM(ras), the rescue of their tion for 1 h at 100 000 g at 108C, the supernatant was di€erentiative potential by exogenous myogenin is extensively extracted with acid phenol and chloroform and linked to restoration of endogenous myogenin expres- ethanol precipitated. 12 mg of RNA were resolved in 0.8% sion. Although we could not eciently correct the agarose-2.2 M formaldehyde gel and transferred to di€erentiation defective phenotype of QM(ras)by nitrocellulose membrane. High-stringency hybridization stable transfection of a MyoD expression vector, it is and washings were carried out according to standard possible that other myogenic bHLH factors, alone or procedures. in combination with other factors such as E12 and For detection of muscle-speci®c and constitutive tran- MEF2 (see Molkentin et al., 1995), might also restore scripts, inserts were excised with the appropriate restriction enzymes from the following plasmids and used as probes: endogenous myogenin expression in QM(ras) and thus cC508, containing a 850 bp cDNA fragment encoding the rescue their di€erentiation potential. In addition, quail homologue of MyoD (qmf1); pQmf3, containing a myogenin-independent pathway(s) toward terminal 1.4 kb cDNA fragment encoding the quail homologue of di€erentiation may also exist, as suggested by the myf-5 (qmf3); cC128, containing a 300 bp quail myosin ability of myogenin null myoblasts to di€erentiate heavy-chain (MHC) cDNA; cC127, containing a 600 bp quail poorly in vivo, but to a substantial extent in vitro myosin light-chain (MLC) cDNA; cC118, containing a (Hasty et al., 1993; Nabeshima et al., 1993). The latter 590 bp fragment of quail a-cardiac-actin cDNA (provided observation is not yet understood, and it is not clear by C Emerson, University of Pennsylvania, PA). pD8, whether such a pathway can be also operating in wild containing a 1 kb chicken desmin cDNA (provided by E type myoblasts. The approach and the model here Lazarides, Merck Research Laboratories, West Point, PA); pCmgn-1, containing a 1.5 kb cDNA fragment encoding the described appear to provide an experimental frame- chicken homologue of myogenin and pck-E12, containing a work that might be useful in understanding the control 2.9 kb cDNA fragment encoding the chicken homologue of of di€erentiation in rhabdomyosarcoma-derived cell E12 (provided by B Paterson, NIH, Bethesda, MD); pck-Id, lines, by de®ning the mechanisms that underlie the containing a 800 bp cDNA fragment encoding the putative regulation and the ®ne tuning of the myogenic chicken homologue of murine Id1 (provided by SA La Rocca, di€erentiative program in normal and transformed UniversitaÁ di Roma, Italy); pEMSV-myogenin (Wright et al., myoblasts. 1989), containing a 1.5 kb cDNA fragment encoding the rat v-ras oncogene and muscle differentiation S Russo et al 72 myogenin under the control of MSV-LTR (obtained from V Transient transfections were performed using the Mam- Sorrentino, DIBIT, Milano, Italy); a plasmid containing a malian Transfection Kit (Stratagene, La Jolla, CA). Brie¯y, 1.2 kb cDNA of the avian glyceraldehyde-3-phosphate- cells were seeded in duplicate at 105 on 35 mm dishes and dehydrogenase (GAPDH) (obtained from C Schneider, CIB, cotransfected with 0.75 mg of reporter construct and 0.75 mg Trieste, Italy). of RSV-lacZ. 15 h after transfection, cultures were fed with either GM or DM and further incubated for 48 h. Cell extracts were prepared by repeated freezing-thawing and the Immuno¯uorescence levels of chloramphenicol acetyltransferase (CAT) protein were determined using an enzymatic immuno-assay (CAT- A mouse monoclonal antibody (MF20) that recognizes the ELISA/Boehringer Mannheim Italia). Cell extracts were skeletal muscle MHC (kindly provided by D Fishman, normalized for protein concentration and CAT expression Cornell University, New York) was used to assess was further normalized to b-galactosidase activity from terminal di€erentiation in normal and transformed quail cotransfected RSV-lacZ (obtained from A Levi, CNR, myoblasts. Cells were ®xed with methanol:acetone (50:50) Rome, Italy), which contains the Rous sarcoma virus and were incubated with the MF20 supernatant for (RSV) long terminal repeat linked to lacZ. Transcriptional 30 min at 378C. After washing with phosphate bu€ered regulation of myogenin expression was analysed using the saline (PBS), a rhodamin-conjugated goat anti-mouse myogenin-CAT reporter gene constructs pMyo-1565CAT, antibody (Cappel, West Chester, PA) was added for containing 1565 bp of the 5' regulatory region of mouse 30 min at 378C. myogenin and pMyo-84(mutMEF2/-E1)CAT, a deletion BrdU-labelled cultures were ®xed for 20 min with 95% mutant containing only the proximal 84 bp of the 5' flanking ethanol/5% acetic acid, treated for 10 min with 1.5 N HCl, sequence with the MEF2-site mutated and the E-box deleted rinsed three times with PBS containing 0.5% Triton X-100 (Edmonson and Olson, 1992). To test the MEF2-box- and stained with an anti-BrdU mAb (Amersham, Amer- dependent transactivating function we used the MHCemb/ sham, UK) for 30 min at room temperature. After several MEF2x2-CAT and MHCemb/MEF2mtx2-CAT constructs, washings with PBS, a rhodamin-conjugated goat anti-mouse where two copies of the normal or mutated muscle-speci®c antibody (Cappel) was added for 30 min at room MEF2-binding site are hooked up to the MHCemb promoter temperature. (Yu et al., 1992). To measure transactivation by MyoD To detect MyoD in QM(ras), cells, cultivated in GM, were family factors, the 4R-CAT, containing the tk minimal ®xed for 10 min with 3% para-formaldehyde in PBS, promoter and four copies of the right E-box from the permeabilized with 0.25% Triton X-100 and incubated for MCK enhancer (Weintraub et al., 1990), and the tkCAT 1 h at 378C with a 1:100 dilution of a rabbit polyclonal constructs were used. The ability to activate transcription antibody raised, in collaboration with S AlemaÁ (ICB, CNR, driven by complex muscle-speci®c regulatory regions was Rome, Italy) and M Crescenzi (IRE, Rome, Italy), against measured by transfecting CAT constructs containing either murine MyoD expressed in bacteria. This antiserum, R4B4, the a-cardiac actin (Eldridge et al., 1985) or the Troponin I cross-reacts with quail MyoD, as assessed by Western blotting. (Yutzey et al., 1989) regulatory sequences, referred to as After several washings, a rhodamin-conjugated Goat anti- alpha-CAT or TNI-CAT respectively; a CAT construct rabbit antibody was added for 30 min at room temperature. containing the chicken b-actin 5' regulatory region (De Nuclei were stained for 10 min at room temperature with Ponti-Zilli et al., 1988), referred to as beta-CAT, was used 1 mg/ml Hoechst 33258 dye. as a constitutive control.

Transfections and CAT assays Acknowledgements To select stable transfectants, 1.56105 cells were seeded on We thank the following persons for providing valuable 60 mm dishes and transfected using the Transfectam reagents used in this study: H Beug, G Calothy, M Reagent (Promega, Madison, WI) for 7 h, with either Crescenzi, C Emerson, D Fischman, J Ghysdael, T 5 mg of the expression vector MDR1, carrying only the Graf, SA La Rocca, E Olson, B Paterson, V neo-resistance gene (Dotto et al., 1989), MDR1-MyoD Sorrentino, H Weintraub and W Wright. We thank all (kindly provided by SA La Rocca, University of Rome, our colleagues who contributed a critical review of this Italy) or MDR1-myogenin (kindly provided by M manuscript. We also thank A Calconi for the careful Crescenzi, IRE, Roma, Italy), where the full length murine preparation of the ®gures and his continuous technical MyoD or rat myogenin cDNAs were respectively subcloned support. This work has been supported by grants from in the unique EcoR1 site under the control of the SV40- Associazione Italiana per la Ricerca sul Cancro, promoter. 24 ± 48 h later, G418 was added to a ®nal Consiglio Nazionale delle Ricerche (progetto ®nalizzato concentration of 1.5 mg/ml and the cultures passaged Applicazioni Cliniche della Ricerca Oncologica) and until uniformly drug resistant. Fondazione Cenci-Bolognetti.

References

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