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1081Journal of Cell Science 111, 1081-1093 (1998)Printed in Great Britain © The Company of Biologists Limited 1998JCS3710

Reversal of myogenic terminal differentiation by SV40 large T antigen results

in mitosis and apoptosis

Takeshi Endo 1,* and Bernardo Nadal-Ginard 2,†

1Department of Biology, Faculty of Science, Chiba University, Yayoicho, Inageku, Chiba, Chiba 263-8522, Japan2Department of Cardiology, Children’s Hospital, Department of Cellular and Molecular Physiology, Harvard Medical School,Boston, MA 02115, USA*Author for correspondence (e-mail: [email protected])†Present address: 315 Dartmouth Street, Boston, MA 02116, USA

Accepted 11 February; published on WWW 25 March 1998

Terminally differentiated skeletal muscle myotubes arearrested in the G0 phase of the cell cycle, and this arrest isnot reversed by stimulation with serum or growth factors.The myotubes have been shown to be refractory toapoptosis even under low serum conditions. When the SV40large T antigen is induced in the C2SVTts11 myotubes,which stably harbor the T antigen gene linked to aninducible promoter, the terminally differentiated cellsreenter the cell cycle to resume nuclear DNA replicationrepresenting S phase. We show here that the large T-expressing myotubes further proceeded to M phaserepresented by the appearance of mitotic figures withcentrosomes, condensed chromosomes, and mitoticspindles. The myotubes eventually cleaved and midbodieswere formed at the cleavage sites of the cytoplasm. In somecases actin filaments, reminiscent of the contractile rings,accumulated at the cleavage furrows. Thus, terminally

differentiated myotubes remain able to resume at least oneround of the cell cycle and consequently are considered tobe capable of dedifferentiation. A subset of myotubesexpressing large T did not undergo mitosis. Some of themwere degenerative and contained deformed giant nucleiand pulverized nuclei. The others suffered apoptotic celldeath, which was identified by morphological changes ofthe nuclei and the labeling with dUTP at the ends ofchromatin DNA fragments. The induction of apoptosis wasunlikely to be confined to a particular phase of the cellcycle. These results imply that terminally differentiatedmyotubes also retain a complete set of machinery forapoptosis.

Key words: Myogenic differentiation, Myotube, SV40 large Tantigen, Mitosis, Cytokinesis, Apoptosis

SUMMARY

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INTRODUCTION

A number of cell types, including skeletal muscle cellterminally differentiate to be arrested in the G0 phase of thecell cycle (Baserga, 1985). Terminal differentiation of skelemuscle cells is morphologically defined by the formation multinucleated myotubes through cell fusion of myoblasts. Tterminally differentiated myotubes are not released from tcell cycle arrest by extracellular mitogens such as serum growth factors (Konigsberg et al., 1960; Stockdale and Holtz1961; Endo and Nadal-Ginard, 1986). When primary culturof mammalian skeletal muscle myoblasts are infected wpolyomavirus, they retain differentiation ability at early stagafter infection (Fogel and Defendi, 1967; Yaffe and Gersho1967). DNA synthesis and rare mitosis-like figures represenby condensed chromosomes have been detected at the timfusion of the infected myoblasts into myotubes (Fogel aDefendi, 1967; Yaffe and Gershon, 1967; Yaffe, 196Graessmann et al., 1973). However, differentiated myotuare resistant to the polyomavirus infection (Fogel and Defen1967). Although by microinjection of polyomavirus, myotube

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To address the question of whether mature myotubes aunable to reenter the cell cycle and resume DNA synthesis amitosis, we have established the mouse skeletal muscle cline C2SVTts11 (hereafter referred to as C2SVT) (Endo anNadal-Ginard, 1989), which stably harbors the SV40 T antigengene encoding temperature-sensitive (ts) large T and wild-tysmall t antigens under the control of the Zn2+-induciblemetallothioneingene promoter. The induction of the large Tantigen in the myotubes, which are preformed in the absenof large T expression, brings about reentry of the terminaldifferentiated cells in the cell cycle. The myotubes resumchromosomal DNA synthesis and exhibit condensechromosomes, indicating that myotubes remain able progress to S and M phases (Endo and Nadal-Ginard, 19Endo, 1992; Endo and Goto, 1992; Ohkubo et al., 1994Similar results have been obtained by infection of retrovirus

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carrying the ts large T to rat primary skeletal muscle cells(Iujvidin et al., 1990). Reinduction of the DNA synthesis imyotubes has also been demonstrated with adenovirus-infemyotubes, which are still permissive to adenovirus infecti(Crescenzi et al., 1995). Furthermore, the cell cycle procein myotubes deficient in the tumor suppressor protein Rb serum stimulation and activation of the Cdc2-cyclin B compl(Schneider et al., 1994; Novitch et al., 1996). Despite taccumulating proof that terminally differentiated myotuberemain competent to replicate DNA and exhibit mitosis-likfigures, there has been no evidence that the mitosis-like figuin myotubes represent complete mitosis leading to cytokine

Although skeletal muscle cells differentiate efficiently undelow serum conditions, these conditions are also favorableinduce apoptosis. In fact, under these conditions an apprecinumber of undifferentiated myoblasts suffer apoptoswhereas myotubes are refractory to apoptosis (Endo, 19Wang and Walsh, 1996). As a consequence, the question aof whether myotubes never undergo apoptosis under the culconditions.

We show here that the large T-expressing C2SVT myotubproceeded to and completed M phase represented by appearance of mitotic figures with centrosomes, condenchromosomes, and mitotic spindles accommodating chromosomes. The myotubes eventually cleaved, amidbodies as well as putative contractile rings were formedthe cleavage furrows. A subpopulation of the large expressing myotubes did not undergo mitosis but instesuffered apoptosis. Thus, terminally differentiated myotubremain competent either to resume at least one round ofcell cycle or to be subjected to apoptosis.

MATERIALS AND METHODS

Cell cultureThe C2SVTts11 cell line was established by transfecting the moskeletal muscle cell line C2C12 (Yaffe and Saxel, 1977; Blau et 1983) with the recombinant plasmid pMtSVTtsNeo as describ(Endo and Nadal-Ginard, 1989; Endo, 1992). This plasmid contathe early region of the SV40 tsA58mutant gene, which codes for thethermolabile large T antigen and the wild-type small t antigen, fusto the mouse metallothionein-I gene promoter. The proliferatingmyoblasts were maintained at 37°C in Dulbecco’s modified Eagl(DME) medium supplemented with 10% fetal calf serum (FC(growth medium). To induce terminal differentiation, ~2×105 cellswere plated in the growth medium on a 100 mm dish and maintaifor 16-24 hours, and then the medium was replaced with DMmedium supplemented with 5% horse serum (differentiatimedium). To induce large T and small t in the cells, the myoblastspreformed myotubes were shifted to the growth medium contain100 µM ZnSO4 at 33°C.

Immunofluorescence microscopyCells grown on glass coverslips were processed immunofluorescence microscopy as described (Endo, 1992). Primantibodies used to estimate reentry into the cell cycle were monoclonal antibody (mAb) PAb101 (formerly clone 412recognizing large T (Gurney et al., 1980) and the mAb D2D6 raisagainst squid nerve ganglion α-tubulin (Saitoh et al., 1988). The cellswere counterstained with bisbenzimide H33258 (Hoechst 33258detect chromosomal DNA (Endo and Goto, 1992). The ceundergoing cytokinesis were also stained with rhodamine-phalloi

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(Molecular Probes, Inc.) plus affinity-purified rabbit antibody tochicken gizzard tropomyosin (Endo et al., 1996), the mAb MF20 sarcomeric myosin heavy chain (MHC) (Bader et al., 1982), or thmAb NT302 to troponin T (Abe et al., 1986). Specimens werobserved with a Zeiss Axioskop microscope or a ZeisPhotoinvertoscope ICM 405 equipped with phase-contrast aepifluorescence optics.

Detection of apoptotic cellsApoptotic cells were detected by staining with the in situ ApoptosDetection Kit (Takara Suzo, Japan), which utilizes a termindeoxynucleotidyl transferase (TdT)-mediated fluorescein-dUTlabeling of the fragmented chromatin DNA. This is a modification othe TdT-mediated dUTP-biotin nick end labeling (TUNEL) method(Gavrieli et al., 1992). The fixed and permeabilized cells were firincubated with PAb101 and then with the TUNEL reagentsupplemented with rhodamine-conjugated goat anti-mouse Ig(affinity-purified, Cappel Laboratories). The cells were examined bfluorescence microscopy as described above.

Determination of the cell cycle of apoptotic cellsThe cell cycle phase of the apoptotic myotubes was determined byincorporation of 5-bromo-2′-deoxyuridine (BrdU) in combinationwith the TUNEL method. The preformed myotubes were shifted the Zn2+-containing growth medium supplemented with 10 µM BrdUand 1 µM 5-fluoro-2′-deoxyuridine at 33°C. The fixed andpermeabilized cells were first incubated with the TUNEL reagentThey were incubated with the mAb BU-1 specific for BrdUsupplemented with 10 µg/ml DNase I (Cell Proliferation Kit,Amersham) and then with rhodamine-conjugated goat anti-mouIgG. The cells were examined by fluorescence microscopy.

Agarose gel electrophoresis of nucleosomal DNABoth adhering and floating cells (0.5-1×107 nuclei) cultured on 100mm dishes were harvested and washed once with cold PBS. DNA wextracted from the cells as described (Ishida et al., 1992) and treawith 0.1 mg/ml RNase A at 37°C for 30 minutes. The DNA waresolved by electrophoresis on a 2% agarose gel and visualizedethidium bromide staining.

RESULTS

Induction of the large T antigen in the myotubesThe C2SVT myoblasts cultured in the differentiation mediumat 37°C in the absence of T antigen expression easdifferentiated to form multinucleated myotubes within 4 daysas do parental C2C12 cells. When the medium was replacwith the growth medium containing 100 µM ZnSO4, the largeT antigen was induced through the metallothionein genepromoter. Immunofluorescence microscopy with the mAPAb101 specific for large T antigen showed that large accumulated in essentially all the nuclei of these myotub(Fig. 1C and D) as well as of the myoblasts. Large T wainduced neither in the myoblasts nor in the myotubes by tgrowth medium without a Zn2+ supplement (Fig. 1A and B).Immunoprecipitation with PAb101 followed by SDS-PAGEindicated that the presence of Zn2+ in the growth mediuminduced 94 kDa large T protein in both myoblasts anmyotubes (data not shown).

Induction of mitosis in the myotubesThe induction of large T at 33°C, a permissive temperature fthe thermolabile large T, allows the terminally differentiate

1083Induction of mitosis and apoptosis in myotubes

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Fig. 1. Induction of large T antigenin C2SVT myotubes detected byimmunofluorescence microscopy.(A and B) Myotubes formed bymaintaining in the differentiationmedium at 37°C for 96 hours andthen culturing in the growthmedium at 37°C for 24 hours.(C and D) Myotubes shifted to theZn2+-containing growth mediumand cultured at 33°C for 24 hours.Phase-contrast micrographs (A andC) and correspondingimmunofluorescent micrographsdetecting large T with the mAbPAb101 (B and D). Bar, 50 µm.

myotubes to reenter the cell cycle (Endo and Nadal-Gina1989; Endo, 1992; Endo and Goto, 1992; Ohkubo et al., 199These myotubes resume chromosomal DNA synthesis almost all the nuclei 17-28 hours after the large T inducti(Endo and Nadal-Ginard, 1989; Ohkubo et al., 1994). We aothers have shown that mature myotubes or differentiamyocytes, which are transfected with the viral oncogeninfected with adenovirus, or deficient in Rb, exhibit mitosislike figures represented by the appearance of condenchromosomes (Endo and Nadal-Ginard, 1989; Iujvidin et a1990; Endo and Goto, 1992; Schneider et al., 1994; Cresceet al., 1995; Novitch et al., 1996). There has been demonstration, however, that the condensed chromosomesassociated with the mitotic spindles, which are essentialcomplete bona fide mitosis and cytokinesis.

To examine whether mitotic spindles were formed association with the condensed chromosomes in the largeexpressing C2SVT myotubes, the cells were doubly stainwith the mAb D2D6 specific for α-tubulin and bisbenzimideH33258 that labels chromosomal DNA. The quiescemyotubes in the absence of large T induction never showcondensed chromosomes or mitotic spindles (Fig. 2C and Centrosome-like structures were not apparent in the quiescmyotubes by the anti-tubulin staining (Fig. 2C). This consistent with the reports that mature myotubes lack appacentrosomes (Tassin et al., 1985; Connolly et al., 1986). In myoblasts, the centrosomes were not evident either interphase but became visible at prophase (Fig. 2A and About 33-34 hours after the large T induction, condenschromosomes were detected in the myotubes (Figs 2F and 3indicating that the myotubes entered prophase of mitosis. Tchromosomal condensation occurred almost synchronouamong gathering nuclei in a myotube. The centrosomes ofbecame apparent in the vicinity of the nuclei containincondensed chromosomes (Fig. 2E).

About 34 hours after the large T induction, some myotub

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contained the bipolar mitotic spindles harboring chromosomaligned across the middle of the spindle (the metaphase pl(Figs 2G,H and 3B). Other myotubes included the spindlaccommodating chromosomes migrating toward the poles (F2I and J). These figures correspond to metaphase and anaprespectively. When several nuclei gathered, multiple spindderived from individual nuclei fused to form giant spindle(Figs 2K-N and 3C). Each spindle composing the giant spindaccommodated chromosomes.

Induction of cytokinesis in the myotubesEventually some sites of the myotubes cleaved by 34-35 hoafter the large T induction. A thin, bridge-like structurcorresponding to the midbody was formed at the cleaveage (Fig. 2O and P). The cleavage and midbody formation toplace at not only the middle but the periphery of the myotub(Figs 2O,P and 3D-F). When the cleavage occurred at middle, the midbody was formed along the long axis of thmyotubes and both daughter cells contained multiple nuc(Figs 2O,P, and 3D). On the other hand, when it occurredthe periphery, the midbody could be placed tangentially to tlong axis of the myotubes and one daughter cell contained or a few nuclei (Fig. 3E and F).

In mononucleated cells under cytokinesis, the actifilament-based contractile ring assembles at the cleavafurrow to generate a force to propel the cleavage. Acfilaments were concentrated at the cleavage furrow in tmyoblasts as revealed by rhodamine-phalloidin staining (F4A and B). Similarly, actin filaments recognized by rhodaminphalloidin staining accumulated at the cleavage furrows of tmyotubes (Fig. 4C). The cleavage furrows also contained only nonsarcomeric tropomyosin but also sarcomeric myosand troponin T (Fig. 4D-F). This result suggests thpreexisting myofibrils or their precursor, myofilament bundleas well as nonsarcomeric actin cytoskeleton disassemble reorganize this structure at the cleavage furrow of the myotu

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It remains to be determined whether this structure consisof these cytoskeletal proteins is equivalent to the contracring, yet some structure should be required for the myotuunder cytokinesis to generate the cleavage force.

Induction of apoptosis in the myotubesA subset of myoblasts suffer apoptosis in the differentiatimedium with a low mitogen concentration, whereas terminadifferentiated myotubes are resistant to apoptosis under condition (Endo, 1996; Wang and Walsh, 1996). C2SV

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myotubes about 34 hours after the large T induction exhibitthree types of figures: (1) above-mentioned mitotic figures; degenerative cells with deformed giant and pulverized nuc(Endo and Nadal-Ginard, 1989; Iujvidin et al., 1990); and (atrophic cells containing condensed and sometimes fragmennuclei. Since the third type of myotubes appeared to be relato cells undergoing apoptosis (Wyllie et al., 1980), thinduction of apoptosis was analyzed by using the TUNEmethod (Gavrieli et al., 1992) to measure the TdT-mediatfluorescein-dUTP incorporation into the nuclei. Fluorescei


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Fig. 2. Induction of mitosis and cytokinesis in C2SVT myotubes. Myoblasts were cultured in the growth medium for 24 hours (A and B).Myotubes were formed by maintaining in the differentiation medium at 37°C for 96 hours (C and D). Preformed myotubes were cultured in theZn2+-containing growth medium at 33°C for 34 hours (E-P). Cells were doubly stained with the mAb D2D6 to α-tubulin (A,C,E,G,I,K,M,O)and bisbenzimide H33258 (B,D,F,H,J,L,N,P). (A and B) Myoblasts at prophase and interphase. The centrosome (A) and condensedchromosomes (B) are evident in the prophase cell (center) but not in interphase cells. (C and D) A quiescent myotube, in which centrosomes arenot apparent. (E and F) A myotube at prophase. The centrosomes (E) and condensed chromosomes (F) are visible. (G and H) A myotube atmetaphase. A mitotic spindle contains chromosomes aligned at the metaphase plate. (I and J) A myotube at anaphase. Chromosomes aremigrating toward the spindle poles. (K and L) A myotube containing a giant spindle at metaphase. (M and N) A myotube containing a giantspindle at prometaphase. (O and P) A myotube during cytokinesis. The midbody is formed between the daughter myotubes. Bar, 20 µm.

dUTP was incorporated only in condensed and fragmenapoptotic nuclei of undifferentiated myoblasts as well as fibroblasts, and the labeling pattern was similar to th

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produced by H33258 (data not shown). The nuclei of tatrophic myotubes expressing large T were clearly labeledfluorescein-dUTP (Fig. 5). These myotubes are therefore m

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Fig. 3.Mitosis and cytokinesis inC2SVT myotubes. Preformedmyotubes were cultured in theZn2+-containing growth mediumat 33°C for 34 hours. They weredoubly stained with D2D6 (greenfluorescence) and H33258 (bluefluorescence). (A) A myotube atprophase. (B) A myotube atmetaphase. Shown is a singlespindle. (C) A myotubecontaining giant spindles.(D-F) Myotubes undercytokinesis. Cleavage took placeat the middle (D) or theperiphery (E and F) of themyotubes. A phase-contrastmicrograph (E) corresponding toa fluorescent micrograph (F)evidently shows that cytokinesisoccurred within a myotube. Bar,20 µm.

likely to be subjected to apoptosis. The nuclei in some of thmyotubes were labeled relatively evenly (Fig. 5C), whereas more condensed or fragmented nuclei in the other myotuwere peripherally labeled (Fig. 5G). The nuclear labelinpattern in the latter myotubes was also similar to that produby H33258 and near-complementary to the location of large(Fig. 5F-H). In addition, this labeling pattern was typical oapoptotic nuclei, which contain condensed chromatin at thperiphery (Wyllie et al., 1980). Apoptosis seems to haprogressed more in the latter myotubes than in the formFluorescein-dUTP incorporation was scarcely detected C2SVT myotubes cultured in growth medium without a Zn2+

supplement or in C2C12 myotubes cultured in Zn2+-containinggrowth medium (data not shown). Thus, large T is likely to responsible for the induction of apoptosis in the myotubes.

To determine whether these apoptotic myotubes were in quiescent state or whether they also reentered the cell cythe preformed myotubes were shifted to the Zn2+-containinggrowth medium supplemented with BrdU. The fluoresceidUTP-labeled nuclei incorporated BrdU into many of thapoptotic myotubes (Fig. 6A-C). Among fluorescein-dUTPlabeled nuclei in several single myotubes, however, only sobut not the others incorporated BrdU (Fig. 6D-F). The ra

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between BrdU-labeled and -unlabeled nuclei amongfluorescein-dUTP-labeled myotube nuclei was 83:17. Thecentrosomes, mitotic spindles, or midbodies were not detectein the apoptotic myotubes. These results imply that apoptosis induced in the nuclei between S and G2 phases as well as inthose prior to S phase in the myotubes under this condition.

The deformed giant and pulverized nuclei in the second typof myotubes also expressed large T but did not incorporatfluorescein-dUTP (Fig. 7). Thus, these myotubes were noundergoing apoptosis. Some myotubes contained both thmitotic figures and the deformed nuclei in discrete regions ina single cell. Table 1 summarizes the ratio of each type omyotubes.

Internucleosomal cleavage of chromatin DNA, which isdetected as a ladder of multiplication nucleosomal unit (180200 bp) on agarose gel electrophoresis, has widely beeregarded as a biochemical feature of apoptosis (Arends anWyllie, 1991). Under the differentiation conditions,internucleosomal fragmentation of chromatin DNA graduallyoccurred in C2SVT cells (Fig. 8) as has been detected in thparental C2C12 cells (Endo, 1996; Wang and Walsh, 1996). Ithe large T-expressing myotubes, however, the chromatin DNfragmentation was hardly detected even 34 hours after the lar


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Fig. 4.Accumulation of actin filamentsand associated proteins at the cleavagefurrows of C2SVT myotubes. Myoblastswere cultured in the growth medium for24 hours (A and B). Preformed myotubeswere cultured in the Zn2+-containinggrowth medium at 33°C for 34 hours (C-F). (A and B) Myoblasts duringcytokinesis. They were stained withrhodamine-phalloidin. (C-F) Myotubesunder cytokinesis. They were doublystained with rhodamine-phalloidin (C) andanti-chicken gizzard (nonsarcomeric)tropomyosin (D). The cells were alsostained with MF20 to sarcomeric MHC(E) or the mAb NT302 to troponin T (F).Bar, 20 µm.

T induction, when the apoptotic figures were evident (Fig. This is presumably due to the presence of Zn2+ in the mediumto activate the metallothionein promoter because Zn2+ inhibitsthe endonuclease responsible for the internucleosomal Dcleavage but not apoptosis (Cohen and Duke, 1984; Coheal., 1992). When C2SVT cells were cultured in the Zn2+-containing differentiation medium at the nonpermissitemperature (40°C), apoptotic cell nuclei were labeled by

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Table 1. The ratios of various types of C2SVT myotubesexpressing SV40 large T antigen

Given type of myotubes/Myotube type total myotubes Percentage

Interphase 46/163 28.2Mitosis/cytokinesis 61/163 37.4Degeneration with deformed 49/163 30.1

nucleiApoptosis 19/163 11.7

C2SVT myotubes cultured at 33°C for 34 hours in growth mediumcontaining Zn2+ were triply labeled with PAb101, fluorescein-dUTP, andH33258. Since only adhering myotubes were counted, detached cells dueapoptosis and so forth were not included. When a single myotube containboth the mitotic figures and the deformed nuclei, the myotube was countefor both the types. Consequently, the sum of each type of myotubes is larthan the number of counted myotubes.

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TUNEL method, but internucleosomal DNA cleavage was nodetected (data not shown). Actually, it is not theinternucleosomal cleavage but the preceding cleavage chromatin DNA into 50-300 kb large fragments, which are alslabeled by the TUNEL method, that is essential for apoptos(Brown et al., 1993; Oberhammer et al., 1993; Walker et a1994).

DISCUSSION

Terminally differentiated myotubes have been shown to remacompetent to replicate chromosomal DNA and exhibcondensed chromosomes (Endo and Nadal-Ginard, 198Iujvidin et al., 1990; Endo and Goto, 1992; Ohkubo et al1994; Schneider et al., 1994; Crescenzi et al., 1995; Novitet al., 1996). In the present study, we revealed that tmyotubes with the expression of SV40 large T antigecompleted canonical mitosis and cytokinesis. The mitosis wrespresented by the appearance of centrosomes in the vicinof nuclei containing condensed chromosomes and mitotspindles harboring the condensed chromosomes. Cleavagethe myotubes and the formation of midbodies as well as taccumulation of actin filaments and associated proteins at tcleavage furrows indicated that cytokinesis occurred aftmitosis. The cytokinesis gave rise to daughter cells containin

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Fig. 5. Induction of apoptosisin C2SVT myotubes detectedby the TUNEL method.Preformed myotubes werecultured in the Zn2+-containinggrowth medium at 33°C for 34hours. The cells were triplystained with PAb101 (B and F),fluorescein-dUTP by theTUNEL method (C and G),and H33258 (D and H).(A-D) An apoptotic myotube atan earlier stage. (E-H) Anapoptotic myotubes at a laterstage. Phase-contrastmicrographs (A and E) andcorresponding fluorescentmicrographs (B-D and F-H).Bar, 20 µm.

not only multiple nuclei but one or a few nuclei. These resuimply that even terminally differentiated myotubes retaincomplete set of machinery to accomplish at least one rounthe cell cycle if the cells are boosted by large T.

Microsurgical removal of centrosomes including centrioldoes not impede DNA replication and cytoplasmic growth bprevents centriole regeneration and cell reproduction in simian kidney cells (Maniotis and Schliwa, 1991). Thobservation demonstrates that centrosomes are essentiacell reproduction through mitosis and cytokinesis. Thu

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although centrosomes were invisible in the quiescent C2SVmyotubes, as in human myotubes (Tassin et al., 1985; Connoet al., 1986), regeneration of visible centrosomes in the larT-expressing myotubes seems to be required for progressmitosis and cytokinesis. This notion is corroborated by thevidence that the Cdc2-cyclin B complex, which playessential roles in inducing the major events in M phase (Nurs1990; Lamb et al., 1990; Dessev et al., 1991; Lüscher et a1991), is associated with centrosomes as well as the mitoapparatus (Bailly et al., 1989; Riabowol et al., 1989; Rattner


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Fig. 6.Determination of thecell cycle phase of apoptoticmyotube nuclei. Preformedmyotubes were cultured in theZn2+-containing growthmedium supplemented withBrdU at 33°C for 34 hours.They were doubly labeled withfluorescein-dUTP by theTUNEL method (B and E) andthe mAb BU-1 to BrdU (C andF). (A-C) An apoptoticmyotube, in which all thenuclei incorporated BrdU. (D-F) An apoptotic myotube, inwhich a part of the nuclei werelabeled by BrdU. Only threenuclei on the upper right werelabeled (F). Phase-contrastmicrographs (A and D) andcorresponding fluorescentmicrographs (B,C and E,F).Bar, 20 µm.

al., 1990; Pines and Hunter, 1991). In fact, not only tamounts of Cdc2 and cyclin B but also the histone H1 kinaactivity ascribable to the Cdc2-cyclin B complex are reinducin the C2SVT myotubes in G2-M phase (Ohkubo et al., 1994)

Large T is believed to drive the cell cycle in quiescent cethrough inactivation of the tumor suppressor protein Rb binding to underphosphorylated Rb (Ludlow et al., 198Manfredi and Prives, 1994). During myogenic termindifferentiation, Rb protein is induced (Endo and Goto, 199and cooperates with the myogenic basic-helix-loop-hetranscription factor MyoD for cell cycle arrest and expressiof late differentiation markers (Gu et al., 1993; Novitch et a1996). Thus, myotubes lacking in Rb are able to reenter cell cycle and proceed to S and G2 phases by serum stimulation(Schneider et al., 1994; Novitch et al., 1996). These myotubhowever, fail to readily reinitiate mitosis. Activation of thCdc2-cyclin B complex by removing inhibitoryphosphorylation on Cdc2 with caffeine results in mitotcatastrophe in the Rb-deficient differentiated myocyt(Novitch et al., 1996). In the large T-expressing C2SVmyotubes, the expression of Rb and MyoD is extinguish(Endo, 1992; Endo and Goto, 1992) and the activity of Cdc

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cyclin B is markedly elevated (Ohkubo et al., 1994). Taketogether, these findings suggest that the induction of mitosisthe large T-expressing C2SVT myotubes is ascribed not onto the downregulation and inactivation of Rb but also treactivation of Cdc2-cyclin B.

Newt limb myotubes in culture easily proceed to S phase serum stimulation (Tanaka et al., 1997). Furthermore, thgenerate mononucleated cells when they are implanted unthe wound epidermis (Lo et al., 1993), which provides a modfor limb regeneration (Brockes, 1997). These myotubes alexpress Rb, but Rb is phosphorylated after serum stimulatiprobably via active Cdk-G1 cyclin complexes due to lack ofCdk inhibitors (CKIs) such as p16 and p21 (Tanaka et a1997). In contrast, Rb is not phosphorylated by serustimulation in mammalian myotubes (Gu et al., 1993), wheCKIs accumulate at high levels (Guan et al., 1994; Halevy al., 1995; Parker et al., 1995). This difference between the neand the mammlian myotubes is likely to determine whether not they are responsive to serum stimulation. However, tpresent and previous studies have shown that large expression can allow mammalian myotubes to complete DNreplication, mitosis, and cytokinesis. Although the fate of th

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Fig. 7.Absence of fluorescein-dUTP incorporation indegenerative myotubes withdeformed nuclei. Preformedmyotubes were cultured in theZn2+-containing growthmedium at 33°C for 34 hours.The cells were triply stainedwith PAb101 (B and F),fluorescein-dUTP by theTUNEL method (C and G),and H33258 (D and H). Phase-contrast micrographs (A and E)and corresponding fluorescentmicrographs (B-D and F-H).Bar, 20 µm.

divided daughter cells at later stages remains to be examinit is plausible that the mononucleated daughter cells furthproliferate and then differentiate to form myotubes undappropriate conditions. C2SVT cells may therefore providemammalian model system for the differentiationdedifferentiation cycle.

Under low mitogen conditions favorable for myogenidifferentiation, a considerable number of undifferentiatemyoblasts suffer apoptosis, whereas myotubes are refractorapoptosis (Endo, 1996; Wang and Walsh, 1996). Despite th

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facts, the incidence of apoptosis has been documented degenerating tadpole tail muscle (Nishikawa and Hayash1995) and dystrophic mouse skeletal muscle (Matsuda et a1995; Smith et al., 1995; Tidball et al., 1995). We showed hethat apoptosis was induced in a part of large T-expressinC2SVT myotubes, in which condensed chromatin was labeleby the TUNEL method. Thus, even myotubes in culture araccessible to apoptosis, when large T is expressed, aconsequently also preserve a complete set of machinery fapoptosis. The BrdU incorporation in combination with the


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Fig. 8. Internucleosomal cleavage of chromatin DNA in C2SVTcells. C2SVT myoblasts were cultured in the differentiation mediuat 37°C (Mb → Mt) for the times indicated. Preformed myotubeswere cultured to induce large T in the Zn2+-containing growthmedium at 33°C (Mt (T)) for the times indicated. Chromatin DNAwas prepared from each culture and subjected to 2% agarose geelectrophoresis. DNA was detected by ethidium bromide stainingsize marker.

TUNEL method indicated that the apoptotic nuclei in thmyotubes were not restricted to a particular phase of the cycle but that they were unlikely to be in M phase. This resis consistent with the previous notion that apoptosis in certnonmyogenic cells is induced in various phases of the ccycle (Cotter et al., 1992).

There have been contradictory observations with regardthe effects of large T on apoptosis. Some observations hindicated that large T antagonizes apoptosis in somammalian cells (Yanai and Obinata, 1994; McCarthy et a1994; Jung and Yuan, 1997). They postulate roles for largin the sequestering of p53, which is a potential inducer apoptosis (Yonish-Rouach et al., 1991; Levine, 1997), orinhibiting a certain caspase, a protease leading to apopt(Cohen, 1997). In contrast, other studies have shown that laT is responsible for apoptosis (Li et al., 1995; Allemand et a1995; Lenahan and Ozer, 1996). So far, we do not know wthese contradictory results have been obtained, yet difference in cell types or in expressed genes might responsible for the apparently reverse effects of large T apoptosis. Both mitosis/cytokinesis and apoptosis take placthe large T-expressing C2SVT myotubes. The large expression results in inactivation of Rb by its downregulatiophosphorylation, or binding of large T (Endo and Goto, 199Gu et al., 1993) and induction of c-Jun (Endo, 1992). Themay play essential roles in the induction of apoptosis as was of the cell cycle in the myotubes because Rb is an inhibof apoptosis (Morgenbesser et al., 1994; Haas-Kogan et 1995; Zacksenhaus et al., 1996) and c-Jun is a transcrip

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factor required for apoptosis (Colotta et al., 1992; BossyWetzel et al., 1997). This postulation is corroborated by thfindings that viral oncoproteins that bind to and inactivate Rbsuch as adenovirus E1A and human papillomavirus E7, alsinduce apoptosis (Debbas and White, 1993; Howes et al., 199Pan and Griep, 1994). Loss of cell-matrix contact, which iregulated through certain signal transduction pathways, leato apoptosis in anchorage-dependent cells (Frisch anRuoslahti, 1997; Meredith and Schwartz, 1997). Thus, loss oanchorage to the substrate in the large T-induced C2SVmyotubes by the signaling proteins might alternatively explainthe induction of apoptosis. Examination of the expressiolevels and interaction of these and other regulatory proteins fapoptosis and cell cycle progression in individual myotubes anecessary to elucidate the mechanisms of the large T-inducapoptosis and cell cycle in the myotubes. This might alscontribute to the unveiling not only of the mechanisms oapoptosis in dystrophic muscle but also of those of thdivergence to apoptosis and differentiation under low mitogeconditions.

We are grateful to Dr Takao Arai for the generous gift of the mAbD2D6 and to Dr Takashi Obinata for the mAb NT302. This study wapartly supported by research grants from the Ministry of EducationScience, Sports, and Culture of Japan and from the Ministry of Healand Walfare of Japan for Nervous and Mental Disorders (2A-1 an8A-1).

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