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© The Rockefeller University Press,
0021-9525/1997//155 $5.00
The Journal of Cell Biology, Volume 136, Number 1,
, 1997 155-165
Article |
Newt Myotubes Reenter the Cell Cycle by Phosphorylation of the Retinoblastoma Protein
Department of Biochemistry and Molecular Biology, University College London, London W1P 8BT, United Kingdom; and Division of Biological Sciences, Lancaster University, Lancaster LA1 4YQ, United Kingdom
Withdrawal from the cell cycle is an essential aspect of vertebrate muscle differentiation and requires the retinoblastoma (Rb) protein that inhibits expression of genes needed for cell cycle entry. It was shown recently that cultured myotubes derived from the Rb–/–mouse reenter the cell cycle after serum stimulation (Schneider, J.W., W. Gu, L. Zhu, V. Mahdavi, and B. Nadal-Ginard. 1994. Science (Wash. DC). 264:1467– 1471). In contrast with other vertebrates, adult urodele amphibians such as the newt can regenerate their limbs, a process involving cell cycle reentry and local reversal of differentiation. Here we show that myotubes formed in culture from newt limb cells are refractory to several growth factors, but they undergo S phase after serum stimulation and accumulate 4N nuclei. This response to serum is inhibited by contact with mononucleate cells. Despite the phenotypic parallel with Rb–/– mouse myotubes, Rb is expressed in the newt myotubes, and its phosphorylation via cyclin-dependent kinase 4/6 is required for cell cycle reentry. Thus, the postmitotic arrest of urodele myotubes, although intact in certain respects, can be undermined by a pathway that is inactive in other vertebrates. This may be important for the regenerative ability of these animals.
Abbreviations used in this paper: BrdU, bromodeoxyuridine; CDI, cyclin-dependent kinase inhibitor; CDK, cyclin-dependent kinase; CIP, calf intestinal phosphatase; GST, glutathione-S-transferase; Rb, retinoblastoma.
Terminal differentiation is associated with stable withdrawal from the cell cycle. In mammalian systems, the return of mature, differentiated tissue to an undifferentiated proliferating state is blocked since, in many tissues, a return to the cell cycle could in principle result in tumor formation or cell death. In contrast, in urodele amphibians such as the adult newt, reversal of differentiation is an integral part of their ability to regenerate limbs and other structures (Brockes, 1994; Okada, 1991). After amputation, epidermal cells from around the wound surface migrate across it to form the wound epidermis. The mesenchymal tissues beneath the wound epidermis dedifferentiate to produce blastemal cells, the proliferating and undifferentiated cells that are the progenitors of the new limb (Steen, 1968; Hay, 1959; Kintner and Brockes, 1984; Casimir et al., 1988). The capacity of newt myotubes to dedifferentiate was demonstrated directly by purifying myotubes formed from cultured newt limb cells, labeling them by injection of a lineage tracer, and implanting them beneath the wound epidermis of an early blastema (Lo et al., 1993). 1–3 wk after implantation, labeled mononucleate cells were found in the blastema, and their number increased with time, indicating that the cells were proliferating. This experiment suggests that the local environment of the blastema stimulates newt myotubes to reenter the cell cycle and to reverse their differentiated state, thus raising a number of issues concerning the identity of the signals that stimulate dedifferentiation, as well as the underlying mechanisms that allow newt cells but not their mammalian counterparts to undergo this process.
Muscle has been a particularly informative system for studying how cells maintain the nondividing, differentiated state (Lassar et al., 1994; Olson, 1992). During differentiation, myoblasts exit from the cell cycle in the G1 phase and fuse to form a multinucleate syncitium that expresses muscle-specific proteins and no longer responds to mitogens. It has been shown that this insensitivity is not caused solely by the down-regulation of cell surface receptors, nor by an irreversible alteration in the capacity of the nucleus to undergo DNA synthesis. The addition of mitogens such as EGF after cell cycle arrest but before receptor down-regulation provokes various intracellular responses, but it does not stimulate cell division (Endo and Nadal-Ginard, 1986; Olwin and Hauschka, 1988; Hu and Olsen, 1990). On the other hand, if myotubes are transfected with transforming viral proteins such as SV-40 large T antigen or adenovirus E1A protein, the myotube nuclei are induced to enter S phase (Endo and Nadal-Ginard, 1989; Iujvidin et al., 1990; Crescenzi et al., 1995).
These experiments with viral oncogenes suggest that the retinoblastoma (Rb)1 protein might have a critical role in maintaining the postmitotic state because mutants of T antigen that are unable to bind Rb do not promote cell cycle reentry (Gu et al., 1993). This role of Rb has recently been demonstrated directly: myoblast cells derived from the Rb homozygous null (Rb–/–) mouse form myotubes that express muscle-specific proteins, but they reenter S phase in response to serum (Schneider et al., 1994). The Rb protein is a regulator of the G1-S restriction point of the cell cycle and acts through the E2F family of transcription factors that control the expression of several genes whose products are required for entry into S phase (Nevins, 1992; LaThangue, 1994; Riley et al., 1995; Weinberg, 1995). Current models indicate that Rb inhibits entry into S phase at least in part by binding E2F and inhibiting transcriptional activation. When cells sensitive to mitogen stimulation are treated with serum, Rb is phosphorylated, thus losing its ability to bind E2F and allowing activation of S-phase entry genes.
Mounting evidence suggests that there is a serum-responsive pathway dedicated to the regulation of Rb phosphorylation in which the kinase complex cyclin-dependent kinase (CDK) 4/6–cyclin D is the mitogen-responsive kinase of Rb (Sherr, 1994; Hunter and Pines, 1994). This kinase activity is regulated in part by the proteins of the INK4 class of cyclin-dependent kinase inhibitors (CDIs) (Sherr and Roberts, 1995), which specifically bind to and inhibit CDK4 and 6 (Serrano et al., 1993; Hannon and Beach, 1994; Sheaff and Roberts, 1995; Koh et al., 1995). p16INK4 can only inhibit entry to S phase or colony formation in cells that express Rb, while inhibition of cyclin D1 is effective in blocking S-phase entry only in cells containing Rb, thus establishing a functional link between CDK4 and Rb in cell cycle control (Guan et al., 1994; Medema et al., 1995; Lukas et al., 1994, 1995). In normal muscle, the CDIs p21 and the p16INK4-related protein p18 are highly expressed (Parker et al., 1995; Guan et al., 1994), with p21 being induced by MyoD expression (Halevy et al., 1995; Guo et al., 1995). These high levels of CDIs presumably inhibit the mitogeninduced phosphorylation of Rb. In view of the plasticity of newt cells, a possible difference between urodele and other vertebrate cells may lie in the regulation of Rb function.
We are attempting to induce myotube dedifferentiation in cultured newt cells where it may be possible to analyze the underlying mechanism. This should provide insight into the process of regeneration and also shed light on how the differentiated state is regulated. Here we report that in contrast with those of other vertebrates, cultured newt myotubes formed from myogenic newt cells enter and traverse S phase when stimulated with serum. In comparison to the behavior of mammalian Rb–/– myotubes (Schneider et al., 1994), an attractive explanation for the response of newt myotubes is that they lack Rb, but we show that these cells express Rb and that phosphorylation of Rb is an endpoint of the serum stimulation pathway. These results suggest that the regulation of Rb phosphorylation is likely to be a key difference that distinguishes the behavior of newt and mammalian myotubes.
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Bromodeoxyuridine Labeling and Immunofluorescence
To induce DNA synthesis, serum was raised to 10–20% at 1–2 d after purified myotubes were plated. After 4 d in serum, myotubes were labeled for 24 h with 1 µl/ml 5-bromo-2 deoxyuridine/5-fluoro-2-deoxyuridine using the proliferation kit (Amersham Corp., Arlington Heights, IL) according to instructions. Cells were fixed for 5 min with methanol, stained for bromodeoxyuridine (BrdU) as previously described (Barres et al., 1994), and then stained for muscle-specific myosin using mAb against muscle-specific myosin heavy chain (A4.1025), a kind gift of Dr. Simon Hughes (Randall Institute, Kings College London, UK). FITC rabbit anti–mouse antibody was used as a secondary antibody, and all antibodies were diluted in 10% goat serum in PBS. For [3H]thymidine labeling, cultures were exposed to 1 µCi of [3H]thymidine per ml for 24 h, fixed with methanol, processed for antibody staining, refixed with methanol, and then coated with photographic emulsion (1:1, K-5; Ilford, Knutsford, Cheshire, UK) and developed after 2 d.
Determination of DNA Content
A more detailed account of the quantitative microscopy will be published elsewhere. In brief, cells were fixed with methanol, washed with PBS, treated with 2 N HCl for 5 min, washed with PBS, and stained with 10 µM propidium iodide for 30 min. Propidium iodide was also included in the mounting medium. The laser-scanning microscope (Biorad MRC-500; Zeiss, Oberkochen, Germany) was used with a x5 objective, with the diaphragm fully open, and aligned for a maximally even field of illumination. Image analysis routines were used to threshold nuclei and to eliminate all background so that the intensity of individual nuclei could be measured in a semi-automated fashion. G1-, G2-, and S-phase peaks were determined by using mononucleate cells that had been labeled for 2 h with BrdU. Nuclei with no BrdU labeling clustered around two peaks: a large peak representing G1 cells with 2N DNA, and a small peak, twice as intense as the first peak, representing G2 cells with 4N DNA. BrdU+ nuclei displayed intensities between these peaks. 4N DNA was also confirmed by measuring the intensity of mitotic cells.
Growth Factor Assay
Myotubes were purified and plated in low serum (0.5%) media. Purified growth factors were added after 24 h, and cells were labeled 4 d later with BrdU for 24 h. Assays were performed in triplicate and at least 300 nuclei were counted per well. Mononucleate cells were plated at low density in low serum (1%) media for 24 h, before growth factor addition, and BrdU uptake was assayed 4 d later. Assays were performed in triplicate and at least 500 cells were counted per well. The growth factors were a kind gift of Dr. Mark Noble (Huntsman Cancer Institute, Salt Lake City, UT). Serum was delipidated as described (Rothblat et al., 1976).
Contact Inhibition Experiments
To form myotubes at varying densities, 
1.3 x 105, 2 x 105, or 4 x 105 A1 cells were plated onto 6-cm-diam tissue-culture dishes. After 24 h, myogenesis was induced by placing the cells in low serum media. After 5 d, serum was added to 20%, and then BrdU uptake and staining were assayed as described above.
To vary the number of mononucleate cells, myotubes were purified in low serum media as described above and plated onto 3 x 3-cm-diam tissue-culture dishes. After 24 h, mononucleate A1 cells ranging in number from 3 x 104 to 26 x 104 were plated onto the plates in low serum media. After 48 h, serum was added to 20%, and then BrdU uptake and staining were assayed as described above.
Tilt Experiment.
Myotubes were purified in low serum media as described above and plated onto two 6-cm-diam tissue-culture dishes. After 24 h, 2.5 x 105 A1 cells were added, and the plates were tilted to allow cells to settle in a gradient of densities on the plate. After 48 h, serum was added to 20%, and then BrdU uptake and staining were assayed as described above.
Rb Cloning and RNase Protection
Degenerate oligonucleotides to the E1A binding region of Rb were used in a PCR reaction to obtain a 570-bp fragment from a 
ZAP retinoic acid– treated newt limb blastema cDNA library. This was used to probe a gt11 limb blastema cDNA library (Brown and Brockes, 1991) from which two positive clones were isolated and sequenced. Both contained the 3' stop codon but lacked 600 bp of 5' sequence, which was obtained from the ZAP library by PCR using antisense oligonucleotides to sequences from within the gt11 clones together with ZAP primers. The amplified fragment was cloned in pBSK (Stratagene, La Jolla, CA). Total RNA was prepared and RNase protection was performed essentially as described (Brown and Brockes, 1991). Total RNA (10 µg) was annealed with a 330bp probe from the E1A binding region to give the predicted 280-bp protected fragment. The gel was exposed for 2–3 d at –70°C.
Generation of Affinity-purified Polyclonal Antibody SK70
A glutathione-S-transferase (GST) fusion protein containing the newt Rb sequence from amino acids 269–447 was produced in bacteria, solubilized using sarcosyl (Frangioni and Neel, 1993), and purified using glutathione– Sepharose (Pharmacia, Uppsala, Sweden) followed by thrombin cleavage. Antibodies against the purified protein were produced in rabbits by Eurogentec (Seraing, Belgium). For affinity purification, IgG was precipitated from serum with ammonium sulfate and passed through a GST-myf-5 affinity column to remove anti-GST antibodies, then passed over an Affigel matrix coupled to the newt Rb fragment, and subsequently washed and eluted with glycine/HCl.
Immunoprecipitation and Western Blotting
Immunoprecipitation was performed essentially as described in Hu et al. (1991). Briefly, in experiments with mononucleate cells, 800,000 cells were rinsed with PBS and lysed for 30 min on ice with 2 ml of lysis buffer. Cellular debris was removed by centrifugation for 1 min at 10,000 g. In initial experiments, extracts were precleared with rabbit serum and fixed Staphylococcus aureus as described (Harlow and Lane, 1988), but this was later found to be unnecessary. For immunoprecipitation with rabbit antibody, 1 ml of lysate was incubated with 1 µl SK70 on ice for 1 h, and then incubated 1 h with 10 µl protein A–Sepharose beads (Pharmacia). For immunoprecipitation with XZ56 (Hu et al., 1991), 1 ml of lysate was incubated for 2 h with 10 µl protein A–Sepharose beads that had been crosslinked with XZ56 antibody, using dimethyl pimelimidate as described in Harlow and Lane (1988). Phosphatase treatment of immunoprecipitates was essentially as described in Ludlow et al. (1989). Immunoprecipitated beads were washed twice with wash buffer (155 mM NaCl, 20 mM Tris, pH 8, 5 mM EDTA, 0.05% NP-40, 0.02% NaN3), and then washed once with CIP buffer (100 mM Tris, pH 8, 100 mM NaCl, 5 mM MgCl2). Beads were incubated with 40 U calf intestinal phosphatase (Boehringer Mannheim Biochemicals, Indianapolis, IN) per 60 µl buffer plus 2 mM PMSF, 20 µg/ml aprotinin, and 10 µg/ml leupeptin with or without phosphatase inhibitors (15 mM NaF, 150 µM sodium orthovanadate) for 15 min at 37°C, and then washed twice in wash buffer and solubilized in Laemmli sample buffer for 10 min at 80°C.
For detection of Rb in myotubes, myotubes were purified from 17 x 100-mm dishes plated onto two 100-mm dishes and kept in 0.5% serum (low serum) or 20% serum (high serum) for 4 d. One 100-mm plate of purified myotubes that contained 50,000 myotube nuclei, with <10% mononucleate contamination, was lysed in 1 ml buffer and centrifuged as above. Samples were incubated with 1 µl SK70 for 1 h, then with 10 µl XZ56 for 1 h with rocking, and further with 10 µl protein A–Sepharose for 1 h. The samples were then washed three times with wash buffer before solubilization in Laemmli sample buffer for 10 min at 80°C. Samples were resolved on 7% SDS polyacrylamide gels, Western blotted onto nitrocellulose, blocked in 1% dried milk in TBS/0.1% Triton X-100 for 30 min, and then probed with SK70 at a 1:200 dilution followed by HRP-conjugated donkey anti–rabbit secondary antibody (Amersham Corp.) and enhanced chemiluminescence (ECL; Amersham Corp.).
34 and p16 Expression
The p16 expression plasmid (pcDNA3WTp16) was a kind gift of Drs. David Parry and Gordon Peters (Imperial Cancer Research Fund, London, UK). For p16 and control microinjections, plasmid (0.2, 0.6, or 1.0 µg/ml) was microinjected into myotube nuclei using an Eppendorf microinjection apparatus at 24 h after purified myotubes were plated in low serum media. Microinjection into a single nucleus per myotube was sufficient to generate expression of protein that spread throughout the cell cytoplasm. Myotubes were transferred to medium with 20% serum 24 h after injection, and BrdU uptake was assayed after 4 d as described above. The p16 protein was detected using mAb DCS-50.2 (a kind gift of Dr. Gordon Peters) at 2.6 µg/ml. In parallel control experiments, 0.2 or 1 mg/ ml alkaline phosphatase expression plasmid (pCAP) (Schilthuis et al., 1993) was microinjected and BrdU uptake was assayed as above. Alkaline phosphatase was visualized with 5-bromo-4-chloro-3-indoyl-phosphate, and BrdU was detected using an HRP-conjugated secondary antibody.
The 34 expression plasmid was a kind gift of Dr. N. Jones (Imperial Cancer Research Fund, London, UK), and Rb plasmid pJ3WHRbc was a kind gift of Dr. P. Jat (Ludwig Institute, London, UK). The DNA was microinjected at 0.5 mg/ml at 48 h after plating, and cells were placed in high serum media after an additional 48 h. [3H]Thymidine uptake was assayed as described above. This protocol, which uses longer times for plating and expression as compared with the p16 experiments, was adopted after the p16 experiments to optimize the number of myotubes obtained. Newt myotubes frequently require 48 h to attach fully, so this latter protocol allowed more cells to be injected. Rb protein expression was detected using mAb G3-245 (Pharmingen, San Diego, CA).
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10% serum (Fig. 1 C). In this experiment, 25% of nuclei incorporated BrdU in a 24-h period on day 3, but overall at least 75% of the cells undergo DNA synthesis after stimulation. Such a response was not found in mouse myotubes. Myotubes formed from the myoblast cell line, C2C12, were purified in a similar way, and BrdU incorporation was assayed up to 72 h after serum stimulation. Cell cycle reentry was not seen in these myotubes even after examination of several thousand nuclei.
To eliminate the possibility that the newt myotubes undergo an aberrant DNA synthesis or activate DNA repair, we determined if the nuclei traverse an S phase of normal duration by executing the protocol outlined in Fig. 2 A. Multiple plates of myotubes were labeled with an 8-h pulse of [3H]thymidine 2 d after serum addition, thus identifying those myotubes that had begun to synthesise DNA at this point. At varying times after the exposure to [3H]thymidine, individual plates were labeled with BrdU for 24 h and then fixed and processed for immunohistochemistry and autoradiography. Nuclei that were [3H]thymidine+ and BrdU+ were those that had started DNA synthesis on day 2 and were still undergoing DNA synthesis at the time of the BrdU labeling. Nuclei that were [3H]thymidine+ but BrdU– were those that had started DNA synthesis on day 2, but had completed it before the second labeling. As seen in Fig. 2 B, of the cells given the BrdU pulse immediately after the [3H]thymidine, almost all [3H]thymidine+ nuclei were also BrdU+, but of those labeled with BrdU 2 d after the [3H]thymidine pulse (4 d after serum), the number of [3H]thymidine+BrdU+ cells had decreased, and it was only 1% by 4 d after the [3H]thymidine pulse (6 d after serum). This indicates that S phase lasts 48–72 h in the myotube nuclei, which is similar to the length of S phase in mononucleate newt cells (Wallace and Maden, 1976). Significantly, this experiment also shows that the total number of [3H]thymidine+ cells present remained relatively constant throughout the experiment, demonstrating that there was little cell death as a consequence of reentry.
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Newt Myotubes Are Refractory to Several Common Polypeptide Growth Factors
Myoblasts divide in response to mitogens such as bFGF and EGF, and thus it was important to determine if the return of newt myotubes to the cell cycle was due to the retention of sensitivity to such growth factors. A number of polypeptide growth factors including PDGF, EGF, IGF, and FGF were tested (Table I), and although they each stimulated division of mononucleate newt A1 cells, none elicited cell cycle reentry of the newt myotubes. These factors were also assayed in the presence of 5% bovine plasma and no response was observed. It is thus apparent that the urodele cells are as refractory to these growth factors as their mammalian counterparts (Olson, 1992).
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4 and 6 kb in normal limb tissue (not shown). As discussed further below, immunological detection of Rb indicated that the protein is expressed in the myotubes (Fig. 5 B). It is clear, therefore, that the myotubes are not Rb negative.
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34 Rb mutant (Hamel et al., 1992), in which all eight of the CDK consensus phosphorylation sites have been mutated, blocked entry into S phase. This mutant should compete with endogenous Rb and therefore inhibit any events that require Rb phosphorylation. Myotubes maintained in low serum media were transfected by microinjection with a plasmid encoding either the 34 Rb or wild-type Rb as a control. 2 d after microinjection, myotubes were transferred to high serum, and, after 4 d, myotubes were assayed for [3H]thymidine incorporation, with Rb expression detected by immunofluorescence using mAb G3-245. As seen in Table IV, expression of 34 Rb resulted in 88% inhibition of DNA synthesis as compared with only 30% inhibition by wild-type Rb.
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To compare the phosphorylation state of Rb in myotubes maintained in low serum vs high serum, extracts from highly purified myotubes (where the mononucleate contamination was <10%) were immunoprecipitated with both SK70 and XZ56, to obtain all forms of Rb, and then detected by Western blotting with SK70. The Rb that was immunoprecipitated from extracts of myotubes maintained in low serum migrated as a single band at 110 kD, indicating that none of the Rb was phosphorylated (Fig. 5 B, lane 1). In contrast, 50% of Rb from myotubes maintained in high serum for 4 d had retarded mobility (Fig. 5 B, lane 2). At this time, 25–50% of myotubes are expected to be in S phase. This profile was identical to the profile of Rb that was immunoprecipitated from proliferating mononucleate cells on which the antibodies were characterized (Fig. 5 B, lane 3), and, as with the mononucleate cells, phosphatase treatment of the immunoprecipitate caused the Rb to migrate as a single band (data not shown). Detection of rabbit IgG confirmed that equivalent amounts of SK70 were immunoprecipitated in each of the samples (data not shown). Thus, like other vertebrate cells that undergo a transition from the G0 or resting state to a proliferating state, the Rb in myotubes maintained in low serum is solely in the nonphosphorylated form but becomes phosphorylated after stimulation by serum to enter S phase.
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Here we have shown that newt myotubes are different from their mammalian counterparts. In culture, newt limb cells withdraw from the cell cycle in low serum medium and form myotubes, which are refractory to growth factors, but respond to elevated serum by entering S phase. This response is striking because although it is known that nuclei within mammalian myotubes are capable of synthesizing DNA in response to the expression of certain viral oncogenes (Endo and Nadal-Ginard, 1989; Iujvidin et al., 1990; Crescenzi et al., 1995), deletion of the Rb gene (Schneider et al., 1994), or infection by the parasitic nematode Trichinella spiralis (Jasmer, 1993), this is the first demonstration in which multinucleate myofibers undergo S phase in response to serum stimulation without the need for direct manipulation of internal cellular components. Furthermore, it is significant that not only do these newt myotubes enter S phase in response to serum, they do so without apparent trauma or evoking apoptosis. This is in contrast with the degeneration seen when SV-40 large T induces reentry in mammalian myotubes (Endo and Nadal-Ginard, 1989; Iujvidin et al., 1990).
It seems likely that the reentry response of newt myotubes underlies their ability to dedifferentiate during regeneration. Histological examination of muscle fibers in early regenerating limbs labeled with [3H]thymidine has identified some fibers containing labeled nuclei (Hay, 1959). Although these experiments could not definitively distinguish between cells that had newly fused to form fibers as opposed to nuclei returning to the cell cycle within fibers, they are consistent with entry into S phase being a very early step in endogenous dedifferentiation.
If induction of S phase is the initial step in muscle dedifferentiation during limb regeneration, then, in vivo, these muscle cells must go on to resolve into mononucleate cells. Indeed, Lo et al. (1993) showed that the same myotubes as those used here do form mononucleate cells when implanted into the regenerating limb. The execution of mitosis with attendant cytokinesis seems a likely means by which the nuclei in the syncytial myotube could resolve into individual cells, although other mechanisms that do not rely on reentering the cell cycle are possible. It is most likely that in culture an additional signal is required to overcome the G2-M arrest, and this is not present in FCS. It is also possible that a separate signal inducing further reversal of differentiation is required before the G2-M block can be relieved. A third possibility is that the configuration that the myotubes assume in culture, in which the nuclei cluster in the center rather than disperse along the fiber, is not conducive to resolution into mononucleate cells.
What is the molecular basis of plasticity in the newt myotubes? Experiments in mammalian myotubes where elimination of Rb function, either by genomic deletion or inactivation by viral proteins, results in S-phase entry by myotube nuclei indicate that differences in regulation of Rb may underlie the ability of newt myotubes to retain a serum response after differentiation. In normal cycling cells, phosphorylation of Rb appears to be the predominant means of inactivating Rb function at the G1 to S transition. In differentiated cells such as mammalian muscle, cell cycle arrest is not relieved by serum stimulation presumably because the kinases such as CDK4 that normally phosphorylate Rb in response to mitogens are inhibited from doing so by CDIs present at high levels in these cells (Guan et al., 1994; Parker et al., 1995). In Rb–/– mouse myoblasts, the Rb-like protein p107 appears to substitute for Rb to allow exit from the cell cycle and differentiation, but, unlike Rb, expression of p107 in the myotubes is downregulated in response to serum, thus bypassing the need for phosphorylation (Schneider et al., 1994). Alternatively, when viral oncogenes are expressed in a myotube, they bind and sequester Rb (Gu et al., 1993), thus driving the cells into S phase.
The situation in the newt cells is rather different. We have shown that newt myotubes express Rb, which is phosphorylated upon cell cycle reentry. Furthermore, expression of the CDI p16 as well as expression of a mutant Rb lacking all CDK consensus phosphorylation sites inhibits the serum response, indicating that phosphorylation of Rb is a critical step in cell cycle reentry in these cells. The dramatic inhibitory effect of p16 expression is consistent with previous work indicating that high levels of p16 effectively inhibit the ability of cyclin D to bind cdk4 and cdk6 both in vivo and in vitro (Xiong et al., 1993; Parry et al., 1995). In this case, the complete inhibition of kinase should result in none of the endogenous Rb becoming phosphorylated by CDK4/6. In contrast, when the 34 Rb mutant is expressed, the endogenous Rb is presumably still subjected to mitogen-stimulated phosphorylation. This competition between the mutant and the endogenous Rb for binding to factors such as E2F may explain the incomplete inhibition seen in the 34 Rb expression experiments.
It appears that in newt myotubes serum stimulation induces S phase through activation of a cdk4/6-cyclin D or related kinase that phosphorylates Rb. The simplest model would posit that p16 or other CDIs are either not produced in the urodele cells, or are downregulated or inactivated by the serum pathway. Of course, we do not know if the effect is direct, or if other factors might be involved between serum stimulation and Rb phosphorylation. The newt myotubes illustrate nonetheless that irreversible inactivation of the Rb phosphorylation pathway is not a prerequisite of muscle differentiation.
It will be of interest to identify the extracellular factors that regulate S-phase entry in the newt myotubes. The present results distinguish two classes of factors—a stimulatory soluble activity that is present in serum, and a dominant inhibitory factor on the surface of other cells that results in contact inhibition. These two contrasting activities may explain how specificity of the response is achieved in vivo. In general, newt muscle must be as firmly locked in the differentiated state as its mammalian counterpart, with dedifferentiation only being triggered under the special circumstances of amputation. In this context, it is perhaps not surprising that the reentry response is not stimulated by standard growth factors and that this aspect of the postmitotic arrest is intact. It is an attractive hypothesis that amputation relieves the inhibition of cell–cell contact, thus allowing muscle fibers and other differentiated cells at the amputation plane to respond locally to soluble serum factors.
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Acknowledgments |
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34 plasmid; and P. Jat for the human Rb plasmid. We also thank A. Akpan and D. Stark for help, David Drechsel for advice, and P. Jat and C. Hill for helpful comments on the manuscript.
Submitted: 22 April 1996
Revised: 18 September 1996
E.M. Tanaka was supported by fellowships from the Muscular Dystrophy Association, Inc. and The Helen Hay Whitney Foundation.
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