* Ludwig Institute for Cancer Research and 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.
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 Muscle has been a particularly informative system for
studying how cells maintain the nondividing, differentiated state (Lassar et al., 1994 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 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 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 Cell Culture
Newt A1 cells were propagated as described (Ferretti and Brockes, 1988 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 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 ×5 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 × 105, 2 × 105, or 4 × 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 × 3-cm-diam tissue-culture dishes. After 24 h, mononucleate A1 cells ranging in number
from ~3 × 104 to 26 × 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 × 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 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 Immunoprecipitation and Western Blotting
Immunoprecipitation was performed essentially as described in Hu et al.
(1991) For detection of Rb in myotubes, myotubes were purified from 17 × 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.).
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 The Cultured Newt Myotubes Undergo S Phase in Response
to Serum
In earlier studies it was shown that A1 newt limb cells stop
dividing and fuse to form myotubes in low (0.5%) serum
medium (Lo et al., 1993
The myotubes entered S phase somewhat asynchronously, starting 2 d after serum addition and with the highest number of nuclei incorporating BrdU at 4 d. Despite
this delay, we found that serum stimulation for as little as 8 h
was sufficient to generate the response. Furthermore, the
uptake of BrdU within a myotube tended to include either
all the nuclei or none at all. A dose response showed that
the myotube response saturated at ~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
There was no evidence of mitotic figures in the newt
myotubes, even at 10 d after serum stimulation, suggesting
that the nuclei arrest in G2 phase. To confirm this finding,
we determined the DNA content of myotube nuclei by fixing and staining the cells with propidium iodide and then
measuring the fluorescence emission of the nuclei by scanning microscopy (see Materials and Methods). We validated the method by comparing the DNA content of serumstarved mononucleate cells where the majority of nuclei
should be 2N (Fig. 3 A, dark bars, asterisk), with mononucleate cells grown in 10% serum where at least 50% of the
cells should have DNA content between 2N and 4N (Fig.
3 A, hatched bars). The value for 4N DNA was confirmed
by measuring the intensity of mitotic nuclei (double asterisk). Before fixation, the cells were labeled for 2 h with BrdU to determine the DNA content of the subset of cells
that were in S phase or had just completed it (Fig. 3 A,
light bars). It is clear that the BrdU-positive nuclei have
fluorescence emissions that lie between the 2N and 4N
peaks. 8% of the serum-starved cells labeled with BrdU
(data not shown).
We compared the distributions of fluorescence intensity
of myotube nuclei maintained in low serum vs those that
had been stimulated with serum for 9 d. To serve as an internal standard for 2N/4N peaks, mononucleate cells were
seeded on the same plate as the myotubes in high serum
media (Fig. 3 B). In low serum almost all myotube nuclei
had intensities corresponding to 2N DNA (Fig. 3 B, dark
bars), as would be expected for nuclei in G0/G1. In marked contrast, a significant number of nuclei in myotubes stimulated with serum had intensities around 4N (hatched bars).
This confirms that the nuclei stimulated to undergo DNA
synthesis were arrested in G2.
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 Table I.
Effects of Serum and Purified Factors on Myotube
Entry into S Phase
; 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.
; 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
).
). 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.
; 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.
/
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.
Materials and Methods
),
myogenesis was induced, and myotubes were purified as described earlier
(Lo et al., 1993
). In brief, myotubes were formed by lowering the serum
concentration from 10 to 0.5%, purified after 4 d by brief trypsinization,
followed by neutralizing with media, sieving through a 100-µm mesh (Cell
MicroSieve; BioDesign Inc. of New York, Carmel, NY), and then sieving
through a 35-µm mesh. Myotubes were retained on this second sieve
while mononucleate cells passed through, and then washed off the sieve
into dishes precoated with 0.75% gelatin. For preparations requiring lowest contamination of mononucleates, the myotubes were sieved onto 35µm meshes twice before plating.
), 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.
).
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.
), 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.
. 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.
34 and p16 Expression
) 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.
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).
Results
). The myotubes contract in response to mechanical stimulation and express markers of
differentiation such as muscle-specific myosin heavy chain
(Fig. 1 A). They could be purified from residual mononucleate cells using 35-µm cell sieves as described previously (Lo et al., 1993
). Such preparations result in <10% contamination by mononucleates as assayed by counting nuclei. When purified myotubes were exposed to medium
containing 10% FCS, they responded by synthesizing
DNA as assayed by incorporation of BrdU. Fig. 1 shows
two myotubes, both of which show positive immunostaining in their cytoplasm for muscle-specific myosin. Hoechst
staining (Fig. 1 A) shows that each myotube contains multiple nuclei; those in the myotube on the right have incorporated BrdU (Fig. 1 B). In the preparation shown in Fig. 1,
additional mononucleate cells were plated with the purified myotubes to illustrate that the BrdU incorporation in
the myotubes is comparable to that in normal, proliferating cells and is unlikely to be due to the activation of DNA
repair mechanisms.
Fig. 1.
Serum stimulates newt myotubes to enter S phase. (A) The two A1 myotubes are stained with muscle-specific myosin (green)
and Hoechst to stain DNA (blue). (B) The same myotubes showing muscle-specific myosin (green) and BrdU-positive nuclei (yellow).
The myotube on the upper right has incorporated BrdU during a 24-h pulse given on day 4 after serum stimulation; the myotube on the
lower left is negative for BrdU. (C, facing page) The graph shows the percentage of myotubes taking up BrdU at various concentrations
of serum. Cells were labeled with BrdU for 24 h at 3 d after addition of serum. Bar, 100 µm.
[View Larger Versions of these Images (34 + 10K GIF file)]
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.
Fig. 2.
Myotube nuclei complete S phase in response to serum.
(A). An outline of the experimental design to determine the duration of S phase in the myotubes (see text for details). Purified
myotubes were labeled with an 8-h pulse of [3H]thymidine 2 d after addition of 20% serum. At various times after this first pulse,
samples were labeled with BrdU for 24 h (lower arrows) and
fixed. (B) The number of myotubes positive for [3H]thymidine is
compared with those positive for both [3H]thymidine and BrdU
as the time between the [3H]thymidine and BrdU pulses increases. The x axis denotes the time as shown in A. (Open squares)
Number of myotubes containing only [3H]thymidine-labeled nuclei. These cells had begun S phase at the time of the [3H]thymidine pulse, but they had completed it before labeling with BrdU.
(Closed diamonds) Number of myotubes containing nuclei positive for both [3H]thymidine and BrdU. These cells had continued
to synthesize DNA from the time of the [3H]thymidine pulse to
the time of the relevant BrdU pulse. Note that the majority of nuclei that had begun S phase on day 2 (when [3H]thymidine was
given) had completed it by day 5.
[View Larger Version of this Image (14K GIF file)]
Fig. 3.
Nuclei with 4N DNA content accumulate in serumstimulated myotubes. (A) A histogram of fluorescence intensity
of propidium iodide-stained nuclei from mononucleate A1 cells.
Intensity profiles from serum-starved cells (dark bars) and cycling cells maintained in high serum (hatched bars) were compared. (Asterisks) Intensities of 2N (*) and 4N (**) nuclei (see
text and Materials and Methods). In addition, the profile generated by the subset of cells in high serum that had incorporated
BrdU in a 2-h labeling is shown (light bars). Intensities fall between the 2N and 4N peaks. (B) The intensity profile of mononucleate cells plated with the myotubes in high serum shown in Fig.
3 C. These cells provided an internal standard to verify the intensities corresponding to 2N (*) and 4N DNA (**). (C) The DNA
content of individual nuclei was measured in myotubes maintained in low serum vs those stimulated with high serum. (Black bars) DNA content of nuclei from myotubes kept in 0.5% serum
medum for 9 d. Most nuclei have 2N DNA content, and very few
have 4N, consistent with withdrawal from the cell cycle in G1.
(Hatched bars) DNA content of nuclei from myotubes kept in
10% serum medum for 9 d. A substantial proportion of cells have
nuclei of 4N DNA content, consistent with passage through S
phase and arrest before mitosis.
[View Larger Version of this Image (25K GIF file)]
).
Cell Cycle Reentry Is Dependent on Cell Density
Unpurified newt myotubes formed from dense cultures of myoblasts did not consistently respond to serum. This suggested that cell cycle reentry is inhibited by contact with other cells, and this possibility was further investigated. First, we examined the reponse of myotubes formed from varying densities (ranging threefold) of myoblasts. Without purification, myotubes formed in dense cultures of myoblasts were unresponsive to serum, whereas those formed in sparse cultures did respond (Table II A). To determine if purified myotubes could also be regulated by cell density, we examined the effect of adding back A1 cells to purified myotubes. 24 h after plating purified newt myotubes, mononucleate cells were added at varying densities and cultured in low serum for 48 h before addition of serum. Myotubes surrounded by a subconfluent population of mononucleate cells responded to serum as well as purified myotubes, while myotubes surrounded by a confluent population of cells were inhibited from cell cycle reentry (Table II B). This inhibition of BrdU uptake into myotubes is significantly greater than the effect of density on the mononucleate A1 cells that appear to be relatively insensitive to contact inhibition. To determine if inhibition reflected direct cell-cell contact rather than the action of a soluble factor released by the mononucleate cells, we performed the following experiment. After plating purified myotubes in a uniform distribution, mononucleate cells were attached in a gradient of density across the plate by tilting it. In the region of the plate where mononucleate cells were sparse, myotubes responded to serum, whereas, on the opposite side of the plate where cells were dense, myotubes were completely inhibited from responding (Table II C). This result provides strong evidence against a diffusible inhibitor and in favor of regulation by local cell contact.
Table II. Cell Cycle Reentry Is Sensitive to Cell Density |
Newt Myotubes Express Rb
As a result of the similarity of newt myotubes and murine
Rb/
myotubes in their response to serum (Schneider et al.,
1994
), we determined if Rb or an Rb-like gene is expressed in the urodele cells. Full-length cDNA of newt Rb
was isolated and sequenced as described in Materials and
Methods. It encodes a protein with a predicted molecular
mass of 103 kD (see Fig. 5) that is 62% identical to mouse
Rb (Bernards et al., 1989
) and 59% identical to Xenopus
(Destree et al., 1992
). It is interesting that the predicted
newt protein, like Xenopus Rb, lacks the proline-rich sequences found at the NH2 terminus of the mouse sequence.
RNase protection analysis revealed that Rb is transcribed in all of the tissues assayed, including A1 myoblasts and myotubes as well as normal limb and blastemal
tissue (Fig. 4), and analysis by Northern blot revealed two
transcripts of ~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.
Phosphorylation of Rb Is Required for Cell Cycle Reentry
It was important to determine if cell cycle reentry of the newt myotubes occurred via the phosphorylation of Rb. To this end, we first asked whether expression of the CDI, p16, which specifically inhibits the CDK4/6 class of kinases, is able to inhibit the serum response. This would implicate Rb phosphorylation in cell cycle reentry, and it would indicate that the phosphorylation of Rb occurs via activation of CDK4/6. When human p16 was expressed in the newt myotubes, it completely inhibited uptake of BrdU (Table III). Control injections with plasmids expressing alkaline phosphatase had normal levels of DNA synthesis.
Table III. Expression of Human p16 in Myotubes Inhibits Reentry to the Cell Cycle |
To test more directly whether phosphorylation of Rb
was required for the serum response, we determined if expression of the 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.
Table IV.
Expression of |
The Phosphorylation of Newt Rb during Serum Stimulation
We next examined the phosphorylation state of endogenous Rb in the newt myotubes. In mammalian cells such as
quiescent fibroblasts and resting lymphocytes, Rb is hypophosphorylated and becomes phosphorylated when the
cells are stimulated to enter S phase. This transition can be
detected by a mobility shift on SDS gels. When an affinitypurified polyclonal rabbit antibody (SK70) prepared against a portion of the pocket region of newt Rb was used
to analyze Rb in extracts of proliferating mononuclate A1
cells by immunoprecipitation and subsequent Western
blotting, a series of bands spanning 110-116 kD was specifically recognized (Fig. 5 A, lane 3). Treatment of the
immunoprecipitate with calf intestinal phosphatase caused
the protein to migrate as a single 110-kD band (Fig. 5 A,
lane 2), a change blocked by inclusion of phosphatase inhibitors (Fig. 5 A, lane 1). The identity of these bands as Rb
was further confirmed by immunoprecipitating extracts of
mononucleate cells with an mAb against human Rb
(XZ56; Hu et al., 1991), which is known to cross-react with
Xenopus Rb, and then by Western blotting with SK70
(Fig. 5 A, lane 6). In this case, a doublet at 110 kD was recognized. Phosphatase treatment of the immunoprecipitate resulted in migration of the protein at the same molecular
mass as the phosphatase-treated protein from the SK70
immunoprecipitate (Fig. 5 A, lanes 5 and 2, respectively).
Based on mobility, the SK70 antibody under immunoprecipitation conditions appears to preferentially recognize
more highly phosphorylated forms of newt Rb as compared with XZ56.
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.
Urodele amphibians have the remarkable ability among
adult vertebrates to regenerate much of the body plan including the limbs, tail, and jaws, as well as ocular tissues
such as the lens. Regeneration proceeds by the local reversal of differentiation of adult tissues to provide the proliferating mesenchymal or epithelial progenitor cells of the
regenerate (Brockes, 1994; Okada, 1991
; Wallace, 1981
).
After removal of the lens, pigmented epithelial cells of the
dorsal iris lose their pigment and proliferate before transdifferentiating into lens, a transition that can be reproduced in clonal cell culture (Eguchi et al., 1974
; Okada,
1991
). In limb regeneration, the blastemal progenitor cells
arise by dedifferentiation of mesenchymal tissues at the
amputation plane (Hay, 1959
; Steen, 1968
; Namenwirth, 1974
; Lo et al., 1993
). It is unclear to what extent urodele
cells are intrinsically different from those of other vertebrates, and to what extent they encounter distinct signals
that evoke reversal.
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 factorsa 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.
Received for publication 22 April 1996 and in revised form 18 September 1996.
E.M. Tanaka was supported by fellowships from the Muscular Dystrophy Association, Inc. and The Helen Hay Whitney Foundation.We thank Alan Entwistle for help in developing the protocol for determination of nuclear DNA content; S. Hughes, G. Peters, and E. Harlow for
antibodies; N. Jones for the 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.
BrdU, bromodeoxyuridine; CDI, cyclin-dependent kinase inhibitor; CDK, cyclin-dependent kinase; CIP, calf intestinal phosphatase; GST, glutathione-S-transferase; Rb, retinoblastoma.