myogenesis. Here we studied the role of the cellular transcription fador. E2F1 on myogenic differentiation. E2F1 expression is irreversibly down- regulated.
Vol.
6,
1299-1306,
October
Cell
1995
Inhibition of in Vitro Transcription Factor’
Jian Wang,2 Kristian Helm, Bernardo Nadal-Ginard
Myogenic
Pei un, and
Department of Cardiology, Children’s Hospital and Harvard Medical School, Boston, Massachusetts 02135 Ii. W., P. J., B. N-G.l, and Division of Cancer Biology, Danish Cancer Society, Copenhagen, Denmark 1K. H.l
Abstract
Terminal differentiation of cultured myocytes requires withdrawal of the cells from the cell cycle. Constitutive overexpression of several oncogenes in myoblasts can inhibit in vitro myogenesis. Here we studied the role of the cellular transcription fador E2F1 on myogenic differentiation.
E2F1
expression
is irreversibly
down-
regulated during differentiation of C2C1 2 myocytes. Furthermore, deregulated E2F1 expression in C2C1 2 cells
prevented
of myogenesis myogenin
myogenic
differentiation.
was associated
expression
This
inhibition
with the repression
and an elevated
cyclin
Differentiation
of
conditions.
Cell Differentiation.
are consistent
with
the notion
that E2F1 can fundion as an oncogene and further suggest that E2F1 down-regulation is required for myogenic differentiation. Introduction Terminal differentiation of cultured skeletal muscle cells requires that they exit from the cell cycle (1 , 2). Growth factors and oncogene products have all been shown to inhibit myogenic differentiation. Growth factors can prevent myogenesis through several mechanisms. For instance, high levels of serum in the medium will induce the expression of Id, a dominant-negative protein of the myogenic bHLH3 regulators (3), while basic fibroblast growth factor activates protein kinase C that, by phosphorylation of a conserved site in the basic region of myogenin, inhibits the DNA-binding activity of myogenin (4). Ectopic expression of oncogenes in myoblasts can also inhibit myogenesis, even when growth factors are withdrawn. ras and c-myc oncogenes both down-regulate myogenic bHLH factor expression and interfere with their function (5, 6). The c-jun gene product associates directly with bHLH factors and inhibits their activity (7, 8). Adenovirus E1A gene product can inhibit MyoD expression (9, 10) and also physically
Received r This
5/4/95; study
was
revised supported
7/1 2/95; by grants
accepted
7/27/95.
to B. N-G.
from
by Cellular
Dl
Results
results
& Differentiation
associate with myogenin (1 1 ). Moreover, both the El A protein and the SV4O large T antigen can bind and inactivate the retinoblastoma gene (RB) family products pRB, p107, and p300, which are also required for muscle cell differentiation (9, 1 2, 1 3). Cellular transcription factor E2F1 is a target of the pRB protein and is involved in cell proliferation regulation (141 7). Deregulated E2F1 in the cells leads to S-phase entry and programmed cell death under low serum conditions (1 8-21 ). Moreover, constitutive E2F1 overexpresssion may induce cellular transformation (22-25). In this study, we demonstrate that E2F1 expression is down-regulated during C2C12 myocyte differentiation and that ectopic E2F1 expression in myoblasts inhibits myogenic differentiation. These results indicate that E2F1 may be involved in regulating cell proliferation and differentiation during muscle development.
expression. Moreover, E2F1 -overexpressing myocytes failed to exit the cell cycle under differentiation These
Growth
the NIH,
the Muscular
Dystrophy Association of America, and the Howard Hughes Medical Institute. K. H. is supported by a grant from the Danish Cancer Society. 2 To whom requests for reprints should be addressed, at Division of Cardiovascular Research, St. Elizabeth Medical Center and Tufts University School of Medicine, 736 Cambridge Street, ACH-3, Boston, MA 021 35. i The abbreviations used are: bHLH, basic helix-loop-helix; CPK, creatine phosphokinase; GST, glutathione S-transferase; RB, retinoblastoma; BrdUrd, 5-hromo-2’-deoxyuridine; FBS, fetal bovine serum.
E2F1 Expression
Is Down-regulated
upon Skeletal
Muscle
E2F1 mRNA level in C2 myocytes was determined by Northern blot analysis (Fig. 1). E2F1 transcription was prominent in exponentially growing myoblasts but was significantly reduced in differentiated myotubes (Fig. 1, Lanes 1 and 2). Moreover, E2F1 transcription in differentiated myotubes was not stimulated by serum (Fig. 1 , Lanes 3 and 4). In contrast, serum stimulation of quiescent fibroblasts induced E2F1 transcription, which peaks at 12 to 16 h after serum stimulation, a time period Corresponding to the G1-S transition during the cell cycle (Refs. 1 7 and 26; Fig. 1 , Lanes 7 and 8). These results are consistent with the fact that differentiated myotubes are unable to reenter the cell cycle in response to growth factor stimulation. Generation of E2F1 -overexpressing Cell Lines. To investigate the effects of E2F1 on muscle cell differentiation, C2 Cell lines that constitutively overexpressed exogenous E2F1 were established. C2 cells were transfected with either the wild-type (E2F1 ) or a COOH terminus truncated form of E2F1 (E2F1 1284; Fig. 2A). E2F1 1-284 lacks the transcription activation and pRB-binding domains in the COOH-terminal of E2F1 (Fig. 2A) and is unable to activate transcription from adenovirus E2 promoter (23). C2 cells transfected with the vector pCMV alone (C2-neo) were also isolated and used as control. Three weeks after transfection, G4l 8-resistant colonies were selected, expanded, and subjected to Northern blot analysis with human E2F1 probe to screening for cells overexpressing exogenous E2F1 or E2F1 1284 (data not shown). Expression of exogenous E2F1 proteins was confirmed by immunoblotting analysis using monoclonal antibody (KH9S) specific to human E2F1 (Fig. 2B). Of 29 G41 8resistant clones analyzed, 4 overexpressed exogenous wildtype E2F1 . Eight of 1 9 G41 8-resistant clones from E2F1 1284 transfected cells overexpressed E2F1 1284#{149} All individual C2-E2F1 clones demonstrated similar differentiation and
1299
1300
E2F1
Inhibition
1234
(if
Myogeriesis
ferentiation (27). The expression of cyclin A and cdc2 also decreased in C2-F2F1 cells cultured in differentiation medium. However, cyclin Dl mRNA level persisted in C2F2F1 cells cultured under differentiation conditions. Of the two cyclin Dl transcripts, the 4.5-kb transcript is still expressed at considerable levels after 72 h in differentiation medium, whereas the 3.8-kb transcript disappeared (Fig. SB).
5678
GAPDH
Fig. 1 . Irreversil)le E2F 1 down-regulation 111)00 (T2( 1 .2 niyocyte differentiatioii. [2F I niRNA levels in replk atirig niyol)lasts ( Lane I (, dlifferentiated riivotulx’s ( L,i,re _0, arid niyotul)(’s restiniulated with high seruni growth rile(Iiunl for 1 2 ,rrid 24 h ( Lanes 3 and 4, res)ectively( were (leternirned by Niirtherri 1)101 with a cDNA I)robe for mouse E2F1 . As ,i control, E2F1 niRNA levels in repliatirig NII-13T3 iibrohl,rsts (Larre 5(, quiescent iibrol)lasts(Lane b(, arid dluiescerit til)rol)l,ists stimulated with serurii for 1 2 and 24 h (Lanes 7 ,iii(l 8. resx-ctiv(’ly( svere also nieasured. The blots were also hybridized to gly eraldehyde-3-phosphate dehydrogeriase ( (APL)!-fl rDNA probe as contrril or (‘(111,11 loading.
growth properties. Therefore, the data from one representing clone, clone 18, will be shown. Deregulated E2F1 Overexpression Inhibits Myogenic Differentiation. Differentiation of C2-neo, C2-F2F1 (clone 1 8), and C2-F2F1 (clone 3) cells was induced by transferring subconfluent cell cultures (Fig. 3, a, d, and ) to differentiation medium. After 3 days in differentiation medium, multinucleatecl myotubes were noted in both C2-neo and C2-F2F1 2fn4 cells (Fig. 3, b and Ii). In contrast, no morphological features suggestive of a differentiated phenotype were manifest in C2-F2F1 cells. C2-F2F1 cells continued to grow until confluence and remained single nucleated (Fig. 3e). Muscle-specific structural proteins were assayed in each of these cell cultures. Immunohistochemical analysis demonstrated a strong expression of sarcomeric myosin heavy chain in C2-neo and C2-F2F1 204 cells (Fig. 3, c and i) but not in C2-F2F1 cells (Fig. 31). Biochemical differentiation of these skeletal myocytes was assessed by using the muscle-specific CPK. As shown in Fig. 4, CPK activities increased exponentially upon C2neo and C2-F2F1 cell differentiation and reached a plateau (40 units/pg of DNA) after 3 days in differentiation medium. In contrast, CPK activity in C2-F2F1 cells reniained constant and did not increase over basal level (0.2 units/pg of DNA). These biochemical results confirmed that C2-F2F1 cells did not undergo differentiation. Effects of E2F1 on Myogenin and Cyclin Dl Expression. To gain insight into the molecular mechanism(s) by which F2F1 inhibits myogenic differentiation, we analyzed the expression of rnyogenic factor MyoD and myogenin in C2-F2F1 and control C2-neo cells. F2F1 overexpression did not significantly affect MyoD expression. Although the MyoD mRNA level was slightly reduced in C2-F2F1 cells (Fig. 5B), MyoD protein levels in C2-F2F1 cells remained the same as in C2-neo cells cultured in differentiation medium (Fig. 5A). In contrast, myogenin expression was Severely inhibited in C2-F2F1 cells. Myogenin protein levels remained low in C2-F2F1 cells cultured in differentiation medium (Fig. 5A). Northern analysis confirmed that the inhibition of myogenin expression occurred at the mRNA level (Fig. 5B). We have shown previously that cyclin Dl , cyclin A, and cdc2 mRNA levels are clown-regulated during C2 cell dif-
C2-E2F1
Cells Failed to Exit the Cell Cycle under
Differ-
entiation Conditions. To further investigate the importance of the elevated cyclmn Dl mRNA level in C2-F2F1 cells under differentiation conditions, we compared the cyclin D1-associated cell cycle kinase activity of C2-F2F1 and C2-neo cells by using a GST-RB fusion protein as substrate. Cell lysates from both C2-F2F1 and C2-neo cells grown in growth medium displayed high levels of cyclmn Dl -associated kinase activity. This pRB kinase activity was reduced in C2-neo cells cultured in differentiation medium for 48 h but remained relatively high in C2-F2F1 cells cultured under the same conditions (Fig. 6). We next investigated, by measuring BrdUrd incorporation, if C2-F2F1 cells remained in the cell cycle under differentiation conditions. After a 48-h incubation in the differentiation medium, DNA synthesis was rarely detectable in C2-neo cells (Fig. 7a). However, BrdUrd-positive cells were abundant in C2-E2F1 cells cultured under the same condition (Fig. 7b), suggesting active DNA synthesis. Thus, C2-F2F1 cells failed to exit the cell cycle under differentiation conditions. These results are consistent with the reports that forced F2F1 expression in quiescent cells can induce S-phase entry (18-21). Proliferation Properties of C2-E2F1 Cells. The proliferation properties ofC2-E2F1 cells were investigated (Fig. 8). In high serum conditions, both C2-F2F1 and control C2-neo cells have a similar population doubling time (20-24 h). However, while C2-neo cells ceased to proliferate after reaching confluence, C2-F2F1 cells continued to grow and finally built up a multilayer of cells. Thus, F2F1-overexpressing cells lost cell-cell contact inhibition of cell growth in mitogen-rich medium. As the concentration of serum declined, the population doubling time of both cell types declined in a parallel fashion. C2-neo cells stopped growing after 2 to 3 days in 2% serum, probably because the differentiation process had been initiated. On the other hand, C2-F2F1 cells continue to replicate until confluent. However, they were unable to grow to a multilayer of cells in 2% serum. When the cells were seeded in 0.1% serum media, both C2-F2F1 and control C2-neo cells stopped proliferating. Consistent with previous reports (18, 20, 21), C2-F2F1 cells were able to enter S-phase in O.l% serum media, as measured by BrdUrd incorporation (data not shown). However, the cell number did not increase but decreased under this condition. In the first 24 h after being seeded into DMFM-O.1% serum, both C2-F2F1 and C2-neo cells experienced cell loss. However, C2-E2F1 lost far more cells than C2-neo cells. After 24 h, C2-F2F1 cells continued to diminish in number, while the cell number of C2 cells remained near constant. Examined microscopically, C2F2F1 cells continued to round up and float away in 0.1% serum medium. This observation suggests that C2-E2F1 would die when deprived of growth factors and is consistent with the reports that deregulated F2F1 expression can induce apoptosis under low serum conditions (19-21).
Cell
Growth
,5 tTiifferenitiationi
A
E2F1 Transcription
Fir,’. .2. A. (liagranir of the F2F1 constructs. T lie various functional domains i)i the F..F 1 genie are sliosvn oii lie tJj)(X’r P.tt)(’l. The two E2F I constructs used in this study are shown on the latter pan(‘Is. E2F I .,,, l,rcks the (TOOHterniinial 1 S 3 amino acids, which includes the transcription activaion and I)RB binding domains. B, Western l)li)t analysis i)f E2F I - and F .tF 1 ,,-Over#{128}’XpresSing (el Is. Whole-cell lysates from representative clones of C2-E2F 1 . C2-neo, ,rndl C2-F2F 1 svere analyzed l)y ininiunioblotting using mAb KH2O. This antibody does nut recognize lit’ endogenous mouse E2F 1 protein. Arrosss, positrons of ectopic ally i)veres.I)ress(’(I E2F1 .nnd E2F I 2O proteins. Note that a nonse tic hand (IX’low the F2F 1 lianid( was Present in sam1)1(5 of C_!F21
I ,inid (2-rico
I
I Pro/Ala
Factor
Transactivation
DNA Binding
rich
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Binding
Constructs
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1-284
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Discussion In this report, we have demonstrated that F2F1 expression is down-regulated upon skeletal muscle cell differentiation. We have also shown that ectopic F2F1 overexpression in myoblasts inhibits myogenic differentiation. These results suggest that F2F1 down-regulation is essential for myocyte differentiation. During in vitro myogenesis and embryogenic muscle development, the myogenic bHLH factors are expressed and function in a sequential pattern. Myogenin functions downstream from MyoD (28-30). Moreover, MyoD appears to be directly involved in the activation of myogenin expression (31). Our data show that although deregulated F2F1 expression inhibited both myogenin expression and myotube fusion, it did not significantly affect MyoD expression (Fig. 5). These results suggest a simple mechanism by which F2F1 prevents myogenesis by inhibiting myogenin
expression. However, F2F1 can also induce the expression of some genes that have been shown to inhibit myogenesis (see below). Furthermore, ectopic F2F1 expression can inhibit the transcription activity of both MyoD and myogenm.4 These data suggest that F2F1 may prevent myogenesis not simply by repressing myogenin expression. Instead, it may inhibit MyoD function, including the ability to activate myogenin and other downstream genes. The F2F consensus motif is present in the promoter region of many growth-stimulating genes (32-35), some of which may directly inhibit cell differentiation, including myogenesis. Deregulated F2F1 expression may result in elevated expression of some of these growth-stimulating genes. In F2F-overexpressing fibroblasts, c-myc expression was ele-
4
1. Wang
and
B. Nadal-Ginard,
unpublished
oI)servationi.
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-
S\ (a-c),
C2-E2F1
(clone
1 8; d-t(
and
C2-E2F1
(clone
3: g-i(
were
sul)conflueni( (‘ (.i, Cl, arid g(. (el) differenti,rtioni w.ns iiiitiatedl l)y shifting these cell cultures to differentiation niedium (DMEM/2% horse serum) Multiniucle.ited nivotulies were ( early fornied in C2-nieo and C2-E2F1 2i4 cells (b and h( but not in C2-E2F1 cells (e(. tmniunostainiinig of muscle-specific lit’.ivy ( hOn Iiri)t(’ini is shown on right ;sinrt’ls ( c, t. arid 1). Myosin heavy ham was strongly expressed in the fused myotubes (c and i( hut not in unfused ( ells tL t’liotographs were taken svith a ‘. .0) ol)jective.
vatecl (22), which has been shown to inhibit MyoD function and niyogenesis (6). Our data revealed increased cyclin Dl mRNA level as well as elevated cyclin Dl-dependent pRB kinase activity of C2-F2F1 cells under differentiation conditions (Figs. 5 and 6). Consistent with this result, the E2F motif is also present in the promoter region of the human cyclin Dl gene (36). Fctopic cyclin Dl expression can inhibit MyoD transcription activation (37, 38). Therefore, elevated c-myc and cyclin Dl protein levels in C2-F2F1 cells may be involved in the inhibition of myogenic differentiation in these cells. C2-F2F1 cells failed to exit the cell cycle when cultured in the differentiation medium (Figs. 7 and 8), possibly through the activation of growth-stimulating genes, including c-myc and cyclin Dl , as mentioned above. It has been suggested that withdrawal from the cell cycle is a prerequisite for the terminal differentiation of C2C12 myocytes. Thus, deregulated F2F1 expression may prevent rnyogenic differentiation by inhibiting the cell cycle exit of myocytes under differentiation conditions. F2F1 expression in myotuhes does not appear to be inducible IJy serum. We have observed previously that cyclin A and cdc2 in myotuhes are also refractory to serum restimulation (27). On the other hand, the cyclin-dependent kinase inhibitor p21 ui,t/tt t has been shown to be induced during myocyte differentiation and sustained in
grown for
to
3 days. myosin C2-E2F 1
z 0 00
-0-
C2-E2FI C2-neo
....o....
0)
C2-E2F1
1-284
> (.3
Days Fig. 4.
Inhibition
in differentiation of CPK
by E2F1
medium
overexpression.
C2-neo,
C2-E2F1
(clone
C2-E2F1 r ri-i (clone 3) cells were seeded at 5 x l0 cells/35-mm dish in growth mediuni. When confluent. cells were shifted to differentiation medium. Al the indicated time points, cells were collected, and their CPK activities were measured. Results are per minute CPK activities at 25’C and are related to their DNA contents. Triplet experinients were performed for each cell line and similar results were obtained.
18), and
Cell
A.
_C2-E2F1_ 0
48
_C2-neo
72
0
48
hrs in DM
72
C2-neo
C2-E2F
GMDM
GMDM
S..
,.
.
-
a.
--
Myo D
-
Myogenin
-
Myosin HG
S
Growth
& Differentiation
1
#{149} #{149} -GST-RB
1234 1 .0
0.8 0.6 0.4
B.
C2-neo,
C2-E2F1
0 48 72
0 48 72
0.2 hrs in DM
Relative -MyoD
,
,;
st
s
]cyclin
I cyclin
kinase activities of these protein coniplexes were measured by using a GST-RB fusion protein as substrate. The ‘P-incorporated GST-RB protein was indicated (upperpanel(. The experiment was repeated twice. and similar results were obtained. The mean values from two independent experiments are expressed as relative kinase activities in the histogram ) (cover panel).
Dl A
. ..
,w
e
activity
Fig. 6. Cyclin Dl -dependent pRB kinase activity. Cell lysates were callected from C2-neo myol)lasts (column 1), C2-nieo cells cultured in differentiation media for 48 h (column 2), C2-E2F1 niyohlasts (column 3). and C2-E2F1 cells cultured in differentiation media for 48 h (column 4). Cyclin Dl protein coniplexes from these cell lysates were precipitated with a C yclin Dl -specific antibody and collected on protein-A,/G Plus beads. The pRB
-Myogenin
I.
kinase
- cdc2
myotubes (39-41). These irreversible changes in the expression of cell cycle regulatory genes may represent one mechanism by which terminally differentiated cells permanently exit from the cell cycle. Further study to investigate the transcription regulation of F2F1 in myotubes may help to understand these mechanisms and provide information for skeletal muscle and heart regeneration. Materials
Cell
-GAPDH F7
Fig. 5. E2F1 inhibits myogeniin expression and niaintains cyclin Dl Iranscription in low serum condition. A, inimunoblotting analysis. Whole-cell lysates svere collected from C2-neo and C2-E2F1 cells at 0, 48, and 72 h after shifting to differentiation niediuni (DM). Forty pg cell lysate from each sample were separated by SDS-PAGE gel electrophoresis and transferred to polyvinylidenie difluoride niembranes (Millipore(. The abundance of E2F1, myogenin, MyoD, audi niyosini heavy chain proteins was examined by ininiiunoblotting using the corresponding antibodies (see “Materials and Methods”). B, Northern blot analysis. Total RNAs were collected from C2E2F 1 and C2-neo at different time points as described in A. The mRNA levels of MyoD, rnyogenin, cyclin Dl, cyclin A, and cdc2 were determined by Northern blot. The same blot was also hybridized to the glyceraldehyde-3phosphate dehydrogenase )GAPDH( probe serving as a control for equal loading. Note that two transcripts were detected by both cyclin Dl and (yclin A probes.
and
Culture.
Methods
The C2C1 2 skeletal myoblasts (C2) were maintained in DMEM supplemented with 20% FBS (growth medium). Cell differentiation was initiated by placing 70% confluent cell cultures in DMEM medium supplemented with 2% heat-inactivated horse serum (differentiation medium). To prepare myotube cultures, myoblasts were cultured in differentiation medium for 2 days, then exposed to 1 0 M cytosine 3-D-arabinofuranoside for 48 h to eliminate undifferentiated myoblasts, and then switched back to differentiation medium for two more days. Serum restimulation of myotubes was performed by transferring myotube cultures to growth medium for 12 and 24 h. NIH/3T3 fibroblasts were cultured in DMFM with 10Yo FBS. Quiescent cultures of fibroblasts were obtained by transferring subconfluent cell cultures to DMEM with 0.5% FBS for 60 h. For cell growth curves, S x 1 0 cells were seeded onto 35-mm Petri dishes in DMFM medium supplemented with different concentrations of FBS. Cells were trypsinized, and
1303
1304
E2F1
.i-
Inhibition
.
of Myogeniesis
.-
_c
I
-
Fig. 7. BrdUrd incorporation by C2-neo and C2-E2F1 cells under differentiation conditions. C2-neo and C2-E2F1 cells cultured on coverslips were transferred to differentiation medium for 48 h and then incubated in the same medium containing 10 BrdUrd for 4 h. BrdUrd incorporation was detected by direct immunostaining with a FITC-conjugated anti-BrdUrd mAh. a and c, the same field of C2-neo cells with anti-BrdUrd staining and Hoechst 33258 for nuclear slain, respectively. b and d, the same field of C2-E2F1 cells with anti-BrdUrd and Hoechst stain.
6
1000
Fig. 8. Effects of etopical E2F1 overexpression on ((‘II growth. Cells (5 >( 1 0i) svere Platedl onto IS-nirni
0
(lisIit’s in DMEM sup)leniented with (Iifferent serum I F BS) concentraions, as indicated. Ci’lls were treated with trypsini, ,rnirl (elI nunihers were deterniinedl under a mi(roscope at (lally intervals. Mean values of cell niuniher from triplicate
z
I)lat(’s
with
SFs ,rre
plotted
100
ENeo
E 10 C.) 1
versus
tinie.
C2-E2F1
0.1%
Serum
0 0
2
3
4
Days in Medium
cell numbers were determined under a phase-contrast microscope at daily intervals. Overexpression of E2F1 in Myoblast. For the establishment of F2F1 -expressing cell lines, C2C1 2 myoblasts were transfected with pCMV-F2F1 , pCMV-F2F1 121n4’ and pCMVneoBAM (42), respectively. G4l8-resistant colonies were isolated 14-21 days after transfection and amplified. Cells overexpressing exogenous F2F1 were screened by Northern blot analysis and confirmed by Immunoblotting. Immunohistochemistry. Cells grown on coverslips were fixed by direct immersion in 3.7% paraformaldehyde in PBS for 5 mm. Fixed cells were then permeabilized by immersion in 0.3% Triton X-100 in PBS for S mm, washed three times with PBS, and incubated with monoclonal myosin heavy chain antibody MF2O. The secondary antibody was rhodamine-conjugated rabbit anti-mouse lgG (Sigma Chemical Co.). Cells were examined by immunofluorescent microscopy using a Zeiss X20 objective.
5
6
0123456
0123456
Days in Medium
Days in Medium
Assay of Creatine Kinase Activity and DNA Content. Cells on 35-mm Petri dishes were washed twice with cold PBS and stored at -70#{176}Cin 0.05 M glycylglycine (pH 6.75). Just before enzyme determinations, the cells were thawed, scraped from the dishes with a rubber policeman, and sonicated. Aliquots were taken for determination of creatine phosphokinase activity as well as DNA content. Creatine phosphokinase was measured using the Sigma kits (45-5), and the activity was expressed in units at 25#{176}C/mm/pg of DNA. DNA content was measured by the modified fluorometric assay using a fluorospectrophotometer. Northern Blot Analysis. Total cytoplasmic RNA was isolated from cultured cells by the guanidinium isothiocyanate/CsCI procedure. Twenty pg RNA from each sample were separated on 1 % agarose-formaldehyde gel and transferred to nylon membrane (Hybond-N; Amersham). The membrane was hybridized with 32P-radiolabeled cDNA fragments using standard procedures as described (43). The
Cell Growth
cDNA fragments used for probes were as follows: the 0.9-kb SaII-BamHI fragment of murine E2F1 ;5 the 1 .5-kb EcoRl MyoD coding region from pEMSV-MyoD (courtesy of H. Weintraub); the 1 .6-kb EcoRI myogenin coding region from pEMSV-myogenin (courtesy of E. Olson); the 1 .6-kb EcoRl fragment of human cyclin A cDNA (44); the 1 .0-kb BamHI fragment of mouse CYL1 cDNA coding region (courtesy of C. Sherr); and the 0.9-kb EcoRI fragment of mouse cdc2 cDNA coding region (courtesy of P. Nurse). Immunoblotting. Immunoblotting analysis was performed as described (27). Briefly, cell lysates were prepared in 1 X NP4O lysis buffer [50 mwi Tris (pH 7.6), 250 mi NaCI, 5 mM EDTA, 0.5% NP4O, 2 jg/ml leupeptin, 2 jg/mI aprotinin, and 1 mM phenylmethylsulfonyl fluoridel. Cell lysates (40 pg) from each sample were separated on SDS-PAGE gel and transferred to Immobilon membranes (Millipore) by Semiphor transfer (Hoefer). The membranes were incubated with specific antibodies and developed by with ECL reagents. The antibodies used were: mAb KH2O to human E2F1 (42); mAb 5.8A (45) to mouse MyoDl (courtesy of P. Dias); mAb F5D (46) to myogenin (courtesy of W. Wright); mAb MF2O to myosin heavy (1 2); and mAb to pRB (PharMingen; PMG3-245). In Vitro RB Kinase Assay. In vitro RB kinase assay was performed as described (47) by using as substrate a protein covering the COOH terminus (605-921) of mouse RB protein that was fused to GST (GST-RB; Ref. 1 2). Briefly, cyclin Di-associated kinase was immunoprecipitated from cell lysates (300 pg) with a mAb specific to cyclin Dl (Santa Cruz) and collected on protein A beads. The beads were washed twice with kinase buffer [50 mt.i Tris (pH 8.0), 10 mM MgCI2, 1 mM DTT, 1 m’vi phenylmethylsulfonyl fluoride, and 1 pg/mI each of leupeptin and aprotinini and incubated with 2 ig of GST-RB fusion protein and 4 iCi E’y-32PJATP in 50 p1 of kinase buffer for 30 mm at room temperature. Reactions were terminated by the addition of 20 p1 of 4X SDS sample buffer and by boiling for 5 mm. Samples were separated by electrophoresis on polyacrylamide gels, and the phosphorylated proteins were visualized by autoradiography of dried-slab gels. The GST-RB fusion protein was purified from Escherichia co/i extracts by batch chromatography using glutathioneSepharose 4B beads (Pharmacia). GST-RB was eluted from the beads by incubation in kinase buffer with 5 mtvt reduced glutathione at 4#{176}C. Eluted proteins were visualized by staining with Coomasie blue following electrophoresis on a denaturing polyacrylamide gel. The concentration of GST-RB fusion proteins was estimated by comparing the density of the GST-RB band to that of protein standards of known mass. BrdUrd Incorporation and Detedion. Cells were seeded onto sterile glass coverslips. After shifting to differentiation medium for 48 h, BrdUrd was added to 10 M and incubated for 4 h. The cells were washed with PBS and fixed with 3.7% paraformaldehyde in PBS. BrdUrd-positive cells were detected by immunostaining with a FITC-conjugated anti-BrdUrd mAb (Boehringer-Mannheim) in the presence of DNase A (0.5 pg/mI), counterstained with Hoechst 33258, and mounted. Specimens were examined and photographed using a Zeiss fluorescence microscope.
& Differentiation
1305
Acknowledgments We are grateful to Drs. C. Sherr, E. Harlow, H. Weintraub, P. Nurse, E. Olson, P. Houghton, and P. Dias for valuable materials. We thank Drs. X. Qin and Q. Huang for technical assistance and Drs. H. Skopiki and L. Weir for help in the preparation
of this
manuscript.
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