[CANCER RESEARCH 58, 2042-2049, May I, 1998]

Elevated Cyclins and Cyclin-dependent Kinase Activity in the RhabdomyosarcomaCell Line RD1

Erik S. Knudsen,2 Claudia Pazzagli,2 Teresa L. Born, Bonnie L. Bertolaet, Karen E. Knudsen, Karen C. Arden,Robert R. Henry, and James R. Feramisco3

Departments of Medicine ¡E.S. K., C. P.. T. L B.. B. L. B., K. E. K., K. C. A., R. R. H.. J. R. F.I and Pharmacology [E. S. K., C. P., T. L B., B. L B.. J. R. F.¡,Cancer Center[E. S. K., C. P., T. L B., B. L B.. J. R. F.I. and Ludwig Institute for Cancer Research ¡K.E. K.. K. C. A.], University of California at San Diego, School of Medicine, La Mia,California 92093-0684

ABSTRACT

An important early event in the differentiation of skeletal muscle cellsis exit from the cell cycle, after which full expression of the musclephenotype occurs. Rhabdomyosarcoma (RMS), a tumor of skeletal muscleorigin, expresses a number of muscle-specific proteins, including MyoD;

however, these cells fail to arrest or differentiate when cultured in differentiation medium (DM). To determine the basis for the failure of RMScells to differentiate or arrest, we studied the molecular response of theembryonal RMS cell line, RD, to culture in DM. Under these conditions,the retinoblastoma protein (RB) was primarily in the hyperphosphory-

lated state. This is in contrast to myoblasts cultured in DM, in which thehypophosphorylated form of RB is exclusively present. Measurements ofthe expression and activities of cyclin-dependent kinases (cdks) cdk2 and

cdk4 indicated that RD cells maintained higher levels than do myoblasts,and the activity and abundance of these proteins did not significantlydecrease upon culture in DM in RD cells, as they did in myoblasts.Similarly, elevated expression of cyclins Dl, E, and A was observed in RDcells. Interestingly, cdk inhibitors are expressed in RD cells, with pl6ink4expression markedly elevated relative to myoblasts. Ectopie expression ofpllcipl, pl6ink4, or p27kipl caused a growth arrest of RD cells but notdetectable expression of a myogenic marker. Furthermore, a constitu-

tively active RB protein could also inhibit the growth of RD cells withoutinducing myogenic differentiation. Taken together, these data suggest thatthe elevated levels of cdk2 and/or cdk4 observed in RD cells contribute tothe inability of RD cells to growth arrest when cultured in DM but thatthese activities alone are not responsible for the failure of RD cells todifferentiate.

INTRODUCTION

RMS4 is the most commonly occurring soft tissue sarcoma in

children (1). RMS tumors resemble normal fetal skeletal muscle inmorphology, and the cells of RMS tumors express several well-knownmuscle-specific genes. As such, RMS tumors are believed to arise

from muscle tissue (1, 2). Several distinct histological subtypes ofRMS have been described: alveolar, embryonal, botryoid, and undif-

ferentiated (1, 2). Although survival rates vary with histologicalassignment, overall long-term survival rates of children with RMS areonly 50-70%. Investigations into the molecular basis of RMS have

centered on the chromosomal alterations associated with specifichistological classes of RMS (1, 2). Studies of the alveolar type of

Received 10/21/97; accepted 3/3/98.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by grants from the NIH/National Institute on Aging (to

J. R. F.) and from the California Tobacco Related Diseases Research Program. E. S. K.was supported by a NIH/National Cancer Institute Training Grant T32CA09290 to theUniversity of California at San Diego Cancer Center. C. P. was supported by a postdoctoral fellowship from the Cancer Research Foundation of America.

2 These authors contributed equally to this work.3 To whom requests for reprints should be addressed, at University of California at San

Diego Cancer Center, Mail Code 0684, 9500 Oilman Drive, La Jolla, CA 92093-0684.4 The abbreviations used are: RMS, rhabdomyosarcoma; DM, differentiation medium;

cdk, cyclin-dependent kinase; MHC, myosin heavy chain; CMV, cytomegalovirus;BrdUrd, bromodeoxyuridine; GST, glutathione S-transferase; GFP, green fluorescentprotein; PM, proliferation medium.

RMS revealed the presence of specific chromosomal translocations,t(2;13) and t(l;13). These translocations result in the expression of achimeric protein, encoded by regions of the Pax3 or Pax7 and FKHRgenes (3, 4). The Pax3-FKHR chimeric protein has been shown to

harbor oncogenic activity and is believed to contribute to alveolarRMS tumor formation and progression (5, 6). For embryonal RMS, acommon region of loss of heterozygosity on chromosome 11 has beenidentified (7, 8). However, the exact identity of a putative embryonaltumor suppressor gene has not yet been determined.

Although they have apparently disparate methods of oncogenictransformation, both alveolar and embryonal tumors exhibit similardefects in myogenic differentiation. Most RMS tumor cells are characterized by the expression of several muscle-specific markers, suchas the myogenic-promoting transcription factor MyoD. Although the

expression of such factors typically correlates with myogenic differentiation, RMS cells fail to undergo terminal differentiation intoskeletal muscle (2). The failure of RMS cells to differentiate ishypothesized to be one mechanism through which these cells gain thegrowth advantage necessary for tumor formation.

In culture, normal myoblasts can be induced to differentiate byculture in mitogen-poor medium (DM). Such a culturing condition

leads to an arrest in the G„/G,phase of the cell cycle, which isfollowed by the induction of the differentiated phenotype characterized by the expression of muscle-specific genes and, ultimately, theformation of multinucleated myotubes (reviewed in Refs. 9-11). Cell

cycle withdrawal is required for this process to occur. The expressionof oncogenes that prevent cell cycle arrest also prevents the expression of the differentiated phenotype in myoblastic cell lines (9-11).

The arrest of cells undergoing myogenic differentiation has beenstudied in great detail. The overall process of myogenic differentiationis accompanied by the down-regulation of cdk activity, which is

required for cell cycle progression. In immortalized murine myoblastic cell lines, cdk2 and cdk4 kinase activity is dramatically inhibited.This inhibition occurs through at least two mechanisms. One mechanism is via the down-regulation of cyclin expression, which is

required for cdk activity. For example, cyclin A, which is associatedwith cdk2, is barely detectable in differentiated myotubes (9, 12, 13)A second mechanism is via the induction of cdk inhibitor expression.For example, p21cipl and pl8ink4c proteins are both highly inducedin myoblasts cultured in DM (9, 13, 14). Down-regulation of cdk

activity is important for the expression of the differentiated phenotypebecause ectopie overexpression of cyclins can prevent myogenesis(15-17).

The principal substrates identified for cdks are the retinoblastomatumor suppressor protein, RB, and the related proteins pl07 and pl30(reviewed in Refs. 18 and 19). The RB family of proteins has beenimplicated in both cell cycle arrest and myogenic differentiation. It isbelieved that RB functions to inhibit cell cycle progression by bindingto and inhibiting the activity of the E2F family of transcription factors(18, 19). E2F regulates the expression of a number of genes requiredfor progression through the cell cycle (20). When E2F is repressed bybinding to RB, these genes are not expressed, and the cell cycle is

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GROWTH DEREGULATION IN RHABDOMYOSARCOMA

arrested (18, 19). However, the association of RB with E2F is disrupted by the cdk-mediated phosphorylation of RB, which leads to the

subsequent activation of E2F.The exact role of RB in myogenic differentiation is still somewhat

unclear. RB-deficient myoblasts can form principally normal myo-

tubes (12). However, these cells never become terminally arrested (12,21, 22). Furthermore, in a number of systems, RB has been shown tocooperate with MyoD for full activation of MyoD transcriptionalactivating function (12, 21-23).

To date, no analysis of the cell cycle response of RMS cell lines toculture in DM has been carried out. One of the best studied RMS celllines is the embryonal RMS cell line, RD. We have investigated thecellular response of RD cells to culture in DM by studying the activityof key cell cycle regulatory proteins. Here, we report that RD cells failto undergo a cell cycle arrest in response to culture in DM. These cellsexpress high levels of cdk and cyclin proteins and have elevated levelsof cdk-associated kinase activity relative to that of primary human

myoblasts. Furthermore, cdk activity is not significantly attenuated byculture of RD cells in DM. Overexpression of exogenous pl6ink4,p21cipl, or p27kipl results in the inhibition of cell cycle progressionin RD cells. Likewise, the expression of a constitutively active RBprotein also caused cell cycle inhibition. Although growth inhibited,these cells did not express the myogenic marker, MHC. Thus, weconclude that the elevated levels of cdks may contribute to the growthabnormality of RD cells but that they are not alone responsible for thedifferentiation defect in RD cells.

MATERIALS AND METHODS

Cell Culture. Cryopreserved normal human skeletal muscle cells from asingle donor were purchased from Clonetics Corp. (San Diego, CA) or obtained from muscle biopsies as described previously under approval of theUniversity of California at San Diego Human Subjects Committee (24).Undifferentiated myoblasts were grown in skeletal muscle growth medium[PM (myoblast)] SkGM BulletKit (Clonetics). in which the basal medium wassupplemented with 50 mg/ml insulin, 0.01 mg/ml human epidermal growthfactor, 0.5 mg/ml fetuin, 0.5 mg/ml BSA, 2 mM L-glutamine. 100 units/ml

penicillin. 100 mg/ml streptomycin sulfate, and 2% (v/v) FBS (Gemini. Calabasas. CA) in 5% (v/v) CO2 humidified atmosphere. To induce differentiation,cells were grown to 80-90% confluence, at which point the growth medium

was changed to DMEM (Fisher, Pittsburgh, PA) supplemented with 2 mML-glutamine, 100 units/ml penicillin, 100 mg/ml streptomycin sulfate, and 2%

(v/v) donor horse serum (Gemini; DM). RD cells were obtained from American Type Culture Collection at passage 36 (CCL-136). RD cells were only

cultured up to 10 additional passages, as extended culturing yielded heterogeneity in growth properties. The cells were cultured in DMEM supplementedwith 2 mM L-glutamine, 100 units/ml penicillin. 100 mg/ml streptomycin

sulfate, and 10% (v/v) FBS (PM; RD). To induce differentiation, RD cells weregrown to a 70-80% confluence, and the growth medium was changed toDMEM supplemented with 2% (v/v) donor horse serum (DM) for 4-5 days.

Plasmids. The green fluorescent protein expression plasmid was obtainedfrom commercial sources (Life Technologies, Inc.). The pl6ink4 andPSM.7-LP expression plasmids have been described previously (25). The

p27kipl expression plasmid was a gift of Dr. J. Pietenpol. The p2lciplexpression plasmid was constructed by Dr. Y. Chen. The E2F-luciferase andCMV-ß-galactosidase plasmids have also been described previously (25).

Immunostaining. Cells were fixed in 3.7% formaldehyde in PBS for 10min and permeabilized in 0.3% Triton-PBS for 10 min. Primary antibodies

were diluted appropriately and detected with fluorescently labeled secondaryantibodies. BrdUrd incorporation was detected with a rat monoclonal antibody(Accurate Scientific) as described (23). BrdUrd labeling of RD cells wascarried out for approximately 30 h. MHC expression was detected with F59antibody (generously provided by Dr. Frank Stockdale). Cells were examinedwith a Zeiss Axiophot epifluorescence microscope (Carl Zeiss, Inc.) andphotographed under a X40 objective.

Kinase Assays. Cdk2 kinase assays were carried out as described in detailfor cdc2 (26), with the exception that cdk2 from 200 ftg of total cell lysate wasspecifically immunoprecipitated with an anti-cdk2 polyclonal antibody (Santa

Cruz Scientific, Santa Cruz, CA). Cdk4 kinase assays were carried out asdescribed (27), with the exception that a COOH-terminal GST-RB fusion

protein was used as a substrate.Immunoblot Analysis. Cells were resuspended in radioimmunoprecipita-

tion assay buffer, and 10 fig of total cellular protein from each cell type wereadded to Laemmli sample buffer and separated by PAGE. The proteins weretransferred to Immobilon-P membranes (Millipore) by semidry transfer according to the manufacturer's instructions (Bio-Rad). Membranes were incu

bated with primary and secondary antibodies and visualized by chemilumi-

nescence (ECL. Amersham). RB. pl07. and pl30 proteins were resolved by6.5% SDS-PAGE. and the RB protein was detected with the 851 antibody (28).

whereas pl07 and pl30 were detected with polyclonal antibody from SantaCruz Scientific. Cyclin proteins were resolved on 10-12% SDS-PAGE. Cyclin

A protein was detected with polyclonal antibody (kindly provided by Dr. TonyHunter). Cyclin DI protein was detected with polyclonal antibody from SantaCruz Scientific. The cyclin E antibody has been described previously (29).Cdk4 and pdk2 protein were also detected with polyclonal antibodies obtainedfrom Santa Cruz Scientific. The cdk inhibitors p21cipl, p27kipl, and pl6ink4were all detected with commercially available antibodies (Santa Cruz Scientific, PharMingen. and Signal Transduction Laboratories).

Coimmunoprecipitation. RD cells were lysed in NET-N 1100 mMNaCI, 1mM EDTA, 20 mM Tris (pH 8.0), and 0.5% Nonidet P-40] buffer supplementedwith protease (Complete Inhibitor Cocktail; Boehringer Mannheim) and phos-phatase inhibitors (10 mM NaF and 10 mM sodium pyro-phosphate). Cells were

subjected to sonication, lysates were clarified by centrifugation. and equal totalprotein was subjected to immunoprecipitation with either a nonspecific antibody (mdm-2. Santa Cruz Scientific) or an antibody directed against cdk4,

cdk2, p21cipl, pl6ink4. or p27kipl (all obtained from Santa Cruz Scientific).Immunoprecipitaled proteins were recovered on protein A-Sepharose andwashed four times with NET-N. Immunocomplexes were denatured by boilingin Laemmli buffer, resolved by SDS-PAGE. and transferred to Immobilon-P

(Millipore). Proteins were then detected by immunoblot as described above.Growth Inhibition Assays. RD cells were transfected using calcium phos

phate precipitation. For inhibition of RD cell growth, 5 fig of the effectorplasmid were cotransfected with 1 fig of CMV-GFP expression plasmid.

Thirty h posttransfection, BrdUrd was added to the cells that were harvestedafter 30 h of labeling. Transfected cells were detected by GFP-mediated

fluorescence, and BrdUrd incorporation was measured to indicate progressionthrough S phase. Entire coverslips of transfected cells were counted.

E2F Inhibition Assays. RD cells were cotransfected with 2 fig of theE2F-luciferase reporter, 1 fig of the CMV-ß-galactosidase plasmid, and 12 fig

of the effector plasmid. Cells were harvested 48 h posttransfection and processed for luciferase assays, as described for the Luciferase Assay System(Promega). Luciferase activity was normalized to ß-galactosidase activity for

transfection efficiency.

RESULTS

RD Cells Fail to Arrest or Differentiate in Response to Culturein DM. Primary human skeletal myoblasts and RD cells were grownin culture to allow for the comparison of cell cycle and differentiativeresponses. Skeletal myoblasts can be induced to differentiate byculture in mitogen-poor medium (DM). This culturing condition

causes a cell cycle arrest in G,/G, that can be scored by the inhibitionof BrdUrd incorporation (Table 1). which is followed by the expression of muscle-specific genes such as MHC, which can be monitored

by immunofluorescence staining (Table 1). Following 4 days ofculture in DM, skeletal myoblasts gave rise to many fused myotubes,of which virtually 100% stained positively for MHC expression. Onaverage, greater than 50% of the nuclei in such a culture were withincells that were positive for MHC. In contrast, RD cells failed toeffectively differentiate in response to culture in DM. They failed toform multinucleated myotubular cell structures, and they showed onlya modest increase in MHC staining, with approximately 90% of the

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RD cells in such a culture staining negative for MHC expression(Table 1). Furthermore, the MHC protein was not as well organized inthese RD cells as it was in normal myotubes (data not shown). Thisfinding is consistent with previous studies showing that RD cells donot effectively differentiate in response to culture in mitogen-poor

medium (30). Interestingly, the RD cells also failed to growth arrest inresponse to this culturing condition. As shown in Table 1, after 4 daysof growth in DM, approximately 50% of RD progressed through Sphase, as measured by BrdUrd incorporation, which was similar to thelevel of BrdUrd incorporation of RD cells cultured in PM. Examination of individual RD cells grown in DM showed that those cells thatexpressed MHC did not incorporate BrdUrd (Fig. 1), indicating thatthese cells had both withdrawn from the cell cycle and had activatedmyogenic gene expression. Together, these results show that the bulkof RD cells fail to undergo the cell cycle arrest and myogenicdifferentiation but that a small percentage show both phenotypes.Because differentiation is highly dependent on exit from the prolifer-

ative cycle, we suspected that the failure of the majority of RD cellsto differentiate could be due, in part, to this overall failure to arrest.

RD Cells Grown in DM Contain Hyperphosphorylated RB. Tounderstand the basis for the failure of RD cells to arrest in response toculture in DM, we investigated the abundance and activity of key cellcycle regulatory proteins. Initially, we analyzed the phosphorylation

Table 1 DNA synthesis ami MHC expression in RD and priman- human

mvoblaslic cells

Celltype"Skeletal

myoblastsRDCulture

condition*Proliferation

(PM)Differentiation(DM)Proliferation(PM)Differentiation

(DM)%

DNA synthesisc87186553%MHCa<1>501.310

" Either skeletal primary human myoblasts or the embryonal RMS/RD cells were

cultured.* Cells were cultured for proliferation (PM) or in 2% horse serum to stimulate

myogenic differentiation (DM), as described in "Materials and Methods."c Actively proliferating cells (cultured in PM) or cells that had been cultured in DM for

4 days were labeled with BrdUrd. Cells were fixed and processed for immunofluorescenceincorporation of BrdUrd. All data presented reflects the total number of nuclei analyzedfrom at least two independent experiments. The % DNA synthesis was determined as thepercentage of nuclei that stain positively for BrdUrd incorporation.

d Actively proliferating cells (cultured in PM) or cells that had been cultured in DM for

4 days were stained for the expression of MHC. Following culture in DM, fusion ofskeletal myoblasts gives rise to multinucleated myotubes, of which 100% score positivefor MHC staining. For RD cells, there was no detectable induction of multinucleated cellsfollowing culture in DM. All data presented reflect the total number of cells analyzed fromat least two independent experiments. The % MHC was determined as the percentage ofnuclei that are in MHC-staining cells.

Fig. 1. RD cells fail to differentiate through culture in DM. A, RD cells grown in PMwere labeled for 30 h with BrdUrd and processed for indirect immunofluorescencestaining. Cells were stained with both anti-BrdUrd and anti-MHC antibodies and thenphotographed at X40 magnification. Shown is a double exposure showing both the MHCstaining (cytoskeletal) and BrdUrd staining (nuclear). Note that MHC-positive cells are

BrdUrd negative. B, RD cells grown in DM were labeled for 30 h with BrdUrd andprocessed for indirect immunofluorescence staining. Cells were stained with both anti-BrdUrd and anti-MHC antibodies and then photographed at X40 magnification. Shown is

a double exposure showing both the MHC staining (cytoskeletal) and BrdUrd staining(nuclear). Note that MHC-positive cells are BrdUrd negative.

B

myoblasts

PM DM PM DM

1234

myoblasts RP

PM DM PM DM

ccRBimmunoblot

•- —••- ocp107 immunoblot

1234

myoblasts RP

PM DM PM DM

bai ^9 — api 30 immunoblot

1234Fig. 2. RD cells retain hyperphosphorylated RB. Equal total protein from myoblasts

(Lanes I and 2) or RD cells (Lanes 3 and 4), cultured in PM (Lanes 1 and 3) or DM (Lanes2 and 4), was resolved by 6.5% SDS-PAGE. A, proteins were transferred to Immobilon-P

membrane, and RB protein was detected by immunoblotting. ppRB, hyperphosphorylatedRB; pRB, underphosphorylated RB. B. proteins were transferred to Immobilon-P mem

brane. and p 107 protein was detected by immunoblotting. ppl07, hyperphosphorylatedpl07; pl07, underphosphorylated pl07. C, proteins were transferred to Immobilon-P

membrane, and pl30 protein was detected by immunoblotting. ppl30. hyperphosphorylated pl30; pl30, underphosphorylated pl30.

status of RB and the related proteins p 107 and p 130. Phosphorylationof these proteins is catalyzed by cdk/cyclin complexes and investigation of the behavior of these proteins reflects cdk activity within thecell (18, 19). We found that, as expected, RB existed in both thehyperphosphorylated (ppRB) and hypophosphorylated (pRB) forms inskeletal myoblasts (Fig. 2A, Lane 1). After culture in DM, RB migrated as a single hypophosphorylated band (Fig. 2A, Lane 2). Incontrast, culture of RD cells in DM did not significantly change thephosphorylation status of RB (Fig. 2A, compare Lanes 3 and 4).

The RB-related proteins p 107 and p 130 were also found to be

phosphorylated in myoblasts and hypophosphorylated in myotubes(Fig. 2, B and C, Lanes I and 2). The overall abundance of theseproteins also markedly changed, with the abundance of p 130 increasing (Fig. 1C) and the abundance of p 107 (Fig. 2B) diminishing as cellsdifferentiated. The response of p 107 and p 130 to DM in RD cells wassimilar to that seen in myoblasts because these cells showed anincrease in the hypophosphorylated forms of these proteins; likewise,there was a change in protein abundance following culture in DM(Fig. 2, B and C, Lanes 3 and 4). These results suggest that there is astriking down-regulation of cdk activity in myotubes, whereas RD

cells cultured in DM retain cdk activity, albeit at a lower level thanthat observed in cells grown in PM.

RD Cells Exhibit Elevated cdk/cyclin Activity. On the basis ofthe observation of hyperphosphorylated RB in RD cell culture in DM,we reasoned that elevated cdk/cyclin activity may play a role in thefailure of RD cells to arrest and/or differentiate in response to DM.Both cdk4- and cdk2-associated kinase activity have been shown to

phosphorylate RB (18, 19). Furthermore, both of these kinases arerequired for progression through G, to S phase (31). Following culture

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in DM, myoblasts exhibited a reduction in the amount of cdk2 protein,whereas cdk4 levels remain unchanged (Fig. 3, A and B, Lanes 1 and2). Relative to myoblasts, RD cells expressed more cdk2 and cdk4protein, neither of which was influenced by culture in DM (Fig. 3, Aand ß,Lanes 3 and 4).

It has been previously reported that cdk4 and cdk2 kinase activityin myoblasts is down-regulated in response to culture in DM (13). To

determine the relative kinase activity of cdk4 and cdk2 in RD cells,kinase complexes were isolated by immunoprecipitation and used forin vitro kinase reactions. As shown in Fig. 4A, RD cells exhibitedelevated levels of cdk4-associated kinase activity as compared with

myoblasts (compare Lanes 2 and 4). Likewise, RD cells contained

A myoblasts RP

PM DM PM DM

cdk4- — — fv ^ occdk4immunoblot

1234

B myoblasts Rp

PM DM PM DM

cdk2- — ¿•»" acdk2 immunoblot

1234Fig. 3. RD cells express elevated levels of cdks. Equal total protein from myoblasts

(Lanes 1 and 2) or RD cells (Lanes 3 and 4), cultured in PM (Lanes I and 3) or DM (Lanes2 and 4), was resolved by 12% SDS-PAGE. Proteins were transferred to Immobilon-P,

and cdk4 protein (A) and cdk2 protein (B) were detected by immunoblotting.

ucdk4CM

•BMyoblasts RD

PM DM PM

GST-RB -

cdk4 -

autoradiogram

ucdk4 immunoblot

Bi1

ocdk2

Myoblasts

PM DM

RD

-

cdk2-|

autoradiogram

immunoblot

1Fig. 4. RD cell exhibit elevated cdk activity. Equal total protein from myoblasts (Lanes

2 and 3} or RD cells (Lanes 1, 4, 5, and 6), cultured in PM (Lanes 1, 2, 4, and 5) or DM(Lanes 3 and 6), was immunoprecipitated with either mdm-2 (Lane /) or cdk4 (Lanes 2-6)

antibody. A, resulting immunoprecipitates were used in in vitro kinase assays withGST-RB as a substrate, and kinase reactions were resolved by 12% SDS-PAGE andtransferred to Immobilon-P. Top, autoradiogram of phosphorylated GST-RB; bottom,

cdk4 protein in the immunoprecipitation detected by immunoblotting. ß,resulting immunoprecipitates were used in in vitro kinase assays with histone HI as a substrate, andkinase reactions were resolved by 12% SDS-PAGE and transferred to Immobilon-P. Top,

autoradiogram of phosphorylated histone H l ; bottom. cdk2 protein in the immunoprecipitation detected by immunoblotting.

A myoblasts RP

PM DM PM DM

cycDI- . — acycD! immunoblot

1234

B myoblasts RP

PM DM PM DM•

cycE- 3JB 0p acycE immunoblot

1234

Q myoblasts RP

PM DM PM DM

cycA- — occycAimmunoblot

1234Fig. 5. RD cells express elevated levels of cyclins. Equal total protein from myoblasts

(Lanes I and 2) or RD cells (Lanes 3 and 4), cultured in PM (Lanes I and 3) or DM (Lanes2 and 4}, was resolved by 12% SDS-PAGE. Proteins were transferred to Immobilon-P,

and cycDI protein (A), cycE (B), and cycA protein (O were detected by immunoblotting.

elevated levels of cdk2-associated activity (Fig. 4B, compare Lanes 2

and 4). These results are consistent with the elevated expression of therespective cdks in RD cells (Fig. 4, bottom panels). Myoblasts cultured in DM had reduced cdk4 activity and barely detectable cdk2kinase activity (Fig. 4, compare Lanes 2 and 3). In contrast, cdk4activity in RD cells was only slightly reduced by culture in DM (Fig.4A, compare Lanes 5 and 6), and there was no detectable reduction incdk2 activity in RD cells (Fig. 4B, compare Lanes 5 and 6).

To determine the basis for the enhanced activity of cdk4 and cdk2,we analyzed both the expression of cyclins, which can activate cdkactivity, and the presence of cdk inhibitors. We found that RD cellsexpressed elevated levels of cyclin Dl (Fig. 5A, Lanes I and J), whichnormally associates with cdk4. Unlike myoblasts, in which culture inDM leads to a slight reduction in cyclin Dl expression, we found thatcyclin Dl protein in RD cells was slightly induced by culture condition (Fig. 5A). Similarly, we found that cyclin E and cyclin A wereboth elevated in RD cells and, in the case of cyclin E, unresponsive toculture in DM (Fig. 5B, Lanes 3 and 4). Interestingly, levels of cyclinA were reduced by the culture of RD cells in DM (Fig. 5C, Lanes 3and 4). However, the level of cyclin A protein in RD cells grown inDM was still above that in myoblasts.

Because cdk inhibitors have been implicated in differentiation, cellcycle arrest, and tumorigenesis, we analyzed their expression in RDcells. We found that the p21cipl levels in RD cells were muchreduced relative to myoblasts (Fig. 6/4). Although p21cipl expressionwas stimulated in myoblasts cultured in DM, culture of RD cells inDM had only a minor enhancement on p21cipl protein level (Fig. 6A).As an indirect measure of the activity of the p21cipl expressed in RDcells, we determined if it specifically associated with cdk2. By coim-

munopreciptitation, p21cipl was, indeed, found to interact with cdk2(Fig. 7, A and D). p27kipl was highly induced in both RD andmyoblasts following culture in DM (Fig. 6B). Like p21cipl, p27kiplwas also found to be active in binding to cdk2, as determined by

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myoblasts RD

PM DM PM DM

p2lcip1- »* ^» ap21cip1 immunoblot

1234

g myoblasts

PM DM PM DM

p27kipl - 4» •» ap27kip1 immunoblot

1234

RDmyoblasts

PM DM PM DM

p16ink4- ap16ink4 immunoblot

1234Fig. 6. RD cells express cdk inhibitors. Equal total protein from myoblasts (Lanes I

and 2) or RD cells (Lanes 3 and 4), cultured in PM (Lanes I and 3) or DM (Lanes 2 and4), was resolved by 15% SDS-PAGE. Proteins were transferred to Immobilon-P. and

p21cipl protein (A}, p27kipl protein (B). and pl6ink4 protein (Q were detected byimmunoblotting.

coimmunoprecipitation (Fig. 7, B and E). These finding suggest that,although RD cells express inhibitors for cdk4 and cdk2, the elevatedlevel of cdk2 and cdk4 in RD cells is such that the induced pZlcipland p27kipl are incapable of effectively inhibiting kinase activity.

The pl6ink4 cdk4-inhibitor is a tumor suppressor that is lost in a

number of tumor cell types, including RMS (31, 32). Interestingly, wefound that RD cells expressed high levels of pl6ink4 (Fig. 6), far andabove that of either myoblasts or myotubes. This was somewhatsurprising because RD cells have hyperphosphorylated RB, growreadily in both PM and DM, and also have relatively high cdk4activity. Furthermore, previous reports have indicated that RD cells donot express pl6ink4 (32). We confirmed the expression of pl6ink4 byimmunoblotting, immunoprecipitation, RNase protection, and immu-

nofluorescence (Figs. 6C and 7, C and F; and data not shown).Because pl6ink4 is expressed in such abundance, we determinedwhether it was active for binding to cdk4. As shown in Fig. 7, wefound that pl6ink4 coimmunoprecipitated with cdk4 but not with thenonspecific control mdm-2.

Specific Arrest of RD Cells. As described above, RD cells exhibitelevated levels of cdk kinase activity that were not effectively attenuated by culture in DM. To determine whether this activity is requiredfor the cell cycle progression of RD cells, we ectopically expressedthe cdk inhibitors p21cipl, pl6ink4, and p27kipl. All of these proteins have been previously shown to arrest specific cell types in G,.Cells were cotransfected with a GFP expression plasmid and expression plasmids encoding each of the cdk inhibitors. Cell cycle progression was scored by measuring BrdUrd incorporation of the trans-fected, GFP-positive cells. Vector-transfected RD cells incorporated

BrdUrd, approximately as effectively as did nontransfected cells (Fig.8/4). Therefore, the expression of GFP does not inhibit the growth ofRD cells. Expression of p21cipl and p27kipl effectively inhibited cellgrowth, leading to a reduction in BrdUrd incorporation (Fig. SA).Interestingly, pl6ink4, which is already highly expressed in RD cells,also inhibited the growth of RD cells (Fig. SA). Immunofluorescencestaining of the pl6ink4 transfected cells showed high levels of en

dogenous staining; however, transfected cells exhibited dramaticallyenhanced levels of pl6ink4 (data not shown). These results indicatethat blockade of cdk4 and/or cdk2 activity is sufficient to arrest thegrowth of RD cells.

Because a key substrate of cdk/cyclins is the retinoblastoma tumorsuppressor, we analyzed the effect of ectopie expression of a consti-tutively active RB protein fragment (PSM.7-LP), which cannot beregulated by cdk-mediated phosphorylation (25). We found that thisprotein also effectively inhibited RD cell growth, whereas the wild-type cognate (RB amino acids 379-928: WT-LP) was not efficient at

the inhibition of RD cell growth (Fig. SB). These results suggest thatproliferation of RD cells is due to the activity of cdk/cyclins, whichact through the phosphorylation of RB. A downstream effector for RBis the E2F family of transcription factors. We, therefore, assayed theeffect of either PSM.7-LP or pl6ink4 on the endogenous E2F activitywithin RD cells. Transfection of an E2F-luciferase reporter construct

in RD cells revealed the presence of E2F activity. This activity wasinhibited by the expression of PSM.7-LP, which has been previously

shown to inhibit E2F activity (Fig. SQ. We found that expression ofpl6ink4 also inhibited E2F activity. These results suggest that proliferation of RD cells is dependent on the activity of the E2F family oftranscription factors (Fig. 8Q. Consistent with this idea, the micro-injection of anti-E2F antibodies specifically slowed RD cell growth

(data not shown).Although expression of cdk inhibitors or PSM.7-LP effectively

inhibited cell cycle progression, none of these proteins stimulateddetectable myogenic differentiation. When stained for MHC expression, there was no significant increase incurred by transfection withthe growth-inhibitory proteins (data not shown). The percentage ofpl6ink4a-, p21cipl-, p27kipl-, and PSM.7-LP-transfected cells stain

ing positively for MHC fluctuated between only 1 and 4%, which wassimilar to the staining observed upon vector transfection. Likewise,there was no observed morphological change associated with myogenic differentiation in the transfected cells. Therefore, the cell cyclearrest and differentiation of RD cells are separable processes.

DISCUSSION

The RMS cell line, RD, fails to arrest or differentiate when culturedin DM. This failure to arrest correlates with the persistence of hyperphosphorylated RB. Consistent with this finding, we found that RDcells express elevated levels of cyclins and cdks. Furthermore, the cdkactivity within RD cells was not significantly attenuated throughculture in DM. Somewhat contrary to this result, we found that RDcells expressed elevated levels of pl6ink4. However, ectopie expression of cdk inhibitors or a constitutively active RB protein led to cellcycle inhibition. Interestingly, we find that, although growth inhibited,the cells ectopically expressing cdk inhibitors or the constitutivelyactive RB still fail to differentiate.

Role of Elevated cdk/cyclins in RD Cells. RD cells continued toproliferate when cultured in DM. This occurred although p27kipl washighly induced, the levels of cdk4 activity in the cells were slightlydiminished, cyclin A protein levels were diminished, and the overallphosphorylation of pl07 and pl30 was reduced. Furthermore, there isan increase in the percentage of RD cells that stain positive for MHCexpression. These results suggest that RD cells are responding to thedifferentiation condition. However, due to the cellular context of RDcells, these changes are not sufficient to bring about exit from the cellcycle for the majority of RD cells. A possible explanation for thefailure of RD cells to arrest is the high levels of cdk4, cdk2, and theirassociated cyclins (Dl, E, and A). These high levels of cdk/cyclinscontributed to a dramatically increased cdk4 and cdk2 activity in RDcells. Treatment with DM does result in the modest down-regulation

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GROWTH DEREGULATION IN RHABDOMYOSARCOMA

IP IP

p21 - «—«(/p21 immunoblot

1 2 3

B IP

p27 - ^p ap27 immunoblot

1 2 3

C IP

cdk2 - Gtcdk2 immunoblot

1 2 3

E IP

cdk2- —* ucdk2 immunoblot

1 2 3

F IP

p16 - ap16 immunoblot cdk4-

1 1

/acdk4 immunoblot

Fig. 7. The cdk inhibitors in RD cells are active for binding cdks. A. equal total protein from RD cells cultured in DM was subjected to immunoprecipitation with mdm-2 (Lane/), cdk2 (Lane 2). or p2lcipl (Lane 3} antibodies. Immunoprecipitated proteins were resolved by 15% SDS-PAGE and transferred to lmmobilon-P membrane. p2lcipl protein wasthen detected by immunoblotting. B. equal total protein from RD cells cultured in DM was subjected to immunoprecipitation with mdm-2 (Lane I), cdk2 (Lane 2), or p27kipl (Lane3) antibodies. Immunoprecipitated proteins were resolved by 15% SDS-PAGE and transferred to Immobilon-P. p27kipl protein was then detected by immunoblotting. C. equal totalprotein from RD cells cultured in DM was subjected to immunoprecipitation with mdm-2 (Lane I). cdk4 (Lane 2), or pl6ink4 (Lane 3) antibodies. Immunoprecipitated proteins wereresolved by 15% SDS-PAGE and transferred to Immobilon-P. pl6ink4 protein was then detected by immunoblotting. D, equal total protein from RD cells cultured in DM was subjectedto immunoprecipitation with mdm-2 (Lane I), cdk2 (Lane 2), or p2Icipl (Lane 3) antibodies. Immunoprecipilated proteins were resolved by 15% SDS-PAGE and transferred toImmobilon-P. cdk2 protein was then detected by immunoblotting. E, equal total protein from RD cells cultured in DM was subjected to immunoprecipitation with mdm-2 (Lane I),cdkl (Lane 2), or p27kipl (Lane 3) antibodies. Immunoprecipitated proteins were resolved by 15% SDS-PAGE and transferred to Immobilon-P. cdk2 protein was then detected byimmunoblotting. F. equal total protein from RD cells cultured in DM was subjected to immunoprccipitation with mdm-2 (Lane /). cdk4 (Lane 2), or pI6ink4 (Lane 3) antibodies.Immunoprecipitated proteins were resolved by 15% SDS-PAGE and transferred to Immobilon-P. cdk4 protein was then detected by immunoblotting.

of cdk4 activity. However, this reduced cdk4 activity was still considerably higher than what is observed in myoblasts. Furthermore, thecdk2 activity in RD cells was not affected by culture in DM. Thissuggests that the increased levels of p27kipl are preferentially inhibiting cdk4 and are too low relative to the cdk proteins in the cell toeffectively block kinase activity. Alternatively, the possibility exists,though, that p27kipl is active for binding to cdk2 and cdk4 but is notactive for inhibiting kinase activity. However, taken together, thesefindings suggest that the high levels of cyclins and cdk may contributeto the oncogenic growth of these cells. Consistent with this idea is thefinding that cyclins and or cdks can exhibit oncogenic activity (31).

Interestingly, RD cells expressed high levels of the pl6ink4 protein.This was surprising, given that RD cells proliferated and containedphosphorylated RB. Furthermore, previous analysis of RD cells suggested that the entire locus covering pl6ink4 and pl5ink4 was ho-

mozygously deleted in RD cells (32). By immunoblotting, immunoprecipitation, and immunofluorescence with two different antibodies,we confirmed the presence of the pl6ink4 protein. Furthermore, wealso detected pl6ink4 mRNA expression by RNase protection, whichargues against homozygous deletion. A possible explanation for thediscrepancy could be due to different culture conditions of the RDcells because numerous reports indicate that pl6ink4 expression canbe lost in culture (33, 34). Analysis of the cdk4 binding activity ofpl6ink4 showed that the pl6ink4 expressed in RD cells was active forbinding to cdk4. Although the reason for this high level of expressionis unclear, it is possible that pl6ink4 acts to keep the activity of the

overexpressed cdk4/cyclin Dl proteins in check. This sort of balancehas been observed in tumor cells before and could reflect a selectiveadvantage or a protection against apoptosis. For example, cells thatare deficient for RB seldom express detectable cyclin Dl and oftenoverexpress pl6ink4 (35-37).

Relationship between Cell Cycle Progression and Differentiation in RMS Cell Lines. RMS cell lines exhibit a definite growthadvantage relative to human myoblasts in culture, which is bestvisualized by culturing in DM. In DM, myoblasts undergo withdrawalfrom the cell cycle and the eventual terminal growth arrest associatedwith complete differentiation to myotubes. In contrast, RD cellsshowed no detectable cessation of cell growth in DM. It is known thatcell cycle exit is required for the differentiation phenotype (9-11).

Oncogenes such as Ras, which mimic mitogenic signaling pathways,inhibit cell cycle withdrawal and myogenic differentiation (11). Furthermore, oncogenes that influence cell cycle regulatory proteins suchas SV40 large T-antigen or adenovirus EIA also inhibit myogenic

differentiation (9, 11). The failure of RMS cell lines to arrest whencultured in DM raised the possibility that failure to exit the cell cyclewas the basis for the failure of RD cells differentiate. We tested thishypothesis directly by inducing the cell cycle exit of RD cells by theoverexpression of growth-inhibitory proteins. These proteins effec

tively inhibited RD cell cycle progression; however, these proteins didnot stimulate the expression of muscle-specific genes such as MHC,

nor did they induce the morphological changes associated with myogenic differentiation in RD.

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Vector WT-LP PSM.7-LP

> 40-

Õ 10-

Vector PSM.7-LPpl6ink4

Fig. 8. Specific inhibition of RD cell growth. A and B. RD cells were cotransfectedwith a GFP expression plasmid and the indicated expression ptasmids. BrdUrd was addedto the cells 24 h posttransfection and carried out for a total of 30 h. Data shown is fromthree independent transfections with at least 200 transfected cells counted per experiment.C, RD cells were cotransfected with E2F-luciferase, CMV-ß-galactosidase, and eitherPSM.7-LP or pl6ink4 expression plasmids. Thirty-six h posltransfection, cells wereharvested, and luciferase activity was determined, which was normalized to ß-galacto-sidase for transfection efficiency. Data shown is from two independent transfections.

The induction of cell cycle arrest in G, was not sufficient tostimulate the myogenic differentiation of RD cells. Using eitherectopically expressed cdk inhibitors or constitutively active RB protein, we inhibited the proliferation of RD cells without inducing anydetectable myogenic differentiation. Similar results have been shownwith pharmacological reagents, such as mitomycin C, which block RDcell proliferation without inducing differentiation (38). Interestingly,several agents, such as the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, can induce the differentiated phenotype in RD cells (38).

However, it is unclear whether this differentiation is concomitant withan exit from the cell cycle.

Investigations into the specific differentiation defect involved inRD cells suggested that the defect is associated with a failure ofMyoD to function as a transcriptional activator (30). MyoD is expressed in RD and has DNA-binding activity; however, MyoD does

not stimulate transcription. MyoD activity has been studied in greatdetail and can be modified by a number of factors through either director indirect action.

The cell cycle machinery, particularly cyclins, can inhibit MyoD-

associated activity. Part of this action is manifested through thephosphorylation of RB (15-17). Underphosphorylated RB can func

tion to stimulate MyoD activity, and in the presence of ectopie cyclinexpression, RB is hyperphosphorylated and incapable of stimulatingMyoD-mediated gene expression (15, 16, 21, 22). Our findings that

pl6ink4, which blocks RB phosphorylation and a constitutively activeRB protein, PSM.7-LP, cannot induce myogenic differentiation sug

gest that the blockade of MyoD activity is not mediated through thephosphorylation or inactivation of the RB protein in RD cells. Ectopieexpression of cyclin Dl has been shown to inhibit MyoD activity ina manner independent of RB phosphorylation (15), suggesting that thehighly expressed cyclin Dl could be contributing to the overallinhibition of MyoD activity. Alternatively, it has been shown that, inthe absence of functional p53, myoblastic cells in culture undergo acell cycle arrest yet fail to differentiate (39). Interestingly, RD cellslack functional p53, and this could explain the failure of the arrestedcells to differentiate (40).

Recent investigations into the activity of MyoD has led to additional insights into how the protein is activated and functions as atranscriptional activator (41, 42). Extensive study into the defectsassociated with MyoD transactivation function may reveal a reason(s)why RD cells fail to differentiate. This line of investigation may alsoyield further insight into the interplay of the cell cycle and differen-

tiative processes in RMS.

ACKNOWLEDGMENTS

We thank Carolati Buckmaster and other members of the laboratory ofJ. R. F. for technical assistance. Dr. Steven I. Reed kindly provided theanti-cyclin E antibody. Dr. Frank Stockdale kindly provided the anti-MHC

antibody. Dr. Tony Hunter provided the cyclin A antibody.

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