Elevated Cyclins and Cyclin-Dependent Kinase Activity in the Rhabdomyosarcoma Cell Line RD1

[CANCER RESEARCH 58, 2042-2049, May I, 1998] Elevated Cyclins and Cyclin-dependent Kinase Activity in the Rhabdomyosarcoma Cell 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 Â¡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.Â¡,Cancer Center [E. S. K., C. P., T. L B., B. L B.. J. R. F.I. and Ludwig Institute for Cancer Research Â¡K.E. K.. K. C. A.], University of California at San Diego, School of Medicine, La Mia, California 92093-0684

ABSTRACT RMS revealed the presence of specific chromosomal translocations, t(2;13) and t(l;13). These translocations result in the expression of a An important early event in the differentiation of skeletal muscle cells chimeric protein, encoded by regions of the Pax3 or Pax7 and FKHR is exit from the cell cycle, after which full expression of the muscle genes (3, 4). The Pax3-FKHR chimeric protein has been shown to phenotype occurs. Rhabdomyosarcoma (RMS), a tumor of skeletal muscle origin, expresses a number of muscle-specific proteins, including MyoD; harbor oncogenic activity and is believed to contribute to alveolar however, these cells fail to arrest or differentiate when cultured in differ RMS tumor formation and progression (5, 6). For embryonal RMS, a entiation medium (DM). To determine the basis for the failure of RMS common region of loss of heterozygosity on chromosome 11 has been cells to differentiate or arrest, we studied the molecular response of the identified (7, 8). However, the exact identity of a putative embryonal embryonal RMS cell line, RD, to culture in DM. Under these conditions, tumor suppressor gene has not yet been determined. the retinoblastoma protein (RB) was primarily in the hyperphosphory- Although they have apparently disparate methods of oncogenic lated state. This is in contrast to myoblasts cultured in DM, in which the transformation, both alveolar and embryonal tumors exhibit similar hypophosphorylated form of RB is exclusively present. Measurements of the expression and activities of cyclin-dependent kinases (cdks) cdk2 and defects in myogenic differentiation. Most RMS tumor cells are char acterized by the expression of several muscle-specific markers, such cdk4 indicated that RD cells maintained higher levels than do myoblasts, and the activity and abundance of these proteins did not significantly as the myogenic-promoting transcription factor MyoD. Although the decrease upon culture in DM in RD cells, as they did in myoblasts. expression of such factors typically correlates with myogenic differ Similarly, elevated expression of cyclins Dl, E, and A was observed in RD entiation, RMS cells fail to undergo terminal differentiation into cells. Interestingly, cdk inhibitors are expressed in RD cells, with pl6ink4 skeletal muscle (2). The failure of RMS cells to differentiate is expression markedly elevated relative to myoblasts. Ectopie expression of hypothesized to be one mechanism through which these cells gain the pllcipl, pl6ink4, or p27kipl caused a growth arrest of RD cells but not growth advantage necessary for tumor formation. detectable expression of a myogenic marker. Furthermore, a constitu- In culture, normal myoblasts can be induced to differentiate by tively active RB protein could also inhibit the growth of RD cells without culture in mitogen-poor medium (DM). Such a culturing condition inducing myogenic differentiation. Taken together, these data suggest that the elevated levels of cdk2 and/or cdk4 observed in RD cells contribute to leads to an arrest in the Gâ€ž/G,phase of the cell cycle, which is the inability of RD cells to growth arrest when cultured in DM but that followed by the induction of the differentiated phenotype character these activities alone are not responsible for the failure of RD cells to ized by the expression of muscle-specific genes and, ultimately, the differentiate. formation of multinucleated myotubes (reviewed in Refs. 9-11). Cell cycle withdrawal is required for this process to occur. The expression of oncogenes that prevent cell cycle arrest also prevents the expres INTRODUCTION sion of the differentiated phenotype in myoblastic cell lines (9-11). RMS4 is the most commonly occurring soft tissue sarcoma in The arrest of cells undergoing myogenic differentiation has been children (1). RMS tumors resemble normal fetal skeletal muscle in studied in great detail. The overall process of myogenic differentiation morphology, and the cells of RMS tumors express several well-known is accompanied by the down-regulation of cdk activity, which is muscle-specific genes. As such, RMS tumors are believed to arise required for cell cycle progression. In immortalized murine myoblas from muscle tissue (1, 2). Several distinct histological subtypes of tic cell lines, cdk2 and cdk4 kinase activity is dramatically inhibited. RMS have been described: alveolar, embryonal, botryoid, and undif- This inhibition occurs through at least two mechanisms. One mech anism is via the down-regulation of cyclin expression, which is ferentiated (1, 2). Although survival rates vary with histological assignment, overall long-term survival rates of children with RMS are required for cdk activity. For example, cyclin A, which is associated only 50-70%. Investigations into the molecular basis of RMS have with cdk2, is barely detectable in differentiated myotubes (9, 12, 13) centered on the chromosomal alterations associated with specific A second mechanism is via the induction of cdk inhibitor expression. histological classes of RMS (1, 2). Studies of the alveolar type of For example, p21cipl and pl8ink4c proteins are both highly induced in myoblasts cultured in DM (9, 13, 14). Down-regulation of cdk

Received 10/21/97; accepted 3/3/98. activity is important for the expression of the differentiated phenotype The costs of publication of this article were defrayed in part by the payment of page because ectopie overexpression of cyclins can prevent myogenesis charges. This article must therefore be hereby marked advertisement in accordance with (15-17). 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by grants from the NIH/National Institute on Aging (to The principal substrates identified for cdks are the retinoblastoma J. R. F.) and from the California Tobacco Related Diseases Research Program. E. S. K. tumor suppressor protein, RB, and the related proteins pl07 and pl30 was supported by a NIH/National Cancer Institute Training Grant T32CA09290 to the University of California at San Diego Cancer Center. C. P. was supported by a postdoc (reviewed in Refs. 18 and 19). The RB family of proteins has been toral fellowship from the Cancer Research Foundation of America. implicated in both cell cycle arrest and myogenic differentiation. It is 2 These authors contributed equally to this work. 3 To whom requests for reprints should be addressed, at University of California at San believed that RB functions to inhibit cell cycle progression by binding Diego Cancer Center, Mail Code 0684, 9500 Oilman Drive, La Jolla, CA 92093-0684. to and inhibiting the activity of the E2F family of transcription factors 4 The abbreviations used are: RMS, rhabdomyosarcoma; DM, differentiation medium; (18, 19). E2F regulates the expression of a number of genes required cdk, cyclin-dependent kinase; MHC, myosin heavy chain; CMV, cytomegalovirus; BrdUrd, bromodeoxyuridine; GST, glutathione S-transferase; GFP, green fluorescent for progression through the cell cycle (20). When E2F is repressed by protein; PM, proliferation medium. binding to RB, these genes are not expressed, and the cell cycle is 2042

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1998 American Association for Cancer Research. GROWTH DEREGULATION IN RHABDOMYOSARCOMA arrested (18, 19). However, the association of RB with E2F is dis Kinase Assays. Cdk2 kinase assays were carried out as described in detail rupted by the cdk-mediated phosphorylation of RB, which leads to the for cdc2 (26), with the exception that cdk2 from 200 ftg of total cell lysate was subsequent activation of E2F. specifically immunoprecipitated with an anti-cdk2 polyclonal antibody (Santa The exact role of RB in myogenic differentiation is still somewhat Cruz Scientific, Santa Cruz, CA). Cdk4 kinase assays were carried out as unclear. RB-deficient myoblasts can form principally normal myo- described (27), with the exception that a COOH-terminal GST-RB fusion protein was used as a substrate. tubes (12). However, these cells never become terminally arrested (12, Immunoblot Analysis. Cells were resuspended in radioimmunoprecipita- 21, 22). Furthermore, in a number of systems, RB has been shown to tion assay buffer, and 10 fig of total cellular protein from each cell type were cooperate with MyoD for full activation of MyoD transcriptional added to Laemmli sample buffer and separated by PAGE. The proteins were activating function (12, 21-23). transferred to Immobilon-P membranes (Millipore) by semidry transfer ac To date, no analysis of the cell cycle response of RMS cell lines to cording to the manufacturer's instructions (Bio-Rad). Membranes were incu culture in DM has been carried out. One of the best studied RMS cell bated with primary and secondary antibodies and visualized by chemilumi- lines is the embryonal RMS cell line, RD. We have investigated the nescence (ECL. Amersham). RB. pl07. and pl30 proteins were resolved by 6.5% SDS-PAGE. and the RB protein was detected with the 851 antibody (28). cellular response of RD cells to culture in DM by studying the activity whereas pl07 and pl30 were detected with polyclonal antibody from Santa of key cell cycle regulatory proteins. Here, we report that RD cells fail Cruz Scientific. Cyclin proteins were resolved on 10-12% SDS-PAGE. Cyclin to undergo a cell cycle arrest in response to culture in DM. These cells A protein was detected with polyclonal antibody (kindly provided by Dr. Tony express high levels of cdk and cyclin proteins and have elevated levels Hunter). Cyclin DI protein was detected with polyclonal antibody from Santa of cdk-associated kinase activity relative to that of primary human Cruz Scientific. The cyclin E antibody has been described previously (29). myoblasts. Furthermore, cdk activity is not significantly attenuated by Cdk4 and pdk2 protein were also detected with polyclonal antibodies obtained culture of RD cells in DM. Overexpression of exogenous pl6ink4, from Santa Cruz Scientific. The cdk inhibitors p21cipl, p27kipl, and pl6ink4 p21cipl, or p27kipl results in the inhibition of cell cycle progression were all detected with commercially available antibodies (Santa Cruz Scien in RD cells. Likewise, the expression of a constitutively active RB tific, PharMingen. and Signal Transduction Laboratories). Coimmunoprecipitation. RD cells were lysed in NET-N 1100 mMNaCI, 1 protein also caused cell cycle inhibition. Although growth inhibited, mM EDTA, 20 mM Tris (pH 8.0), and 0.5% Nonidet P-40] buffer supplemented these cells did not express the myogenic marker, MHC. Thus, we with protease (Complete Inhibitor Cocktail; Boehringer Mannheim) and phos- conclude that the elevated levels of cdks may contribute to the growth phatase inhibitors (10 mM NaF and 10 mM sodium pyro-phosphate). Cells were abnormality of RD cells but that they are not alone responsible for the subjected to sonication, lysates were clarified by centrifugation. and equal total differentiation defect in RD cells. protein was subjected to immunoprecipitation with either a nonspecific anti body (mdm-2. Santa Cruz Scientific) or an antibody directed against cdk4, cdk2, p21cipl, pl6ink4. or p27kipl (all obtained from Santa Cruz Scientific). MATERIALS AND METHODS Immunoprecipitaled proteins were recovered on protein A-Sepharose and washed four times with NET-N. Immunocomplexes were denatured by boiling Cell Culture. Cryopreserved normal human skeletal muscle cells from a in Laemmli buffer, resolved by SDS-PAGE. and transferred to Immobilon-P single donor were purchased from Clonetics Corp. (San Diego, CA) or ob (Millipore). Proteins were then detected by immunoblot as described above. tained from muscle biopsies as described previously under approval of the Growth Inhibition Assays. RD cells were transfected using calcium phos University of California at San Diego Human Subjects Committee (24). phate precipitation. For inhibition of RD cell growth, 5 fig of the effector Undifferentiated myoblasts were grown in skeletal muscle growth medium plasmid were cotransfected with 1 fig of CMV-GFP expression plasmid. [PM (myoblast)] SkGM BulletKit (Clonetics). in which the basal medium was Thirty h posttransfection, BrdUrd was added to the cells that were harvested supplemented with 50 mg/ml insulin, 0.01 mg/ml human epidermal growth after 30 h of labeling. Transfected cells were detected by GFP-mediated factor, 0.5 mg/ml fetuin, 0.5 mg/ml BSA, 2 mM L-glutamine. 100 units/ml fluorescence, and BrdUrd incorporation was measured to indicate progression penicillin. 100 mg/ml streptomycin sulfate, and 2% (v/v) FBS (Gemini. Cala through S phase. Entire coverslips of transfected cells were counted. basas. CA) in 5% (v/v) CO2 humidified atmosphere. To induce differentiation, E2F Inhibition Assays. RD cells were cotransfected with 2 fig of the cells were grown to 80-90% confluence, at which point the growth medium E2F-luciferase reporter, 1 fig of the CMV-ÃŸ-galactosidase plasmid, and 12 fig was changed to DMEM (Fisher, Pittsburgh, PA) supplemented with 2 mM of the effector plasmid. Cells were harvested 48 h posttransfection and pro L-glutamine, 100 units/ml penicillin, 100 mg/ml streptomycin sulfate, and 2% cessed for luciferase assays, as described for the Luciferase Assay System (v/v) donor horse serum (Gemini; DM). RD cells were obtained from Amer (Promega). Luciferase activity was normalized to ÃŸ-galactosidase activity for ican Type Culture Collection at passage 36 (CCL-136). RD cells were only transfection efficiency. cultured up to 10 additional passages, as extended culturing yielded heteroge neity in growth properties. The cells were cultured in DMEM supplemented with 2 mM L-glutamine, 100 units/ml penicillin. 100 mg/ml streptomycin RESULTS sulfate, and 10% (v/v) FBS (PM; RD). To induce differentiation, RD cells were grown to a 70-80% confluence, and the growth medium was changed to RD Cells Fail to Arrest or Differentiate in Response to Culture DMEM supplemented with 2% (v/v) donor horse serum (DM) for 4-5 days. in DM. Primary human skeletal myoblasts and RD cells were grown Plasmids. The green fluorescent protein expression plasmid was obtained in culture to allow for the comparison of cell cycle and differentiative from commercial sources (Life Technologies, Inc.). The pl6ink4 and responses. Skeletal myoblasts can be induced to differentiate by PSM.7-LP expression plasmids have been described previously (25). The culture in mitogen-poor medium (DM). This culturing condition p27kipl expression plasmid was a gift of Dr. J. Pietenpol. The p2lcipl causes a cell cycle arrest in G,/G, that can be scored by the inhibition expression plasmid was constructed by Dr. Y. Chen. The E2F-luciferase and of BrdUrd incorporation (Table 1). which is followed by the expres CMV-ÃŸ-galactosidase plasmids have also been described previously (25). sion of muscle-specific genes such as MHC, which can be monitored Immunostaining. Cells were fixed in 3.7% formaldehyde in PBS for 10 by immunofluorescence staining (Table 1). Following 4 days of min and permeabilized in 0.3% Triton-PBS for 10 min. Primary antibodies culture in DM, skeletal myoblasts gave rise to many fused myotubes, were diluted appropriately and detected with fluorescently labeled secondary of which virtually 100% stained positively for MHC expression. On antibodies. BrdUrd incorporation was detected with a rat monoclonal antibody (Accurate Scientific) as described (23). BrdUrd labeling of RD cells was average, greater than 50% of the nuclei in such a culture were within carried out for approximately 30 h. MHC expression was detected with F59 cells that were positive for MHC. In contrast, RD cells failed to antibody (generously provided by Dr. Frank Stockdale). Cells were examined effectively differentiate in response to culture in DM. They failed to with a Zeiss Axiophot epifluorescence microscope (Carl Zeiss, Inc.) and form multinucleated myotubular cell structures, and they showed only photographed under a X40 objective. a modest increase in MHC staining, with approximately 90% of the 2043

RD cells in such a culture staining negative for MHC expression myoblasts (Table 1). Furthermore, the MHC protein was not as well organized in these RD cells as it was in normal myotubes (data not shown). This PM DM PM DM finding is consistent with previous studies showing that RD cells do not effectively differentiate in response to culture in mitogen-poor ccRB immunoblot medium (30). Interestingly, the RD cells also failed to growth arrest in response to this culturing condition. As shown in Table 1, after 4 days of growth in DM, approximately 50% of RD progressed through S 1234 phase, as measured by BrdUrd incorporation, which was similar to the level of BrdUrd incorporation of RD cells cultured in PM. Examina B myoblasts RP tion of individual RD cells grown in DM showed that those cells that expressed MHC did not incorporate BrdUrd (Fig. 1), indicating that PM DM PM DM these cells had both withdrawn from the cell cycle and had activated myogenic gene expression. Together, these results show that the bulk â€¢- â€”â€¢â€¢- ocp107 immunoblot of RD cells fail to undergo the cell cycle arrest and myogenic differentiation but that a small percentage show both phenotypes. 1234 Because differentiation is highly dependent on exit from the prolifer- ative cycle, we suspected that the failure of the majority of RD cells myoblasts RP to differentiate could be due, in part, to this overall failure to arrest. RD Cells Grown in DM Contain Hyperphosphorylated RB. To PM DM PM DM understand the basis for the failure of RD cells to arrest in response to culture in DM, we investigated the abundance and activity of key cell bai ^9 â€” api 30 immunoblot cycle regulatory proteins. Initially, we analyzed the phosphorylation 1234 Table 1 DNA synthesis ami MHC expression in RD and priman- human mvoblaslic cells Fig. 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 (Lanes Celltype"Skeletal condition*Proliferation DNA synthesisc87186553%MHCa<1>501.3102 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, hyperphosphorylated myoblastsRDCulture (PM)Differentiation RB; pRB, underphosphorylated RB. B. proteins were transferred to Immobilon-P mem (DM)Proliferation brane. and p 107 protein was detected by immunoblotting. ppl07, hyperphosphorylated (PM)Differentiation pl07; pl07, underphosphorylated pl07. C, proteins were transferred to Immobilon-P (DM)% membrane, and pl30 protein was detected by immunoblotting. ppl30. hyperphosphory " Either skeletal primary human myoblasts or the embryonal RMS/RD cells were lated pl30; pl30, underphosphorylated pl30. 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 immunofluorescence status of RB and the related proteins p 107 and p 130. Phosphorylation incorporation of BrdUrd. All data presented reflects the total number of nuclei analyzed of these proteins is catalyzed by cdk/cyclin complexes and investiga from at least two independent experiments. The % DNA synthesis was determined as the tion of the behavior of these proteins reflects cdk activity within the percentage of nuclei that stain positively for BrdUrd incorporation. d Actively proliferating cells (cultured in PM) or cells that had been cultured in DM for cell (18, 19). We found that, as expected, RB existed in both the 4 days were stained for the expression of MHC. Following culture in DM, fusion of hyperphosphorylated (ppRB) and hypophosphorylated (pRB) forms in skeletal myoblasts gives rise to multinucleated myotubes, of which 100% score positive for MHC staining. For RD cells, there was no detectable induction of multinucleated cells skeletal myoblasts (Fig. 2A, Lane 1). After culture in DM, RB mi following culture in DM. All data presented reflect the total number of cells analyzed from grated as a single hypophosphorylated band (Fig. 2A, Lane 2). In at least two independent experiments. The % MHC was determined as the percentage of contrast, culture of RD cells in DM did not significantly change the nuclei that are in MHC-staining cells. phosphorylation 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 these proteins also markedly changed, with the abundance of p 130 increas ing (Fig. 1C) and the abundance of p 107 (Fig. 2B) diminishing as cells differentiated. The response of p 107 and p 130 to DM in RD cells was similar to that seen in myoblasts because these cells showed an increase 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 a striking down-regulation of cdk activity in myotubes, whereas RD cells cultured in DM retain cdk activity, albeit at a lower level than that observed in cells grown in PM. Fig. 1. RD cells fail to differentiate through culture in DM. A, RD cells grown in PM were labeled for 30 h with BrdUrd and processed for indirect immunofluorescence RD Cells Exhibit Elevated cdk/cyclin Activity. On the basis of staining. Cells were stained with both anti-BrdUrd and anti-MHC antibodies and then the observation of hyperphosphorylated RB in RD cell culture in DM, 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 we reasoned that elevated cdk/cyclin activity may play a role in the BrdUrd negative. B, RD cells grown in DM were labeled for 30 h with BrdUrd and failure of RD cells to arrest and/or differentiate in response to DM. processed for indirect immunofluorescence staining. Cells were stained with both anti- Both cdk4- and cdk2-associated kinase activity have been shown to 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 phosphorylate RB (18, 19). Furthermore, both of these kinases are (nuclear). Note that MHC-positive cells are BrdUrd negative. required for progression through G, to S phase (31). Following culture 2044

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1998 American Association for Cancer Research. GROWTH DEREGULATION IN RHABDOMYOSARCOMA in DM, myoblasts exhibited a reduction in the amount of cdk2 protein, A myoblasts RP whereas cdk4 levels remain unchanged (Fig. 3, A and B, Lanes 1 and 2). Relative to myoblasts, RD cells expressed more cdk2 and cdk4 PM DM PM DM protein, neither of which was influenced by culture in DM (Fig. 3, A and ÃŸ,Lanes 3 and 4). cycDI- . â€” acycD! immunoblot It has been previously reported that cdk4 and cdk2 kinase activity in myoblasts is down-regulated in response to culture in DM (13). To 1234 determine the relative kinase activity of cdk4 and cdk2 in RD cells, kinase complexes were isolated by immunoprecipitation and used for in vitro kinase reactions. As shown in Fig. 4A, RD cells exhibited B myoblasts RP elevated levels of cdk4-associated kinase activity as compared with PM DM PM DM myoblasts (compare Lanes 2 and 4). Likewise, RD cells contained â€¢ cycE- 3JB 0p acycE immunoblot A myoblasts RP 1234 PM DM PM DM cdk4- â€” â€” fv ^ occdk4immunoblot Q myoblasts RP PM DM PM DM 1234 cycA- â€” occycAimmunoblot B myoblasts Rp 1234

PM DM PM DM Fig. 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 (Lanes cdk2- â€” Â¿â€¢Â»"^acdk2 immunoblot 2 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.

1234 Fig. 3. RD cells express elevated levels of cdks. Equal total protein from myoblasts elevated levels of cdk2-associated activity (Fig. 4B, compare Lanes 2 (Lanes 1 and 2) or RD cells (Lanes 3 and 4), cultured in PM (Lanes I and 3) or DM (Lanes and 4). These results are consistent with the elevated expression of the 2 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. respective cdks in RD cells (Fig. 4, bottom panels). Myoblasts cul tured in DM had reduced cdk4 activity and barely detectable cdk2 kinase activity (Fig. 4, compare Lanes 2 and 3). In contrast, cdk4 ucdk4 activity in RD cells was only slightly reduced by culture in DM (Fig. CM â€¢BMyoblasts RD 4A, compare Lanes 5 and 6), and there was no detectable reduction in PM DM PM cdk2 activity in RD cells (Fig. 4B, compare Lanes 5 and 6). To determine the basis for the enhanced activity of cdk4 and cdk2, GST-RB - autoradiogram we analyzed both the expression of cyclins, which can activate cdk activity, and the presence of cdk inhibitors. We found that RD cells expressed elevated levels of cyclin Dl (Fig. 5A, Lanes I and J), which cdk4 - ucdk4 immunoblot normally associates with cdk4. Unlike myoblasts, in which culture in DM leads to a slight reduction in cyclin Dl expression, we found that cyclin Dl protein in RD cells was slightly induced by culture condi B ocdk2 tion (Fig. 5A). Similarly, we found that cyclin E and cyclin A were i both elevated in RD cells and, in the case of cyclin E, unresponsive to Myoblasts RD culture in DM (Fig. 5B, Lanes 3 and 4). Interestingly, levels of cyclin 1 PM DM A were reduced by the culture of RD cells in DM (Fig. 5C, Lanes 3 and 4). However, the level of cyclin A protein in RD cells grown in - autoradiogram DM was still above that in myoblasts. Because cdk inhibitors have been implicated in differentiation, cell cdk2-| immunoblot cycle arrest, and tumorigenesis, we analyzed their expression in RD cells. We found that the p21cipl levels in RD cells were much 1 reduced relative to myoblasts (Fig. 6/4). Although p21cipl expression Fig. 4. RD cell exhibit elevated cdk activity. Equal total protein from myoblasts (Lanes was stimulated in myoblasts cultured in DM, culture of RD cells in 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) DM had only a minor enhancement on p21cipl protein level (Fig. 6A). antibody. A, resulting immunoprecipitates were used in in vitro kinase assays with As an indirect measure of the activity of the p21cipl expressed in RD GST-RB as a substrate, and kinase reactions were resolved by 12% SDS-PAGE and cells, we determined if it specifically associated with cdk2. By coim- transferred to Immobilon-P. Top, autoradiogram of phosphorylated GST-RB; bottom, cdk4 protein in the immunoprecipitation detected by immunoblotting. ÃŸ,resulting immu munopreciptitation, p21cipl was, indeed, found to interact with cdk2 noprecipitates were used in in vitro kinase assays with histone HI as a substrate, and (Fig. 7, A and D). p27kipl was highly induced in both RD and kinase reactions were resolved by 12% SDS-PAGE and transferred to Immobilon-P. Top, autoradiogram of phosphorylated histone H l ; bottom. cdk2 protein in the immunopre myoblasts following culture in DM (Fig. 6B). Like p21cipl, p27kipl cipitation detected by immunoblotting. was also found to be active in binding to cdk2, as determined by 2045

myoblasts RD dogenous staining; however, transfected cells exhibited dramatically enhanced levels of pl6ink4 (data not shown). These results indicate PM DM PM DM that blockade of cdk4 and/or cdk2 activity is sufficient to arrest the p2lcip1- Â»* ^Â» ap21cip1 immunoblot growth of RD cells. Because a key substrate of cdk/cyclins is the retinoblastoma tumor suppressor, we analyzed the effect of ectopie expression of a consti- 1234 tutively active RB protein fragment (PSM.7-LP), which cannot be regulated by cdk-mediated phosphorylation (25). We found that this protein also effectively inhibited RD cell growth, whereas the wild- g myoblasts type cognate (RB amino acids 379-928: WT-LP) was not efficient at PM DM PM DM the inhibition of RD cell growth (Fig. SB). These results suggest that p27kipl - 4Â» â€¢Â» ap27kip1 immunoblot proliferation of RD cells is due to the activity of cdk/cyclins, which act through the phosphorylation of RB. A downstream effector for RB is the E2F family of transcription factors. We, therefore, assayed the 1234 effect of either PSM.7-LP or pl6ink4 on the endogenous E2F activity within RD cells. Transfection of an E2F-luciferase reporter construct RD myoblasts in RD cells revealed the presence of E2F activity. This activity was PM DM PM DM inhibited by the expression of PSM.7-LP, which has been previously shown to inhibit E2F activity (Fig. SQ. We found that expression of p16ink4- ap16ink4 immunoblot pl6ink4 also inhibited E2F activity. These results suggest that prolif eration of RD cells is dependent on the activity of the E2F family of 1234 transcription factors (Fig. 8Q. Consistent with this idea, the micro- injection of anti-E2F antibodies specifically slowed RD cell growth Fig. 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 and (data not shown). 4), was resolved by 15% SDS-PAGE. Proteins were transferred to Immobilon-P. and Although expression of cdk inhibitors or PSM.7-LP effectively p21cipl protein (A}, p27kipl protein (B). and pl6ink4 protein (Q were detected by immunoblotting. inhibited cell cycle progression, none of these proteins stimulated detectable myogenic differentiation. When stained for MHC expres sion, there was no significant increase incurred by transfection with coimmunoprecipitation (Fig. 7, B and E). These finding suggest that, the growth-inhibitory proteins (data not shown). The percentage of although RD cells express inhibitors for cdk4 and cdk2, the elevated pl6ink4a-, p21cipl-, p27kipl-, and PSM.7-LP-transfected cells stain level of cdk2 and cdk4 in RD cells is such that the induced pZlcipl ing positively for MHC fluctuated between only 1 and 4%, which was and p27kipl are incapable of effectively inhibiting kinase activity. similar to the staining observed upon vector transfection. Likewise, The pl6ink4 cdk4-inhibitor is a tumor suppressor that is lost in a there was no observed morphological change associated with myo number of tumor cell types, including RMS (31, 32). Interestingly, we genic differentiation in the transfected cells. Therefore, the cell cycle found that RD cells expressed high levels of pl6ink4 (Fig. 6), far and arrest and differentiation of RD cells are separable processes. above that of either myoblasts or myotubes. This was somewhat surprising because RD cells have hyperphosphorylated RB, grow DISCUSSION readily in both PM and DM, and also have relatively high cdk4 activity. Furthermore, previous reports have indicated that RD cells do The RMS cell line, RD, fails to arrest or differentiate when cultured not express pl6ink4 (32). We confirmed the expression of pl6ink4 by in DM. This failure to arrest correlates with the persistence of hyper immunoblotting, immunoprecipitation, RNase protection, and immu- phosphorylated RB. Consistent with this finding, we found that RD nofluorescence (Figs. 6C and 7, C and F; and data not shown). cells express elevated levels of cyclins and cdks. Furthermore, the cdk Because pl6ink4 is expressed in such abundance, we determined activity within RD cells was not significantly attenuated through whether it was active for binding to cdk4. As shown in Fig. 7, we culture in DM. Somewhat contrary to this result, we found that RD found that pl6ink4 coimmunoprecipitated with cdk4 but not with the cells expressed elevated levels of pl6ink4. However, ectopie expres nonspecific control mdm-2. sion of cdk inhibitors or a constitutively active RB protein led to cell Specific Arrest of RD Cells. As described above, RD cells exhibit cycle inhibition. Interestingly, we find that, although growth inhibited, elevated levels of cdk kinase activity that were not effectively atten the cells ectopically expressing cdk inhibitors or the constitutively uated by culture in DM. To determine whether this activity is required active RB still fail to differentiate. for the cell cycle progression of RD cells, we ectopically expressed Role of Elevated cdk/cyclins in RD Cells. RD cells continued to the cdk inhibitors p21cipl, pl6ink4, and p27kipl. All of these pro proliferate when cultured in DM. This occurred although p27kipl was teins have been previously shown to arrest specific cell types in G,. highly induced, the levels of cdk4 activity in the cells were slightly Cells were cotransfected with a GFP expression plasmid and expres diminished, cyclin A protein levels were diminished, and the overall sion plasmids encoding each of the cdk inhibitors. Cell cycle progres phosphorylation of pl07 and pl30 was reduced. Furthermore, there is sion was scored by measuring BrdUrd incorporation of the trans- an increase in the percentage of RD cells that stain positive for MHC fected, GFP-positive cells. Vector-transfected RD cells incorporated expression. These results suggest that RD cells are responding to the BrdUrd, approximately as effectively as did nontransfected cells (Fig. differentiation condition. However, due to the cellular context of RD 8/4). Therefore, the expression of GFP does not inhibit the growth of cells, these changes are not sufficient to bring about exit from the cell RD cells. Expression of p21cipl and p27kipl effectively inhibited cell cycle for the majority of RD cells. A possible explanation for the growth, leading to a reduction in BrdUrd incorporation (Fig. SA). failure of RD cells to arrest is the high levels of cdk4, cdk2, and their Interestingly, pl6ink4, which is already highly expressed in RD cells, associated cyclins (Dl, E, and A). These high levels of cdk/cyclins also inhibited the growth of RD cells (Fig. SA). Immunofluorescence contributed to a dramatically increased cdk4 and cdk2 activity in RD staining of the pl6ink4 transfected cells showed high levels of en cells. Treatment with DM does result in the modest down-regulation 2046

IP IP

p21 - Â«â€”Â«(/p21immunoblot cdk2 - Gtcdk2 immunoblot

1 2 3 1 2 3 B IP E IP

p27 - ^p ap27 immunoblot cdk2- â€”* ucdk2 immunoblot

1 2 3 1 2 3 C IP F IP / p16 - ap16 immunoblot cdk4- acdk4 immunoblot 1 1 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 was then 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 (Lane 3) antibodies. Immunoprecipitated proteins were resolved by 15% SDS-PAGE and transferred to Immobilon-P. p27kipl protein was then detected by immunoblotting. C. equal total protein 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 were resolved 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 subjected to immunoprecipitation with mdm-2 (Lane I), cdk2 (Lane 2), or p2Icipl (Lane 3) antibodies. Immunoprecipilated proteins were resolved by 15% SDS-PAGE and transferred to Immobilon-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 by immunoblotting. 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 con overexpressed cdk4/cyclin Dl proteins in check. This sort of balance siderably higher than what is observed in myoblasts. Furthermore, the has been observed in tumor cells before and could reflect a selective cdk2 activity in RD cells was not affected by culture in DM. This advantage or a protection against apoptosis. For example, cells that suggests that the increased levels of p27kipl are preferentially inhib are deficient for RB seldom express detectable cyclin Dl and often iting cdk4 and are too low relative to the cdk proteins in the cell to overexpress pl6ink4 (35-37). effectively block kinase activity. Alternatively, the possibility exists, Relationship between Cell Cycle Progression and Differentia though, that p27kipl is active for binding to cdk2 and cdk4 but is not tion in RMS Cell Lines. RMS cell lines exhibit a definite growth active for inhibiting kinase activity. However, taken together, these advantage relative to human myoblasts in culture, which is best findings suggest that the high levels of cyclins and cdk may contribute visualized by culturing in DM. In DM, myoblasts undergo withdrawal to the oncogenic growth of these cells. Consistent with this idea is the from the cell cycle and the eventual terminal growth arrest associated finding that cyclins and or cdks can exhibit oncogenic activity (31). with complete differentiation to myotubes. In contrast, RD cells Interestingly, RD cells expressed high levels of the pl6ink4 protein. showed no detectable cessation of cell growth in DM. It is known that This was surprising, given that RD cells proliferated and contained cell cycle exit is required for the differentiation phenotype (9-11). phosphorylated RB. Furthermore, previous analysis of RD cells sug Oncogenes such as Ras, which mimic mitogenic signaling pathways, gested that the entire locus covering pl6ink4 and pl5ink4 was ho- inhibit cell cycle withdrawal and myogenic differentiation (11). Fur mozygously deleted in RD cells (32). By immunoblotting, immuno thermore, oncogenes that influence cell cycle regulatory proteins such precipitation, and immunofluorescence with two different antibodies, as SV40 large T-antigen or adenovirus EIA also inhibit myogenic we confirmed the presence of the pl6ink4 protein. Furthermore, we differentiation (9, 11). The failure of RMS cell lines to arrest when also detected pl6ink4 mRNA expression by RNase protection, which cultured in DM raised the possibility that failure to exit the cell cycle argues against homozygous deletion. A possible explanation for the was the basis for the failure of RD cells differentiate. We tested this discrepancy could be due to different culture conditions of the RD hypothesis directly by inducing the cell cycle exit of RD cells by the cells because numerous reports indicate that pl6ink4 expression can overexpression of growth-inhibitory proteins. These proteins effec be lost in culture (33, 34). Analysis of the cdk4 binding activity of tively inhibited RD cell cycle progression; however, these proteins did pl6ink4 showed that the pl6ink4 expressed in RD cells was active for not stimulate the expression of muscle-specific genes such as MHC, binding to cdk4. Although the reason for this high level of expression nor did they induce the morphological changes associated with myo is unclear, it is possible that pl6ink4 acts to keep the activity of the genic differentiation in RD. 2047

tion to stimulate MyoD activity, and in the presence of ectopie cyclin expression, RB is hyperphosphorylated and incapable of stimulating MyoD-mediated gene expression (15, 16, 21, 22). Our findings that pl6ink4, which blocks RB phosphorylation and a constitutively active RB protein, PSM.7-LP, cannot induce myogenic differentiation sug gest that the blockade of MyoD activity is not mediated through the phosphorylation or inactivation of the RB protein in RD cells. Ectopie expression of cyclin Dl has been shown to inhibit MyoD activity in a manner independent of RB phosphorylation (15), suggesting that the highly expressed cyclin Dl could be contributing to the overall inhibition of MyoD activity. Alternatively, it has been shown that, in the absence of functional p53, myoblastic cells in culture undergo a cell cycle arrest yet fail to differentiate (39). Interestingly, RD cells lack functional p53, and this could explain the failure of the arrested cells to differentiate (40). Recent investigations into the activity of MyoD has led to addi tional insights into how the protein is activated and functions as a transcriptional activator (41, 42). Extensive study into the defects associated with MyoD transactivation function may reveal a reason(s) why RD cells fail to differentiate. This line of investigation may also yield further insight into the interplay of the cell cycle and differentiative processes in RMS. Vector WT-LP PSM.7-LP

ACKNOWLEDGMENTS

> 40- We thank Carolati Buckmaster and other members of the laboratory of J. R. F. for technical assistance. Dr. Steven I. Reed kindly provided the anti-cyclin E antibody. Dr. Frank Stockdale kindly provided the anti-MHC antibody. Dr. Tony Hunter provided the cyclin A antibody.

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Erik S. Knudsen, Claudia Pazzagli, Teresa L. Born, et al.

Cancer Res 1998;58:2042-2049.

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