Pediatr Blood Cancer 2012;58:877–884

Retinoic Acid Fails to Induce Cell Cycle Arrest With MyogenicDifferentiation in Rhabdomyosarcoma

Alaa Al-Tahan, MD,1 Omar Sarkis, BS,1 Mohamad Harajly, MS,1 Omar Kebbe Baghdadi, BS,1 Kazem Zibara, PhD,2

Fouad Boulos, MD,3 Dipti Dighe, MD,4 Steven Kregel, BS,4 Ali Bazarbachi, MD, PhD,5 Marwan El-Sabban, PhD,6

Stephen X. Skapek, MD,4 and Raya Saab, MD1*

INTRODUCTION

Rhabdomyosarcoma (RMS) is the most common soft tissue

sarcoma in children [1]. Despite multimodality therapy, approxi-

mately 30% of treated children will experience relapse, with

overall survival being particularly poor in those originally diag-

nosed with high risk disease (reviewed in Ref. 2). By definition,

rhabdomyosarcoma cells express some skeletal myogenic pro-

teins, such as MyoD and Myogenin, and certain structural pro-

teins like desmin and vimentin [3–5]. Despite this lineage

commitment, the normal myogenic differentiation program is

stalled in RMS cells because many of the later aspects of differ-

entiation (e.g., expression of functional proteins, morphological

changes, cell fusion) are absent and, unlike terminally differenti-

ated myocytes, rhabdomyosarcoma cells proliferate [6]. There has

been a long-standing interest in promoting differentiation in can-

cers such as RMS because such differentiation could, in principle,

irreversibly arrest cell proliferation to control the disease with less

side effects than conventional therapies [7,8].

Retinoic acid is a morphogen that has been shown to induce

differentiation of normal myoblasts, neuroblasts, and epithelial

cells [9–11]. Pre-clinical studies show that retinoic acid fosters

differentiation in neuroblastoma, another primitive and aggressive

childhood tumor [12]. When used in the setting of minimal resid-

ual disease, retinoic acid improves disease-free survival for chil-

dren with metastatic neuroblastoma [13]. Whether such an

approach is useful for children with other poorly differentiated

tumors is not known. Several in vitro studies have demonstrated

that retinoic acid influences cell proliferation and muscle gene

expression in human RMS cell lines [14,15]. However, in vivo

studies of retinoic acid therapy in rhabdomyosarcoma are lacking.

We assessed the effect of retinoic acid on RMS in vitro and

using an in vivo minimal residual disease model, using two cell

lines representing the major subtypes of RMS: embryonal and

alveolar histology. We report that All-Tans Retinoic Acid

(ATRA) slowed RMS cell proliferation and cell accumulation in

culture. In addition, prolonged in vitro exposure to ATRA altered

cell morphology and augmented the expression of Myosin Heavy

Chain (MyHC), an indicator of terminal differentiation. However,

maintenance therapy following chemotherapy-induced remission

of RMS xenografts augmented the expression of a more differen-

tiated myogenic phenotype in vivo, but this did not preclude

continued cell proliferation and eventual relapse. When ATRA

and Cis-Retinoic Acid (CRA) were applied to normal myoblasts

in vitro, only an early marker of muscle differentiation was

enhanced, and RB protein activation and cell cycle arrest did

not occur. These findings suggest that retinoic acid alone is

unlikely to be beneficial as a single agent in inducing RMS

differentiation. Further, they provide new molecular evidence as

to why retinoids fail to fully promote myogenic differentiation

and cell cycle arrest, thereby forming a foundation from which

Background. Rhabdomyosarcoma (RMS) is the most commonsoft tissue sarcoma in children. Current treatment strategies do notcure most children with recurrent or high-risk disease, underlyingthe need for novel therapeutic approaches. Retinoic acid has beenshown to induce differentiation in a variety of cells including skele-tal myoblasts and neuroblasts. In the setting of minimal residualdisease, retinoic acid improves survival in neuroblastoma, anotherpoorly differentiated childhood tumor. Whether such an approachis useful for rhabdomyosarcoma has not yet been investigated.Several in vitro studies have demonstrated an appreciable effectof retinoic acid on human RMS cellular proliferation and differenti-ation. Procedure. We assessed the efficacy of ATRA on rhabdomyo-sarcoma, in vitro and in vivo, using cell lines and xenografts.Results. ATRA slowed RMS cell proliferation, and promoted a

more differentiated myogenic phenotype in both alveolar and em-bryonal RMS cell lines. Treatment of cultured murine myoblastswith retinoids increased Myogenin expression, but did not inducecell cycle arrest. Despite the favorable in vitro effects, ATRA failedto delay relapse of minimal residual disease using human RMSxenografts in immuno-suppressed NOD-SCID (NSG) mice. Interest-ingly, tumors that recurred after ATRA treatment showed evidenceof enhanced muscle differentiation. Conclusion. Our results indi-cate that ATRA could increase the expression of some genesassociated with muscle differentiation in rhabdomyosarcoma cells,but there was no benefit of single-agent therapy in an MRD model,likely because cell cycle arrest was uncoupled from the pro-differentiation effects of retinoids. Pediatr Blood Cancer 2012;58:877–884. � 2011 Wiley Periodicals, Inc.

Key words: differentiation; minimal residual disease; retinoic acid; rhabdomyosarcoma; therapy; xenograft

1Department of Pediatrics, American University of Beirut, Beirut,

Lebanon; 2Lebanese University, Beirut, Lebanon; 3Department of

Pathology and Laboratory Medicine, American University of Beirut,

Beirut, Lebanon; 4Division of Pediatric Hematology-Oncology,

University of Chicago, Chicago, Illinois; 5Department of Internal

Medicine, American University of Beirut, Beirut, Lebanon;6Department of Anatomy, Cell Biology and Physiological Sciences,

American University of Beirut, Beirut, Lebanon

Grant sponsor: The Lebanese National Council for Scientific

Research (CNRS). Alpha-Omega-Alpha (AOA) Carolyn L. Kuckein

Student Research Fellowship award (Alaa Al-Tahan). The American

Lebanese Syrian Associated Charities (ALSAC) and International

Outreach Program at St Jude Children’s Research Hospital, Memphis,

TN (Dr Saab’s laboratory).

Conflict of interest: Nothing to declare.

*Correspondence to: Raya Saab, MD, Department of Pediatrics,

American University of Beirut, Riad El Solh Street, Beirut 1107

2020, Lebanon. E-mail: rs88@aub.edu.lb

Received 27 April 2011; Accepted 26 May 2011

� 2011 Wiley Periodicals, Inc.DOI 10.1002/pbc.23246Published online 13 July 2011 in Wiley Online Library(wileyonlinelibrary.com).

additional pharmacological strategies can be applied to attempt to

fully activate skeletal muscle differentiation as a therapy for

RMS.

MATERIALS AND METHODS

Cell Lines, Growth, and Treatment Conditions

Human rhabdomyosarcoma cell lines of alveolar (Rh30) and

embryonal (JR-1) histology were generously provided by Peter

Houghton; both have been previously characterized [16,17].

Mouse C2C12 myoblasts were obtained from the ATCC. Cells

were cultivated at 378C and 5% CO2 in 10% fetal bovine serum

(FBS) in DMEM (Invitrogen, Carlsbad, CA). Cells were treated

with 5 mM ATRA (Sigma–Aldrich, St. Louis, MO), or an equal

volume of DMSO vehicle. Culture medium (with fresh ATRA or

DMSO) was changed every 3 days. Because ATRA is light-

sensitive, all handling of ATRA stocks, working solution and

ATRA treatment were done in subdued light.

Cell Accumulation Assay

JR1 and Rh30 cells were seeded onto six-well plates at

1 � 103 cells per well and cultured at 378C and 5% CO2 in

10% FBS in DMEM. Six sets of triplicate wells were plated for

each cell line. The following day (day 0), half of the wells of each

cell line were refed with culture medium containing ATRA in

DMSO or an equivalent volume of vehicle. On days 3, 6, and 9,

triplicate wells were harvested, and the total number of cells was

counted via hemocytometer; remaining wells were refed with

medium containing either ATRA or DMSO as above. Phase-

contrast photomicrographs to assess cellular morphology were

taken using AxioCam HRC (Zeiss, Thornwood, NY) camera.

Data shown are representative of three or more independent

experiments.

Senescence-Associated Beta Galactosidase (SABG)Assay

SABG staining was done as described [18]. In summary, cells

were fixed using 2% paraformaldehyde, then washed and stained

in SABG staining solution (1 mg/ml X-gal, 40 mM Citric acid/

NaPhos buffer pH 6.0, 5 mM K3CN, 5 mM K4CN, 150 mM

NaCl, 2 mM MgCl2) at 378C overnight, washed in PBS, and

counterstained with eosin.

TUNEL Assay

Terminal deoxynucleotidyl transferase-mediated dUTP nick

end labeling (TUNEL) was done using the DeadEnd Cell Death

Labeling kit (Roche, Indianapolis, IN). Nuclei were labeled with

DAPI and then cover-slipped. The percentage of TUNEL-positive

cells was determined by counting representative 200� fields using

a fluorescence microscope.

BrdU Treatment and Immunostaining of CulturedRMS Cells

Rhabdomyosarcoma cells were cultured as detailed above in 8-

wells chamber slides, with ATRA or an equal volume of DMSO

vehicle, for 9 days. Cells were then treated with bromodeoxyur-

idine (BrdU) (Sigma–Aldrich) at 50 mmol/L, for 15 minutes or

4 hours. Cells were fixed by methanol/acetone (1:1), and

immunostained for either BrdU alone, or BrdU and Myosin

Heavy Chain (MyHC). For BrdU staining, fixed cells were treated

with 2N HCL for 10 minutes, then neutralized by Borate buffer

for 12 minutes. After blocking, slides were probed with anti-BrdU

antibody (Santa Cruz Biotechnology, Santa Cruz, CA), anti-

MyHC antibody (Millipore, Billerica, MA), or both. Cy3-

conjugated secondary antibody (Jackson Immunoresearch, West

Grove, PA) and/or Alexa 488-conjugated antibody (Invitrogen)

were used for detection. Stained cells were covered with aqueous

mounting medium containing DAPI (Vector Laboratories, Burlin-

game, CA), and visualized by immunofluorescence microscopy

using an Axiovert 135M microscope equipped with an Axiocam

digital camera (Zeiss). BrdU-positive cells were quantified by

counting positive cells in five high-power fields in each condition

and normalizing to total number of cells (DAPI-stained) in each

field. Statistical analysis was done using Student’s t-test. Data

shown are representative of three or more independent experi-

ments with duplicate or triplicate samples.

Proliferation and Differentiation AssaysUsing Myoblasts

C2C12 cells were cultivated as previously described [19] in

10% FBS in DMEM (Growth Medium or GM), in 2% FBS

in DMEM (differentiation medium or DM) or differentiation

medium with insulin (10 mg/ml) prior to harvest. In some experi-

ments, all-trans retinoic acid (ATRA) (5 mM) or cis-retinoic acid

(CRA) (5 mM) (or equal volume of vehicle) was added with the

GM. At harvest, cells were fixed in 4% paraformaldehyde in PBS

and processed for BrdU incorporation as described above,

or lysates of non-fixed cells were used for Western blotting as

previously described [19].

Xenograft Studies

All animal experiments were approved by the American

University of Beirut’s Institutional Animal Care and Use

Committee. NSG mice (Reference 005557, NOD.Cg-

Prkdc<scid>Il2rg<tm1Wjl>/SzJ) were obtained from the Jack-

son Laboratory (Bar Harbor, Maine). All animals were handled

under pathogen-free sterile conditions, maintained under micro-

isolators, and fed sterile food. To form xenografts, Rh30 cells

were injected subcutaneously into the right flank, at 1 � 107 cells

per injection. Mice were then monitored until tumors grew to a

volume of at least 300 mm3, assessed using the following formu-

la: [length � (width)2]/2. Mice were then treated with vincristine

(Gedeon Richter Ltd, Budapest, Hungary), at a dose of 1 mg/kg

once weekly by intra-peritoneal injection. Mice were examined

twice weekly until complete tumor regression, then randomized to

receive ATRA (3.5 mg/kg by intra-peritoneal injection once daily,

5 days/week for 4 weeks), or an equal volume of vehicle alone

(DMSO). A total of 37 mice were treated: 19 with ATRA; 18 with

vehicle. Mice were examined twice weekly to detect tumor recur-

rence. Kaplan–Meier survival estimates and box-plots were used

in statistical analyses of tumor relapse rate.

Xenograft Harvesting and ImmunohistochemicalStaining

Mice with relapsed xenografts were monitored until a tumor

volume of at least 300 mm3, at which time they were euthanized.

878 Al-Tahan et al.

Pediatr Blood Cancer DOI 10.1002/pbc

Tumors were dissected, fixed in 4% paraformaldehyde, and

embedded in paraffin. Standard histology procedures were

followed to prepare 3 mm sections for immunohistochemical

staining with the following antibodies: anti-Myogenin (Lab

Vision, Fremont, CA), anti-MyoD (Dako, Glostrup, Denmark),

anti-MyHC (BioGenex, Fremont, CA). Digital photomicrographs

were obtained using an Olympus DP12 camera and software.

Western Blot Analysis

Cells or tissue samples were lysed on ice for 20 minutes in

Universal Lysis Buffer 100 mM Tris pH 7.5, 300 mM NaCl,

4 mM EDTA, 4 mM EGTA, 0.4% Triton X-100, 1% NP40,

50 mM NaF, 20 mM b-glycerophosphate, 1 mM phenylmethyl-

sulfonyl fluoride, 10 mg/ml leupeptin, 10 mg/ml aprotinin, 1 mM

DTT. Lysates were clarified by centrifugation and protein was

quantified by Bradford assay (Bio-Rad Laboratories, Hercules,

CA). Equivalent amounts of protein (50–80 mg) were fractionatedby 10% SDS–PAGE and transferred to polyvinylidene difluoride

membranes (Bio-Rad Laboratories, Hercules, CA). Blotted

proteins were detected using the following primary antibodies:

anti-Myogenin (BD Biosciences Pharmingen, San Diego, CA);

anti-MyHC (Millipore, Billercia, MA); anti-phosphorylated RB

at Ser780 (Cell Signaling Technology, Danvers, MA); anti-

p18Ink4c, anti-p21Cip1, anti-p27Kip1, anti-HSC 70, and anti-

Cyclin A (all from Santa Cruz Biotechnology, Santa Cruz, CA).

Primary antibodies were detected with species-specific horserad-

ish peroxidase-coupled secondary antibodies (Santa Cruz

Biotechnology, Santa Cruz, CA) and visualized by enhanced

chemiluminescence (Roche, Indianapolis, IN). The level of

expression of different proteins was analyzed by using the public

Image J software. Exposures in the linear range of the X-ray film

were selected. The relative band intensity was assessed by densi-

tometric analysis of digitalized autoradiographic images, and the

ratio of the band intensity of the protein of interest (MyHC) to the

loading control protein (HSC70) was calculated.

RESULTS

ATRA decreases RMS cell proliferation in vitro: We treated

alveolar (Rh30) and embryonal (JR1) RMS cell lines with ATRA

at a final concentration of 5 mM. ATRA treatment decreased RMS

cell accumulation by day 6, and the effect was maintained at

day 9 (Fig. 1A). To investigate the mechanism by which ATRA

resulted in less cell accumulation, we evaluated treated cells for

evidence of senescence, apoptosis, or cell cycle arrest. There was

no evidence of senescence after 9 days of treatment with ATRA,

detected by staining for acidic senescence-associated beta-

galactosidase (Fig. 1B). Increased cell death was not observed

in ATRA-treated culture plates, and TUNEL staining showed less

than 1.5% cell death by day 3 of treatment, and less than 0.5%

Fig. 1. ATRA inhibits RMS cell accumulation in culture. A: Rep-resentative charts showing number of the indicated RMS cells after

cultivation for 3, 6, and 9 days in the presence of ATRA (A) or the

drug vehicle DMSO (D). B: Staining for senescence-associated beta

galactosidase (SABG) activity in RH30 and JR1 cells treated for

9 days with ATRA or vehicle (DMSO), as indicated. Central inset

demonstrates positive SABG staining in oncogene-expressing murine

primary cells. C: Percentage of Rh30 and JR1 cells that are BrdU-

positive, after 9 days of treatment with either ATRA or drug vehicle

DMSO, in high-serum (10% FBS) or low serum (1%FBS) conditions,

as indicated. Cells were treated with short pulses (15 minutes) of

BrdU. Asterisk indicates statistical significance with P-value <0.05

(student t-test).

RA Fails to Induce Cell Cycle Exit in RMS 879

Pediatr Blood Cancer DOI 10.1002/pbc

cell death by day 6 (standard deviation 0.22% and 0.003%,

respectively). However, ATRA treatment significantly decreased

the proportion of cells progressing through the S-phase of the cell

cycle, in both Rh30 and JR1 cells, assessed by pulse-labeling of

cells with BrdU. This effect was apparent regardless of whether

cells were cultivated in medium with high (10% FBS) or low

serum (1% FBS) (Fig. 1C). We conclude that ATRA treatment

in vitro slows cell proliferation in both RMS cell lines.

ATRA treatment enhances myogenic features of RMS cells in

vitro: In addition to slowing cell proliferation, we observed

morphological changes suggesting skeletal muscle differentiation,

in that spindle-shaped cells accumulated when either the

embryonal (JR1) or alveolar (Rh30) RMS cell line was exposed

to ATRA. This was evident by day 6 of treatment and was

pronounced by day 9 (Fig. 2A and B, upper panels). To investi-

gate if these morphological changes represented skeletal muscle

Fig. 2. Evidence of differentiation in ATRA-treated RMS cells. Representative images for (A) JR1 and (B) Rh30 cells after culture for 9 days

with ATRA or vehicle alone (DMSO), showing cellular morphology by phase-contrast imaging (upper panels); immunofluorescence staining

for Myosin Heavy Chain (MyHC; middle panels) and corresponding nuclear stain by 4,6-diamidino-2-phenylindole (DAPI; lower panels). C:Quantitative data from a representative experiment showing the fraction of RMS cells with MyHC detectable by immunofluorescence staining

in four representative fields. Asterisk denotes statistical significance (P < 0.05, t-test). D: Representative images for JR1 cells after culture for

9 days at differential densities, such that cellular density is similar by 9 days of treatment with ATRA or vehicle alone (DMSO). Shown is

immunofluorescence staining for Myosin Heavy Chain (MyHC; upper panels) and corresponding nuclear stain by 4,6-diamidino-2-phenyl-

indole (DAPI; lower panels). E, Upper panel: Representative Western blot for Myosin Heavy Chain (MyHC), and loading control HSC70, of

Rh30 and JR1 cells treated for 9 days with either ATRA or vehicle (DMSO), as indicated. Lower panel: Relative MyHC band intensity

corrected to the loading control (HSC70) band intensity in the different lanes of the above Western blot, as indicated.

880 Al-Tahan et al.

Pediatr Blood Cancer DOI 10.1002/pbc

differentiation, we assessed the expression of Myosin Heavy

Chain (MyHC), a protein expressed late in the differentiation

program. We found that individual RMS cells expressing MyHC

were much more apparent following exposure to ATRA, in both

Rh30 and JR1 lines (Fig. 2A and B, middle and lower panels; and

Fig. 2C). To account for the different cell density in ATRA-treated

versus vehicle-treated cells, these experiments were also done

with differential plating such that cell density was similar at

day 9 of treatment, and the results were reproducible (representa-

tive Fig. 2D). Western blotting confirmed that MyHC expression

was augmented in ATRA-treated cells (Fig. 2E). Thus, prolonged

exposure to ATRA induced morphological and molecular

evidence of greater skeletal muscle differentiation in both embry-

onal and alveolar RMS cell lines, and this correlated with the

decreased proliferation.

Prolonged exposure to ATRA enhanced the expression of

MyHC but did not prevent relapse in a model of minimal residual

disease: Given the apparent success of the in vitro studies, we

evaluated differentiation therapy by ATRA in a model of minimal

residual disease (MRD) in vivo. We injected RMS cells to form

human RMS xenografts in immunodeficient NSG mice. After

tumors reached a volume of at least 300 mm3, we treated the

mice with vincristine once weekly until tumors resolved. Once

the mice achieved a clinical remission (i.e., the tumors were no

longer palpable), we instituted maintenance therapy for presumed

minimal residual disease. Animals were treated with either ATRA

or vehicle, 5 days/week for 4 weeks. Approximately 10 weeks

after start of maintenance therapy, 47% (9 out of 19) of ATRA-

treated and 50% (9 out of 18) of vehicle-treated mice had devel-

oped local tumor relapse. We found no significant difference in

tumor-free survival or mean time to relapse (Fig. 3A,B). Interest-

ingly, tumors that relapsed after ATRA treatment expressed the

late differentiation marker MyHC, while those that recurred after

vehicle alone did not (Fig. 3C,D). As expected, all tumors

expressed MyoD (Fig. 3D). In addition, expression of the early

muscle protein Myogenin decreased (Fig. 3C), an event that also

occurs during normal myoblast differentiation [20]. Thus, ATRA

treatment for 4 weeks in mice bearing minimal amounts of resid-

ual RMS did not prevent tumor relapse even though it enhanced

the expression of MyHC, a marker of terminal muscle

differentiation.

ATRA-induced differentiation is not sufficient to engage

terminal cell cycle arrest in RMS: Since tumors that recurred after

ATRA treatment expressed the differentiation marker MyHC, we

evaluated whether MyHC-expressing, ATRA-treated RMS cells

were still able to proliferate. This would be surprising because

Fig. 3. ATRA-treated mice develop recurrent tumors that show evidence of differentiation. A: Kaplan–Meier survival analysis of tumor

relapse from time of maintenance therapy initiation, in mice treated with maintenance therapy using ATRA or vehicle (DMSO); P ¼ 0.87. B:Box-plot analysis of median time to relapse in ATRA and DMSO (vehicle)-treated mice, from time of initiation of maintenance therapy;

P ¼ 0.58. C, Upper panel: Representative Western blot for Myogenin, Myosin Heavy Chain (MyHC), and loading control HSC70 in two

representative relapsed tumors after ATRA (RT-ATRA), two tumors relapsing after DMSO vehicle (RT-DMSO), and an untreated tumor (T).

Lower panel: Relative MyHC band intensity corrected to the loading control (HSC70) band intensity in the different lanes of the above

Western blot, as indicated. D: Representative immunohistochemical staining for the early myogenic marker MyoD (upper panel), and the late

differentiation marker Myosin Heavy Chain (MyHC) at low (middle panel) and high magnification (lower panel), in indicated tumors. Arrows

indicate MyHC-positive cells with brown immunostaining.

RA Fails to Induce Cell Cycle Exit in RMS 881

Pediatr Blood Cancer DOI 10.1002/pbc

terminal muscle differentiation is normally coupled to cell cycle

arrest in G0/G1 phase [21]. To evaluate whether ATRA simply

delayed progression through S-phase, we exposed the cells to

longer pulses of BrdU (4 hours), then evaluated the percentage

of cells in S-phase. We found that the difference between ATRA-

treated and vehicle-treated RMS cells persisted (Fig. 4A).

We then evaluated whether ATRA-treated cells that continued

to proliferate showed evidence of differentiation. Dual staining for

BrdU as a cell proliferation marker and MyHC revealed that,

although most ATRA-treated RMS cells were either BrdU-

positive alone or MyHC-positive alone, some cells showed

positivity for both markers (Fig. 4B). This indicated that

ATRA-induced differentiation did not preclude continued cell

cycle progression and cellular proliferation in cultured RMS cells.

Previous work using genetically engineered mice has shown

that myoblasts that lack the retinoblastoma protein (RB�/� cells)

can express some terminal skeletal muscle differentiation

proteins, but can re-enter the cell cycle upon growth factor stim-

ulation [22–24]. This is due to the failure of RB to repress genes

activated by members of the E2F family of transcription factors

[25]. Thus lack of RB prevents the engagement of the terminal

differentiation program and concomitant cell cycle exit.

We considered that the RB protein may remain functionally

inactive in ATRA-treated RMS cells to foster continued prolifer-

ation. Previous studies have shown that ATRA can induce expres-

sion of CDK-inhibitors, leading to decreased phosphorylation of

RB in several types of cells [26–28]. By Western blotting, we

found that ATRA treatment increased the expression of the

CDK4-inhibitor p18Ink4c in both Rh30 and JR1 cells, and

increased the expression of the CDK2-inhibitors p21Cip1 and

p27Kip1 in JR1 cells (but not Rh30 cells; Fig. 4C). However,

this did not translate into CDK4 inhibition because the RB protein

was still phosphorylated at CDK4-dependent sites in RMS cells

regardless of ATRA treatment (Fig. 4D). In addition, Cyclin A, a

known E2F target, was still expressed in ATRA-treated RMS cells

(Fig. 4C) further demonstrating that the RB pathway was func-

tionally inactive. Thus, we conclude that, even though ATRA

treatment induced several CDK-inhibitors, this was neither suffi-

cient to repress Cyclin A nor block RB protein phosphorylation.

This provides a molecular explanation for continued cellular

proliferation despite expression of late muscle differentiation

markers.

Retinoids fail to fully engage the terminal differentiation

program in normal myoblasts: We next sought to examine the

pro-differentiation effects of retinoic acid compounds in the well-

established C2C12 murine myoblast model. The withdrawal of

serum-derived mitogens (differentiation medium, DM) and the

addition of insulin (DM þ I) is well-established to activate myo-

genic differentiation, resulting in cell elongation and myoblast

fusion, irreversible cell cycle arrest, and increased expression of

Myogenin and MyHC within 24–72 hours (Fig. 5A,C). When

either ATRA or CRA were added to mitogen-rich cell culture

medium (GM), the expression of Myogenin increased to nearly

the same level as that achieved by DM þ I (Fig. 5A). However,

we observed no induction of MyHC in this timeframe. Both DM

and DM þ I also increased the fraction of Myogenin expressing

cells by more than 10- and 25-fold over GM (Fig. 5B, left panel).

However, even though Myogenin induction by CRA

Fig. 4. ATRA-induced differentiation does not prevent RMS cellular proliferation. A: Percentage of BrdU-positive Rh30 and JR1 cells, after

9 days of treatment with either ATRA or drug vehicle DMSO; cells were treated with long pulses of BrdU (4 hours). B: Representativephotomicrograph showing dual immunofluorescence staining for Myosin Heavy Chain (MyHC) and BrdU, and corresponding DAPI staining, in

JR1 cells treated with ATRA for 9 days in culture. Small arrowhead indicates a double-positive cell; large arrowhead shows an MyHC-only

positive cell, and long arrows indicate BrdU-only positive cells. C: Representative Western blot for the indicated cell cycle proteins in Rh30 and

JR1 cells treated for 9 days with either ATRA (A) or vehicle (DMSO; D). HSC70 is a loading control. D: Representative Western blot for the

retinoblastoma (RB) protein phosphorylated at Ser780 (CDK4-dependent site) in Rh30 and JR1 cells treated for 9 days with either ATRA (A) or

vehicle (DMSO; D). HSC70 is a loading control; mature skeletal muscle (SkM) was used as a negative control; proliferating cells were used as

positive control.

882 Al-Tahan et al.

Pediatr Blood Cancer DOI 10.1002/pbc

approximated that in DM þ I when measured by Western blotting

(Fig. 5A), CRA had a very small effect on the faction of Myo-

genin-expressing cells (Fig. 5B).

We considered whether the discrepancy between the two

results was due to a failure of retinoic acid to arrest cell prolifer-

ation. Indeed, the total number of cells and the fraction of cells

incorporating BrdU were the same in cells treated with either

CRA, ATRA, or vehicle (Fig. 5B, right panel and Fig. 5C) where-

as cell number and BrdU incorporation decreased in DM or

DM þ I. These data demonstrate that retinoic acid treatment

enhanced Myogenin expression in normal myoblasts, but this

was not accompanied by the robust cell proliferation arrest that

normally accompanies myoblast differentiation.

DISCUSSION

Previous studies have shown that retinoids can slow RMS cell

proliferation and/or promote differentiation in vitro, and in certain

skeletal myoblast cell lines [9,14,15,29,30]. This prompted us to

further explore its therapeutic potential in vivo. We used a model

that closely reflects the clinical scenario in which retinoids are

likely to be applied: maintenance therapy following chemothera-

py-induced minimal residual disease. We chose the JR1 and Rh30

cell lines because previous studies with these lines had yielded

promising results [14,15] and they represented the two major

subtypes of RMS. Our results demonstrate that, despite enhancing

muscle gene expression and slowing progression of RMS cells

through the cell cycle in vitro, retinoic acid does not completely

inhibit RMS cell proliferation in vivo nor does it delay disease

recurrence when applied to minimal residual disease.

Of course, caution must be applied when drawing conclusions

from pre-clinical models, including ours. For example, it is

formally possible that longer duration of ATRA therapy could

have delayed tumor recurrence. We think this is unlikely because

some tumors had already recurred by the end of the four weeks of

treatment. Also, although the cell lines we examined represented

both major RMS subtypes, the conclusions are based on results

from only two cell lines. We chose not to further pursue this line

of investigation using a larger panel of cell lines because our

analysis of normal myoblasts revealed a disconnection between

retinoid-induced induction of myogenic proteins and cell cycle

exit. Since retinoic acid did not foster the coordinated induction of

muscle genes and cell cycle arrest in normal myoblasts, it seemed

unlikely to induce permanent cell cycle arrest in other RMS cell

lines as a single agent.

Our analyses provide some insight into why retinoic acid did

not have a more robust effect on tumor recurrence. It is well-

established that cell cycle exit in G0 phase is essential for normal

skeletal muscle differentiation, and the RB protein plays a partic-

ularly important role [21]. In cultured myoblasts, the G0 arrest

occurs approximately 24 hours after muscle differentiation begins

and is coupled to induction of Myogenin [31]. The arrest

correlates with repression of G1-cyclins such as Cyclin D1, and

induction of Cdk inhibitors like p21Cip1 and p18Ink4c [32,33].

The net effect is to activate the RB protein by preventing its

phosphorylation. Hypophosphorylated, active RB augments the

ability of muscle-specific transcription factors MyoD, Myogenin

and MEF2 to increase muscle gene expression [23,34,35] and

fosters the accompanying cycle arrest by repressing E2F-

dependent gene expression [23,24]. With these numerous layers

of control, DNA synthesis is never observed in a normal, maturing

myocyte unless RB function is compromised by phosphorylation

or mutation [23,24,36].

Our studies reveal that retinoic acid fails to orchestrate cell

cycle arrest, RB activation, and muscle gene induction in RMS

cell lines. One possible explanation is that the RMS cell lines

used may carry a mutated RB allele. This seems unlikely because

pharmacological inhibition of CDK4/6 using PD 0332991 arrests

proliferation in both JR1 and Rh30 cells [19], and cell cycle arrest

by PD 0332991 depends on wild-type RB [37]. Retinoic acid was

also unable to couple cell cycle arrest with differentiation in

normal myoblasts, supporting the conclusion that failure to link

cell cycle arrest to muscle gene induction has to do with the drug

effect rather than a defect intrinsic to the RMS cell lines.

Fig. 5. Retinoids induce Myogenin but fail to arrest cell prolifera-

tion in cultured myoblasts. A: Representative Western blot showing

Myosin Heavy Chain (MyHC), Myogenin, and Heat Shock Complex

70 (HSC70) (as a loading control) in C2C12 myoblasts cultivated for

48 hours in the indicated medium with or without 9-cis-retinoic acid

(CRA) or ATRA. B: Quantitative data showing the fraction of C2C12

cells with Myogenin detectable by immunofluorescence staining (leftpanel) and the total cells visible by DAPI staining (right panel) innine representative field. Cells harvested by fixation following

48 hours with or without CRA treatment. Data represent mean and

standard deviation from four separate wells. All values are statisti-

cally significant when compared to control (GM) (P < 0.05, t-test).

C: Quantitative data showing the fraction of C2C12 cells incorporat-

ing BrdU, measured by direct counting of 10 representative fields.

Values represent mean and standard deviation. Decreased BrdU in-

corporation in DM þ I was significantly different from GM baseline

(�P < 0.05) whereas the percent of BrdU positive cells in

GM þ ATRA and GM þ CRA was not (#P > 0.05; t-test).

RA Fails to Induce Cell Cycle Exit in RMS 883

Pediatr Blood Cancer DOI 10.1002/pbc

Treatment with retinoids enhanced Myogenin but not MyHC

in normal myoblasts, whereas it enhanced MyHC in RMS. If

retinoids were triggering a critical pro-differentiation pathway,

we would expect them to have similar effects in both models.

Instead, we suspect that a pharmacological dose of retinoic acid

likely acts as a transcriptional regulator to enhance the expression

of certain myogenic genes without activating a central regulatory

process. For example, in the C2C12 cells which are committed to

the myogenic lineage by virtue of MyoD expression, retinoic acid

may push them slightly more toward a differentiated myocyte by

enhancing Myogenin expression, but MyHC is not induced

because the RB pathway is not engaged and the cells do not

arrest. In contrast, the RMS cells already display a baseline phe-

notype that is more differentiated (i.e., expression of Myogenin)

and one in which the normal coordination of the cell cycle and

differentiation is already deranged because proliferating tumor

cells express Myogenin. In this setting, retinoic acid fosters

some maturation with increased MyHC but stops short of terminal

differentiation. Further studies identifying genes bound by reti-

noic acid receptors in different cell types seem likely to provide

insight into the differential activity of retinoids in these two cell

types.

Even though there was no obvious delay in tumor recurrence,

the RMS xenografts arising following ATRA treatment displayed

enhanced MyHC and decreased Myogenin, both of which occur in

terminal muscle cell differentiation [20]. Recent data indicate that

human rhabdomyosarcoma tumors with a differentiated pheno-

type, based on RNA expression profiling, are associated with a

better clinical outcome [38,39]. It is therefore possible that the

seemingly small effect that retinoids have on myogenic differen-

tiation in RMS may translate into substantially improved survival

when combined with standard, cytotoxic therapy. Such a possibil-

ity needs to be investigated, and may be studied using preclinical

models.

Finally, it is important to note that differentiation therapy in

RMS may still be a sound goal. Given the importance of RB

in the terminal differentiation process, the approach may not

work in the subset of embryonal RMS in which the RB gene is

mutated or deleted [40]. However, differentiation therapy may be

especially practical in the majority of alveolar RMS where the RB

gene is intact [40] but the protein is functionally compromised

such as by increased CDK4 activity [41]; or loss of CDK4/6-

inhibitors, such as p16INK4a, p15INK4b, and p18INK4c [42].

Treatment of RMS cell lines with CDK4/6 inhibitors has shown

some promise in decreasing cellular proliferation, and a positive

effect on cellular differentiation [19]. By achieving a greater

understanding of exactly how retinoids foster increased muscle

gene expression, it may be possible to rationally apply additional

targeted agents to fully engage the terminal differentiation

program.

ACKNOWLEDGMENT

The authors thank Dr Peter Houghton for providing the RMS

cell lines.

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