a2a-interleron-induced differentiation ofhumanalveolar rhabdomyosarcoma cells...

Vol. 7, 531-541, April 1996 Cell Growth & Differentiation 531

a2a-Interleron-induced Differentiation of Human AlveolarRhabdomyosarcoma Cells: Correlation with Down-Regulation of the Insulin-like Growth FactorType I Receptor’

Ramachandran Thulasi, Peter Dias,2Peter J. Houghton, and Janet A. Houghton3Department of Molecular Pharmacology, St. Jude Children’s Research

Hospital, Memphis, Tennessee 38101

Abstract

Rhabdomyosarcoma, a tumor of skeletal muscle origin,appears developmentally arrested at an early stage inthe myogenic differentiation pathway. The proliferationof an alveolar rhabdomyosarcoma cell line Rh30 isdependent on the insulin-like growth factor (IGF) lI/IGF-I receptor (IGF-IR) signaling pathway and is highlysensitive to recombinant human IFN-a2a, whichinduces growth arrest and differentiation of thesemalignant myoblasts. IFN-a2a-induced growth arrest ofRh30 cells was observed within 48 h, and reduction incolony formation was obtained with an ICea of 0.81 lU/ml for 72 h exposure. Down-regulated expression ofIGF-IR was apparent by 24 h after Initiation of IFN-a2atreatment. Furthermore, an initial increase followed byreduced expression of MyoD, in concert with elevatedexpression of myogenin, increased frequency ofskeletal muscle myosin-positive cells, and theformation of multinucleated cells, indicated anenhancement of differentiation of Rh30 cells in thepresence of IFN-a2a. To probe the role of IGF-lR In thedifferentiation of Rh30 cells along the myogenic

lineage, the effect of antisense RNA-medlatedreduction of endogenous IGF-lR on growth andexpression of muscle-specific proteins wasdetermined. Rh30 cells transfected to stably expressantisense IGF-IR (clone AS#23) showed significantreduction in growth rate, decreased expression ofIGF-lR protein, increased expression of MyoD,myosin heavy chain, and an increased number ofmultinucleated cells in comparison to the parental line.

Received 12/2/95; revised 1/29/96; accepted 2/1/96.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to mdi-cate this fact.I This work was supported by NCI Grants CA23099 and CA21765 and bythe American Lebanese Syrian Associated Charities.2 � address: Pharmingen, 10975 Torreyana Road, San Diego, CA92121.3 To whom requests for reprints should be addressed, at Department ofMolecular Pharmacology, St. Jude Children’s Research Hospital, 332North Lauderdale, Memphis, TN 38105-2794. Phone: (901) 495-3440;Fax: (901) 521-1668.

These data are consistent with overexpression of IGF-lR inhibiting differentiation. IFN-a2a treatment of AS#23

cells further induced both MyoD and myogeninexpression, thereby allowing cells to proceed furtherdownstream of the differentiation pathway.

Introduction

RMS,4 tumors with embryonal or alveolar histology arising denovo from skeletal muscle precursors, comprise the mostcommon soft tissue sarcoma of childhood. The clinical out-come and modality of therapy vary according to the tumortype. Embryonal tumors that occur at specific sites are

treated with a combination of surgery and chemotherapy,

and this results in long-term survival of a high proportion ofpatients; in contrast, alveolar AMS tend to be larger, metes-tasize earlier, and are more difficult to treat, with survival ratebeing <20% (1). Phenotypically, AMS appear as immature,undifferentiated forms of the skeletal muscle-forming cells,express MyoD, and hence represent cells committed to myo-

genesis but that are arrested at an early stage of differenti-ation in the myogenic lineage. Skeletal muscle differentiationinvolves withdrawal of committed proliferating myoblastsfrom the cell cycle, followed by fusion of mononucleatedmyoblasts to form multinucleated myotubes (2). Normalmyogenic differentiation is governed by the coordinate ex-

pression of muscle-specific, myogenic-determining factors

that include MyoD, myogenin, Myf5, and MRF4 (3). Severalstudies in vitro and in vivo have demonstrated the expressionof MyoD as a marker for the committed myoblast state (3, 4).Myogenin appears to be downstream of MyoD or Myf5 andis expressed only when myoblast cells are induced to differ-entiate. Furthermore, the disruption of myogenin using geneknock-out mice resulted in the lack of skeletal muscle de-velopment, whereas disruption of MyoD or Myf5 individuallydid not affect myogenesis (5-8). These data suggest thatfunctional myogenin is essential for myogenic differentiation.

IGF II is known to stimulate normal skeletal myoblast pro-liferation and differentiation (9). The IGF-IR is considered tomediate these actions (10). IGF II is expressed at high levelsin normal fetal skeletal muscle and declines in adult muscle(1 1-1 5). A number of growth factors and oncogene productshave been shown to prevent myogenic differentiation by

4 Thaabbraviations used are: RMS, rhabdomyosarcomas; IGF, insulin-likegrowth factor� IGF-IR, IGF type I receptor� CAT, chloramphenicol acetyl-trans/erase; RFI, relative fluorescence index; PDGF, platelet-derivedgrowth factor� EGF, epidermal growth factori FBS, fetal bovine serum;�3-gal, f3-galactosidase; IFN-lR, IFN type I receptor.

72 h

80

1C50 = 0.81 lU/mI

60

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Fig. 1. IFN-a2a sensitivity of Rh30 cells. Rh30 cells grown in RPMI 1640with 10% FBS were planted at cloning densities of 3000 cells/well andtreated following overnight attachment with increasing concentrations ofIFN-a2a(0 to 1000 lU/mI) for 72, 96, and 120 h, respectively. The resultingcolonies were counted using an Artek colony counter 7 days after the endof treatment. The dlonogenic potential is represented as the percentage ofsurvivors (ordinate) with respect to control. The IC�s calculated were at50% survival. The data shown at 72 h were obtained from three experi-ments, each conducted in triplicate; bats, SE. The IC�s at 72, 96, and 120h were 0.81 , 0.43, and 0.26 lU/mI.

532 IFN Induction of RMS Differentiation

ated cells in the presence of IFN-a2a indicate enhancement

inhibiting or interfering with the expression of MyoD or myo-

genin (16-21). Florini et a!. (22, 23) suggested that IGFsstimulate myogenesis by regulating the expression of myo-

genin. Antisense oligonulceotides complementary to myoge-

nm mANA blocked IGF I-induced muscle differentiation (22).In normal mouse myoblasts, it has been shown that IGF IIexpression increases transiently during the early phase ofdifferentiation and then decreases as cells fuse to form myo-tubes (24, 25). Antisense oligonucleotides complementary to

IGF II inhibited spontaneous differentiation of myoblasts indifferentiation medium but not the stimulation of differentia-

tion by exogenous IGF II (23).

Recently, it has been shown that both embryonal and alve-olar RMS cell lines express high levels of IGF II mRNA, secrete

IGF II peptide in conditioned media, and grow in mitogen-free,serum-free conditions. These data suggest that IGF II functionsas an autocrine growth factor in AMS (26). Interestingly, RMScell lines express cell surface-specific IGF-IR. aIR3, an antibodythat neutralizes IGF-lA (27), inhibited the growth of AMS inserum-free medium, suggesting that the mitogenic actions ofIGF II are mediated by IGF-IA (26-28). Furthermore, in vWo

treatment with the alA3 antibody suppressed the growth ofhuman RMS xenografts in mice (29). The decrease in tumor

growth was associated with a decrease in a cell cycle-regulat-

ing protein p34cdc2, suggesting an arrest of proliferating cells inthe tumor (29). Moreover, alveolar AMS cells transfected with

an antisense IGF-IA expression vector showed decreasedIGF-IR expression, marked decrease in growth in vitro, impairedcolony formation in soft agar, and failure to form tumors in

immunodeficient mice (30). In mouse skeletal muscle,

Rosenthal eta!. (31) reported that differentiation from myoblaststo myocytes was associated with a decrease in IGF-IR bindingsites. The exogenous addition of IGF II to undifferentiated myo-blasts also decreased IGF-IR expression in BC3H-1 mousemuscle cells (31).

IFN-a, a member of the cytokine superfamily, is a secretedpolypeptide that exhibits a wide range of biological effectshaving antiviral, antiparasitic, antiproliferative, and antitu-

morigenic properties (32). IFN-a2a mediates intracellular

events by binding to IFN-lRs, thereby initiating a cascade of

intracellular signals via the Jak/Stat protein kinases (33-35).Evidence for the involvement of IFNs in the growth arrest and

differentiation of human/mouse skeletal muscle cells in cul-

ture has been reported (36, 37). Elevated levels of IFN-in-

duced enzymes, such as 2-5A synthetase and double-stranded RNA-activated protein kinase, have been shown toaccompany or precede the appearance of major muscle-specific differentiation factors during myogenesis (37). IFNhas also been shown to induce acceleration of myotubeformation, enhance the accumulation of creatine kinase, and

inhibit accumulation of acetylcholine esterase in normal hu-

man myoblast cultures (36). However, the effect on malig-

nant muscle cells has not been investigated.

In this study, we have shown for the first time that IFN-a2acauses down-regulation of the expression of IGF-IR andinduces growth arrest. Furthermore, the elevation of muscle-

specific genes, including MyoD, myogenin, and skeletalmuscle myosin, and the enhanced formation of multinucle-

of differentiation of malignant myoblasts. We also demon-strate that Rh30 cells expressing antisense IGF-lR acquiresome of the morphological and biological characteristics ofenhanced differentiation along the myogenic lineage. Thesedata suggest that down-regulation of IGF-IR is involved, inpart, in enhancing the differentiation of RMS.

Results

IFN-a2a-induced Growth Arrest of Human RMS Cell LineRh30. The alveolar RMS cell line, Rh30, was highly sensitive

to IFN-cr2a. Following exposure for 72 h, IFN-a2a sensitivitywas determined by colony formation (Fig. 1). IC50s for Rh30cells decreased from 0.81 to 0.26 lU/mI IFN-a2a, as theduration of exposure was increased.

To determine whether IFN-a2a effects were cytostatic orcytotoxic, dose-response and time course assays were con-ducted on the growth of Rh30 cells. Cells were treated withIFN-a2a at concentrations ranging between 0 and 300 lU/mI.Cell nuclei counts were determined at intervals for up to 144h. Increasing concentrations of IFN-a2a or increasing expo-sure times resulted in a significant decrease in the percent-age of cells relative to untreated cultures (Fig. 2A). A com-parison of IFN-a2a-treated cells with the number of cellspresent at the time of initiation of treatment (Fig. 2B) mdi-

cated that cell numbers were similar at time zero and 72-96

h after treatment, with no reduction in the number of cellsplated. Furthermore, 1 00 lU/mI IFN-a2a treatment resulted in59.8% (± 2.3%) inhibition in the incorporation of �H]thymi-dine within 24 h. These data suggested that IFN-a2a induceda cytostatic rather than a cytotoxic response.

Down-Regulation of IGF-lR by IFN-a2a in Rh30 Cells.Since Rh30 cells are highly dependent for their proliferation

on IGF Il/IGF-IR-mediated mitogenic signaling, we were in-terested in studying the effect of IFN-a2a on the expression

1 10 100IFNa-2a (lU/mI)

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20 144h

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100 150300

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Fig. 2. IFN-a2a induces growth arrest of Rh30 cells. Rh30 cells (50,000cells/well) were treated with increasing concentrations of IFN-a2a (0 to300 lU/mi). Cell nuclei counts were determined using a Coulter counter atintervals up to 144 h. A, the dose-response and time course effects ofIFN-a2a on the growth of Rh30 cells. B, to demonstrate the cytostaticrather than cytotoxic effects of IFN-a2a, the actual cell numbers at timezero were taken as 100%, and the number of cells at each time point ordosage of IFN-a2a were calculated as,relative ratios with respect to timezero and expressed as percentages (ordinate). The data shown are av-erages from a representative experiment conducted in triplicate. The SEobtained was <5%.

50 100HOURS

Cell Growth & Differentiation 533

of IGF-IR in increasing concentrations of IFN-a2a for 72 h.The expression of IGF-IR (cell surface staining), as deter-mined by immunoperoxidase staining, decreased with in-creasing concentrations of IFN-a2a [Fig. 3(a)]. Interestingly, atime course analysis of IGF-IR expression by flow cytometry[Fig. 3(b)] indicated that IGF-IA levels were decreased > 30%within 24 h of the addition of 100 lU/mI IFN-a2a, with little orno change at later time points. This time course is consistentwith the decrease in �HJthymidine incorporation.

IFN-a2a Induces Morphological Changes in Rh30 Cells

Indicative of Enhanced Differentiation. Logarithmicallygrowing Rh30 cells treated with IFN-a2a were fixed withice-cold acetone or paraformaldehyde. The nuclei werestained with hematoxylin, and the cytoplasm was coun-terstained using eosin (Fig. 4). Following the addition ofIFN-a2a to Rh30 cells in culture, morphological changes,including increased cell size, enhanced alignment, andformation of spindle-shaped cells, were observed. Fur-thermore, the number of multinucleated cells increased ina dose-dependent manner, with >30% in cultures treated

for 72 h with 100 lU/mI IFN-a2a (Table 1). These morpho-logical changes were indicative of enhanced differentia-tion of malignant myoblasts along the myogenic lineagewhich in normal myoblasts terminates with the formationof multinucleated myotubes.

IFN-a2a Induces Biological Changes in Human RMSCells Characteristic of Differentiation along the Myo-genic Lineage. Differentiation involves a transition frommyoblasts (undifferentiated, proliferating, mononucleatedstate) to myotubes (tubular, differentiated, multinucleatedstate). The initial event in myoblast fusion is withdrawal fromthe cell cycle. Upon differentiation, myotubes express mus-cle-specific genes associated with the synthesis and orga-nization of the contractile structure. MyoD and MyfS arepostulated to be responsible for determining the myoblaststate, whereas myogenin is expressed only when cells areinduced to differentiate. Having shown that IFN-a2a treat-ment gives rise to morphological changes characteristic ofdifferentiation, we examined whether the expression of mus-cle-specific genes, including MyoD, myogenin, and skeletalmuscle myosin, were up-regulated.

The expression of MyoD and myogenmn in Ah30 cells wasexamined by immunoperoxidase staining using monoclonal an-

ti-MyoD [Fig. 5(a), MyoD (1)] and anti-myogenin [Fig. 5(a� Myo-genin (10]antibodies following treatment for 72 h with increasingconcentrations of IFN-a2a. Concomitant with the elevated ex-pression of myogenin (nuclear staining), the expression ofMyoD (nuclear staining) decreased with increasing concentra-tion of IFN-a2a. Moreover, immunoperoxidase staining for skel-etal muscle myosin heavy chain, a marker for muscle differen-

tiation, showed higher levels of expression in Rh30 cells treatedwith IFN-a2a [Fig. 5(a), Skeletal Muscle Myosin (#{238}#{252})].The timecourse for changes in MyoD and myogenin expression wasnext determined by Western blot analysis. Myogenin expres-sion increased with exposure time, reaching a maximum by 96h [Fig. 5(b)] with no further increase at 120 h (data not shown).However, MyoD protein levels initially increased (5 h) in IFN-a2a-treated cells (50-100 lU/mI), followed by decreased ex-pression, reaching a minimum by 72 h [Fig. 5(b)].

It was suggested by Tapscott et a!. (38) that RMS were

deficient in factor(s) required for MyoD activity since MyoD inAMS, although capable of binding DNA, was relatively nonfunc-

tional as a transcriptional activator. To test whether MyoD func-tions were activated in IFN-a2a-treated Rh30, cells were trans-fected with muscle creatine kmnase-dnven CAT reporterconstructs (MCK-CA1) and simultaneously with pSVI3-gai tonormalize transfection efficiency. At 48 h aftertransfection, cellswere treated with 50 lU/mI of IFN-a2a, and CAT activities weremeasured at various time intervals. Exposure to IFN-a2a for 96

h resulted in a reproducible 2-4-fold increase in the expressionof MCK-CAT (Fig. 6), suggesting that IFN-a2a may be alteringthe functional activities of myogenic factors.

Rh30 Cells Expressing Antisense IGF-lR DemonstrateReduced Growth Capacity and Elevated Expression ofDifferentiation Markers. It has been demonstrated that thestable reduction of IGF-IR expression by transfection of an-tisense IGF-IR altered the malignant phenotype of Rh30 cells(30). These transfected cells (clone AS#23) had decreasedIGF-IA binding sites per cell, markedly reduced growth rates

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Fig. 3. Down-regulation of IGF-IR by IFN-a2a in Rh30 cells. a, imrnunostaining. Rh30 cells (50,000/well) were planted on two-chamber glass slides andtreated with IFN-a2a at concentrations of 0 (A), 10 (B), and SO lU/mI (C). After 72 h, cells were fixed with ice-cold acetone. Cells were then incubated withmouse monoclonal anti-IGF-IR antibody aIR3 and stained by immunoperoxidase using Vectastain ABC kit and counterstained using light green. The cellsurface staining was observed under a light microscope and photographed at x40. The images were computer generated using ADOBE Photoshop 2.5software. b, flow cytometry analysis. Rh30 cells (1 x 10w) were treated with 0, 100, and 300 lU/mI of IFN-a2a for 24, 48, 72, or 96 h. Cells were harvestedby trypsinization and incubated with the primary IGF-IR-specific antibody alR3 or isotype-matched control mouse immunoglobulin MOPC 21 . Cells weresubsequently incubated with FITC-conjugated secondary antibody and analyzed by flow cytometry. The RFI at each time point was calculated as the ratioof the mean fluorescence for IGF-IR relative to control; bars, SE.


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in vitro, and failed to develop tumors in immune-deprived

mice (30). However, the differentiation of these cells has not

been studied previously.

AS#23 cells were grown in 500 nM methotrexate to allowthe amplification of the plasmid encoding a 700-bp 5’ region

of IGF-IR in the antisense direction. By flow cytometry (Fig. 7),

Rh30 cells expressed significant amounts of IGF-lR with a RFI

of 6.3, whereas the AS#23 cells expressing antisense IGF-IA

had significantly less (< 50%) IGF-IR with an AFI 3.1 . The

doubling time for AS#23 was also somewhat greater, being 38h compared with 30 h for Ah30 cells. Moreover, there was a

linear relationship between the IGF-IR levels and the colony

formation in Ah30 antisense clones (data not shown).

We next examined the effect of antisense IGF-IR ox-

pression on cell morphology and muscle-specific gene

expression. Fig. 4D demonstrates the morphological char-

acteristics of AS#23 cells compared to Ah30 cells (Fig.

4A). AS#23 cells appeared larger with a significant in-

crease in the number of multinucleated cells (>80%) com-

pared with parental Rh30 (2-3%) and 100 lU/mI IFN-a2a-treated Rh30 cells (30%; Table 1).

To confirm whether the morphological changes induced in

AS#23 cells were associated with alterations in the expres-sion of muscle-specific genes, the expression of MyoD,desmin, and myosin heavy chain were studied by immunoper-

oxidase staining in parental Rh30 cells and AS#23. In compar-ison to Ah30, AS#23 cells showed increased expression of themuscle-specific differentiation markers (Fig. 8). We subse-

quently examined by Western blot analysis the effect of IFN-

a2a treatment on the expression of MyoD and myogenin in

AS#23 cells. The time course for changes in MyoD [Fig. 9(a)]and myogenin [Fig. 9(b)] expression in AS#23 cells treated with

1 00 lU/mI IFN-a2a demonstrated that both MyoD and myoge-nm expression were elevated with exposure time. These data

suggest that decreased expression of endogenous IGF-IA inAS#23 cells can result in enhanced differentiation of RMS cells.

Furthermore, IFN-a2a treatment also enhanced the expression

of muscle-specific differentiation marker genes.

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Fig. 4. Increase in the number multinucleated cells indicates enhancement of differentiation. Rh30 cells (50,000/well) were planted on two-chamber glassslides. Following overnight attachment, cells were treated with IFN-a2a at concentrations 0 (A), 50 (B), and 100 lU/mI (C). After 72 h, cells were fixed withice-cold acetone, nuclei were stained with hernatoxylin, and the cytoplasm was counterstained using eosin. Similarly, control AS#23 (D) cells were fixed,and nuclei were stained. The nuclear staining was observed under a light microscope and photographed at x40. The images were computer-generatedusing ADOBE Photoshop 2.5 software.

Table 1 Number of multinucleated cells in Rh30 cells treated for 72 hwith IFN-a2a and control AS#23 cells

Cell linesIFN-a2a

concentration(lU/mI)

Percentage of cells withmore than three

nuclei/cell

Total no. ofcells

counted

Rh30 0 2-3 800

Rh30 10 5-8 800Rh30 SO 25-28 800Rh30 100 30-32 800AS#23 0 80 500

DiscussionRMS are highly malignant childhood tumors of skeletal mus-

cle origin that demonstrate evidence of primitive myogenic

differentiation. Rhabdomyoblasts express MyoD, IGF II, andIGF-IR found in fetal muscle, consistent with the develop-mental arrest at a step after myogenic commitment. The

proliferation of an alveolar RMS cell line, Rh30, is dependentprimarily on autocrine signaling mediated by IGF II binding to

IGF-IR (26). Previously, it was shown that IGF II stimulatestwo different types of response in alveolar RMS: (a) a mito-

genic response through IGF-IR; and (b) a motility response

through type II receptors (26). Furthermore, it has been dem-

onstrated that antisense-mediated reduction of IGF-lR ex-

pression can suppress the malignant phenotype of Rh30

cells both in vitro and in vivo (30).

In this study, we present evidence that: (a) IFN-a2a, a

cytokine with antiproliferative properties, inhibits growth, in-

duces differentiation of alveolar RMS, and down-regulates

the expression of IGF-lR; and (b) the reduction of endoge-

nous IGF-IR using antisense IGF-IR, alone and in the pres-

ence of IFN-a2a, enhances the differentiation stage of thesemalignant myoblasts. IFN-a2a treatment resulted in loss of

clonogenic potential with an IC50 of 0.81 lU/mI following 72

h exposure caused by prolonged cytostasis, not by cytotox-

icity. Furthermore, IFN-a2a treatment also resulted in the

down-regulation of IGF-lR. Various morphological changes

characteristic of differentiation, including the formation of

spindle-shaped, multinucleated cells, and elevated expres-

sion of the muscle-specific genes, myogenin and skeletal

muscle myosin, were observed. These data suggested that

the consequences of IFN-a2a treatment were down-regula-

tion of IGF-lR expression, growth arrest, and enhancement

of myogenic differentiation. Growth arrest appeared to be

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Fig. 5. IFN-cx2a effects on the expression of muscle-specific proteins. a, immunocytochemical staining. Rh30 cells (50,000/well) were planted ontwo-chamber glass slides and treated with IFN-a2a at concentrations of 0 (A), 50 (B), and 100 lU/mi (C). After 72 h, cells ware fixed with 1 %paraformaldehyde and permeabilized with 0.25% Triton X-1 00 in PBS. Cells were than incubated with mouse monoclonal antibody 5.8A to MyoD (i), ratmonoclonal anti-myogenin antibody (ii), and rabbit polyclonal antibody to skeletal muscle rnyosin (iii), and the imrnunoperoxidase reaction was developedusing the Vectastain ABC kit. The staining was observed under a light microscope and photographed at x40. The images were computer generated usingADOBE Photoshop 2.5 software. b, immunoblot analysis. Total cellular proteins extracted (30 jig) from Rh30 (1 x 10�) treated with 100 lU/mI of IFN-a2afor 0, 5, 24, 48, 72, and 96 h were subjected to 10% SDS-PAGE, and the separated proteins were transferred to an Immobilon membrane. The membraneswere probed with mouse monoclonal anti-MyoD 5.8A or rat anti-myogenin antibody, and respective goat antimouse or goat antirat secondary antibodieslinked to horseradish peroxidase. The peroxidase reaction was developed on Fuji X-ray film using ECL chemiluminescence kit. The images were computergenerated using ADOBE Photoshop 2.5 software.

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Fig. 6. Induction of MCK-CAT expression by IFN-a2a in Rh30 cells.Rh30 cells (1 X 1 0�) were transfected with 20 �g each of MCK-CAT andpSVI3-gal expression vectors by electroporation. About 48 h after trans-fection, cells were treated with 50 lU/mI of IFN-a2a and assayed for CATactivity by scintillation method and f3-gal expression by colorimetric as-say. The CAT activity data were normalized to the p-gal expression. TheCAT expression (ordinate) was calculated as the ratio of dpm per 106cells/h to the absorbance per 1 06 cells/h. The data shown were obtainedfrom a representative experiment. �, 50 lU/mI IFN-a2a treatment; 0,0 lU/mI IFN-a2a exposure.

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rapid, with >50% decrease in [3H]thymidine incorporation

within 24 h of exposure to IFN-a2a, and was in agreement

with the time course for reduced expression of IGF-IR. Be-

cause the effect of IFN-a2a was cytostatic, it is unlikely that

Fig. 7. Reduced expression of cell surface IGF-IR in cells (clone AS#23)stably expressing antisense IGF-IR. Cells (Rh30 or AS#23; 1 x 10�) wareharvested by trypsinization and incubated with an antibody aIR3 or iso-type-matched, purified immunoglobulin MOPC 21 . Cells were subse-quently incubated with FITC-conjugated secondary antibody and ana-lyzed by flow cytometry. The RFI calculated for Rh30 and AS#23 cellsusing arithmetic histogram statistics are shown.

Rh30 AS23

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Fig. 8. Expression of muscle-specific genes in Rh30 and AS#23 cells by immunostaining. Rh30 or AS#23 cells were fixed and stained for the expressionof MyoD, desmin, and myosin heavy chain using respective antibodies; the immunoperoxidase reaction was developed using the Vectastain ABC kit. Thestaining was observed under a light microscope and photographed at x40. The images were computer generated using ADOBE Photoshop 2.5 software.


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the rapid decrease in IGF-IR was a consequence of cyto-

toxic action to cells with higher levels of receptor expression.

During normal myogenic differentiation, the myogenic de-

termining factors Myf5, MyoD, and myogenin are expressedin a coordinate fashion (3). MyoD or Myf5 are postulated tobe primarily responsible for defining commitment to the myo-blast state and for proper positioning of the cells in the

muscle-forming region of the body (4). Furthermore, MyoD

and MyfS are also the principle sites for negative regulators

such as fibroblast growth factors, oncogenes including fos,fun, myc, and ras, and the inhibitory protein Id (16-21). MyoD

has been shown recently to induce the expression of cyclin-

dependent kinase inhibitor protein p2lC�P�, which causes G1phase arrest (39). Furthermore, expression of MyoD is reg-

ulated by itself and myogenin (3), and MyoD may also stim-

ulated myogenin expression (40). Myogenin is expressed

only in cells induced to differentiate such that an initial in-

crease, followed by a decrease in MyoD protein levels with a

coordinate increase in the expression of myogenin demon-

strated in this study in the presence of IFN-a2a, is consistent

with enhanced myogenic differentiation of Rh30 cells.

Rh30 cells that stably express antisense IGF-IR (AS#23)

showed morphological and biological changes characteristic

of myogenic differentiation. AS#23 cells have been shown to

exhibit impaired colony formation in soft agar linearly related

to IGF-IR levels, marked decrease in growth in vitro, and

decreased formation of tumors in immunodeficient mice,

suggesting a critical role for IGF-lR in mitogenic signaling.

These data are consistent with overexpression of IGF-IRinhibiting differentiation (41-43) and the involvement of


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Fig. 9. IFN-a2a induces MyoD and myogenin expression in AS#23 cells. Total cellular protein (30 �g) from AS#23 cells treated with 1 00 lU/mI of IFN-a2afor 0, 24, 48, 72, and 96 h was analyzed on 12% SDS-PAGE and probed with anti-MyoD 5.8A antibody (a) or anti-myogenin antibody (b). The filters werenormalized for protein loading with anti-tubulin antibody. The peroxidase reaction was developed using the ECL chemiluminescence kit. The images werecomputer generated using ADOBE Photoshop 2.5 software.

IGF-IR in the IFN-a2a-induced differentiation of Rh30 cells.Furthermore, the increased number of multinucleated cells in

clone AS#23, in comparison to the IFN-a2a-treated Rh30

cells, may be due to the greater length of time that AS#23,

cells have been under selection pressure. In this study, Rh30

cells were treated with IFN-a2a for 72 h prior to staining,

whereas AS#23 were selected for stable expression of anti-sense IGF-IR and further maintained in culture in the pres-ence of methotrexate. Hence, morphological differentiation

induced by stable down-regulation of IGF-IR alone (AS#23)

and IFN-a2a treatment (Rh30) appeared somewhat different.

Furthermore, lFN-c�2a treatment of AS#23 cells induced bothMyoD and myogenin expression in contrast to the parental

Rh30 cells, where IFN-a2a induced expression of myogeninand reduced the expression of MyoD following an initial

increase. However, we observed that although IFN-ct2a

caused a general decrease in the expression of MyoD in

Rh30 cells, the decrease was in most but not all cells [Fig.

5(a), MyoD (i)]. Hence, the differences in the expression of

MyoD in AS#23 may be due to the clonal selection of a single

population of cells already expressing higher levels of MyoD

(Fig. 8). These data suggest that IFN-a2a may act in asso-ciation with IGF-IR, thereby allowing cells to proceed further

downstream of the differentiation pathway. Furthermore, the

induction of differentiation of Rh30 cells was observed under

mitogenic conditions in medium containing 10% FBS. In

contrast, most studies for myoblast fusion are conducted

under conditions that could induce differentiation. Interest-

ingly, culturing of Rh30 cells in serum free, low serum, or in

differentiation medium did not have a significant effect on the

morphology of the cells, although the growth rate was

slightly reduced. Failure to differentiate under these condi-

tions may be a consequence of autocrine stimulation of

growth by endogenous IGF II secretion.

It was suggested by Tapscott et a!. (38) that RMS are

deficient in factor(s) required for MyoD activity since MyoD in

RMS, although capable of binding DNA, was relatively non-

functional as a transcriptional activator. We have shown that

IFN-a2a treatment induces expression of a reporter gene,

CAT, driven by muscle creatine kinase (MCK-CAT) in Rh30

cells, suggesting that the functional activity of myogenic

regulatory factors may be activated by IFN-cr2a. Moreover,

the finding that IFN-a2a increases expression of myogenin

suggests that either expression of this myogenic factor isinduced directly, or alternatively, negative functional regula-

tion of MyoD is suppressed. Alternatively, in IFN-a2a-treated

Rh30 or AS#23 cells, MyoD function may be activated by a

cascade of events that include dephosphorylation of threo-

nine 1 15 in the DNA-binding domain (44, 45), which com-

prises a negative regulatory site.

It is possible that the two events, i.e., growth arrest and

myogenic differentiation of RMS cells, may be mediated by

different pathways. Growth of Rh30 cells may be dependentprimarily on IGF-IR function, whereas differentiation of Rh30

cells along the myogenic lineage may be dependent primarily

on the coordinate expression of muscle-specific genes suchas MyoD and myogenin. Hence in AS#23 cells, decreasedIGF-IR levels reduced the growth rate, and as a conse-

quence, some of the morphological and biological changes

were observed. In an embryonal RMS cell line RD, phorbol

ester-induced differentiation has been shown to be mediated

by the transient activation of an isoform protein kinase C a,

which activates events necessary for myogenic differentia-

tion; in addition, constitutively active protein kinase C � and� are required for the maintenance of cell growth (46). The

role of IGF-IR in cell growth and transformation in vivo and invitro has been well established (47, 48). NIH3T3 and human

diploid fibroblast cell growth depend on cooperation be-

tween two growth factors, IGF I and PDGF, either of which

alone fails to induce cell proliferation (48-50). Moreover, cells

derived from knock-out mice with disruption of the IGF-IR

gene and the IGFII gene failed to grow in serum-free medium

supplemented with growth factors and were resistant to

transformation by SV4O T antigen (49, 51-53). Overexpres-

sion of either EGF receptor or PDGF receptor in cells dis-

rupted in IGF-IR did not allow cells to grow or be transformed

by the addition EGF or PDGF (49). However, reintroduction ofIGF-IR into these null cells restored EGF or PDGF-mediated


growth and transformation (49). Interference of IGF-lR ex-pression using antisense IGF-IR RNA or oligonucleotidescomplementary to IGF-lR reversed the transformed pheno-type (54, 55). Since RMS express high levels of IGF II, Minnitiet a!. (56) studied the role of IGFII in the pathogenesis of RMSby overexpressing IGF II in normal mouse myoblasts.Expression of high levels of IGF II resulted in morphological

and biological changes, such as impaired ability to differen-tiate, increased proliferative rate typical of the malignantphenotype, although there was no change in the expressionof IGF-IR. They suggested that down-regulation of IGF IIis essential for the completion of the differentiationprocess (56).

IGF-IR transduces mitogenic signals through several path-ways. The most defined pathway involves two substrates,insulin receptor substrate I and Shc, which subsequentlyactivates the signal transduction pathway leading throughRas and Raf to the nucleus to Fos and Jun (57). Other growthfactors, like PDGF and EGF, bind to specific receptors andactivate ras, raf-dependent signal transduction (58). Suchcross-talk between various growth factors has been well

established. IFN signaling through the recently discoveredJak/Stat protein kinases transactivates IFN-responsive

genes (33-35). Recently, the interaction between the IFN-induced growth inhibitory pathway and PDGF-inducedgrowth stimulatory pathway were reported (59). Therefore, inRMS cells, IFN-a2a signaling and IGF IVIGF-IR signalingmight be interacting or interfering at some locus in the signaltransduction pathway. Hence, IGF-lR down-regulation couldbe a cause or a consequence in IFN-a2a-induced differen-tiation of Rh30 cells along the myogenic lineage.

In conclusion, in the presence of IFN-a2a, Rh30 cellsexhibit growth arrest, reduced IGF-lR levels, and enhance-ment of differentiation along the myogenic lineage. Further-more, antisense RNA-mediated reduction of IGF-lR in Rh30cells, alone and in the presence of IFN-a2a, enhanced thedifferentiated state. Hence, down-regulation of IGF-IR is in-volved, in part, in the enhancement of differentiation of thesemalignant myoblasts.

Materials and MethodsCell Cufture. The human alveolar RMS cell line Rh30 was establishedfrom the bone marrow of a patient with metastatic tumor (60). Cells werecultured in RPMI 1640 (BioWhittaker, Walkersville, MD) supplementedwith 10% heat-inactivated FBS, and 2 mp,i glutammna in a humidified

atmosphere containing 5% CO2 at 37#{176}C.Stably transfected Rh30 cells(30) expressing antisense IGF-lR, AS#23 were grown in RPMI 1640 sup-plemented with 10% heat-inactivated FBS, 2 m,.i glutammne, and 500ng/ml methotrexate.

Antibodies and Expression Vectors. The following antibodies were

used: mouse monoclonal antibody, 5.&A., to MyoD (61), rat monoclonal an-tibody to myogenin (a kind gift from Dr. W. E Wright, University of TexasSouthwestem Medical Center, Dallas, TX); mouse monoclonal antibody,alR3, to IGF-IR (a generous gift from Dr. S. Jacobs, Burroughs Weilcorne,Research Triang� Park, NC); rabbit polyclonal antibody to skeletal musclemyosin, affinity-purified mouse immunoglobulmn, MOPC 21, and mousemonoclonal antibody to tubulin (Sigma Chemical Co., St. Louis, MO); and

mouse monoclonal arttibodytodesm,n (DAKO Corporation, Carpinteria, C�.

Secondary antibodies used were goat antirat, antirabbit, and antimouselinked to horseradish peroxidasa (Amersham, Mington Heights, IL). Thefollowing expression vectors were used: CATexpression vector MCK-CAT(agenerous gift from Dr. S. Tapscott, Fred Hutchinson Cancer Research Can-

ter, Seattle, WA); and pSV�3-gal control vector (Promega, MadiSOn, WI).

Clonogenic and Growth Assays. For clonogenic assays, Rh30 cellswere plated in triplicate on 6-well plates (Becton Dickinson Labware,

Franklin Lakes, NJ) at a density of 3000 cells/well. Following ovemightattachment, varying concentrations of recombinant human IFN-a2a(Roche Laboratory, Nutley, NJ) were added. After varied duration ofexposure, cells were washed once with RPMI 1640 without FBS. FreshRPMI 1640 medium with FBS was added to the cells, and plates were

subsequently incubated for a total of 7 days prior to staining with crystalviolet dye and quantitated using an Artek model 880 colony counter.

For growth assays, cells (5 x 104/well) were plated in triplicate on6-well cuiture dishes. The following day, increasing concentrations ofIFN-a2a were added. Cells were lysad at the required time points underhypotonic conditions (10 m� HEPES and 1 .5 m�.i MgCI�), and nuclei werecounted using a Coulter counter ZM.

H&E StaIning. Rh30 cells (50,000/wall) were plated in two-chamberglass slides. The following day, cells were treated with varying concan-trations of IFN-a2a. Cells were fixed with ice-cold acetone or 1 % para-

formaldehyde, parrneabilized with 0.25% Triton X-100, and washed withPBS. Nuclei were subsequently stained with hamatoxylin (Richard AllanMedical, Richland, Ml). Cells were rinsed several times in water, followed

by acidic alcohol (80% ethanol + 20% acetic acid) and neutralized withBlue reagent (Richard Man Medical). Cells were then rinsed with water,

followed by 95% ethanol priorto countarstaining with eosin (Richard Allan

Medical), and finally rinsed with absolute ethanol prior to mounting inpermount as a preservative.

Flow Cytometry Analysis. Cells were harvested by trypsinization fromconfluent flasks, washed twice in �e-coId PBS, and then incubated with

IGF-IR antibody, alR3(1:loodilution of2.2 �/g�l) orcontrol isotype-rnatchedantibody MOPC 21 for 30 mm at room temperature. Cells were washed withPBS and incubated with fluoresceinated affinity-purified goat antimouse im-

munoglobuhn for 30 mm at room temperature. After further washing withPBS, cells were resuspended in DNA staining medium contalning 0.25 m�propidium �iodkIe and analyzed with a EPICS 753 flow cytometer. The fluo-rescence scale was a 256-channeV3-decade log scale. The RFI was dater-

mined by calculating the ratio ofthe mean channel of calls stalned with alR3compared to the same cells treated with MOPC 21.

Immunoperoxidase Staining. Approximately 50,000 cells/well wereplated in two-chamber glass slides. Varying concentrations of IFN-a2awere added the following day. After appropriate durations of exposure,cells were washed once with PBS. For intracellular or nuclear proteins,cells were fixed in 1 % paraformaldehyde for 20 mm at room temperature

and permeabilized in 0.25% Triton X-100 for 20 mm at room temperature.For call surface receptor proteins, cells were fixed in ice-cold acetone, andno further perrneabilization was carried out. After fixation, cells werewashed twice with PBS and incubated with the primary antibody diluted

in PBS for 1 h at room temperature or ovemight at 4#{176}C.Cells were then

washed several times with PBS prior to incubation with appropriatelydiluted Vectastain biotmnylated secondary antibody for 30 mm (VectastainABC kit; Vector Laboratories, Inc., Burlingarne, CA). Calls were againwashed with PBS and incubated with horseradish peroxidase conjugatedwith streptavidin for 30 mm. Finally, cells were washed with PBS, and thehorseradish peroxidase reaction was developed using the substrate dia-rninobenzidina and H2O2. Cells were counterstained with light green

(Fisher Scientific, Pittsburg, PA), where indicated.Immunoblot Analysis. About 1 x 10� cells were harvested by centrif-

ugation after appropriate concentrations and time intervals of exposure toIFN-a2a. Cell pellets were washed with PBS and stored at -70#{176}Cfor immu-noblot analysis. The pellets were reconstituted in appropriate volumes of lysisbuffer containing 50 mM TrisCI (pH 7.5), 20 m� MgCI2, 150 rn�i NaCI, 0.5%

NP4O, 10 �g/rnl each of aprotinin, leupeptin, and antipain, 0.5 m� phenyim-ethylsulfonyl fluoride, and 1 rn� sodium orthovanadate. Total cell lysateswere obtained by sonicating on �e for 2 s. Total protein was assayed usingthe method of Bradford (Bio-Rad Laboratories, Hercules, CA). For irnrnuno-blothng, 30-40 �g protein/sample were mixed with an equal volume 2xSDS-PAGE sample buffer. The samples were heat denatured by heating at100EC for 3 mm, and proteins were separated at 100 V for 1 h using 10% or

12% precast readygels (BiO-Rad). Rainbow-colored protein molecular mark-ers (Amersham) were used as standards. The separated proteins were thentransferred onto an Imrnobilon-P membrane (Millipore Corp., Bedford, MA)

using a sernidryTransblot apparatus(lntegrated Separation Systems, Natick,MA). The membranes were blocked by incubation with 5% nonfat dry milk in

PBS orTris-buffered saline containing 0.2% Tween 20 (lBS-i) at 37#{176}Cfor 30


mm to 1 h. Membranes were incubated with primary anthody overnight at

4EC or at room temperature for 2-3 h and washed three times, 5 mm each,

with PBS 0rTBS-T and incubated with 1:3000 dilution ofsecondary antibody

linked to horseradish peroxidase at room temperature for 1 h. Membranes

were finally washed three times with PBS or TBS-T, and the reaction was

developed on Fuji X-ray film using the ECL chemiluminescence kit (Amer-sham).

Transient Transfection by Electroporation. Logarithmically growingcells were trypsinized and resuspended in 10 ml of PBS, and about 1 x1o� cells were used per transfection. Twenty �g of expression vectorswere mixed with the cells in electroporation cuvettes (Bio-Rad Labs). Themixture of calls and DNA was pulsed at room temperature with a setting

of 200 V and 960 �F capacitance. The cells were incubated at roomtemperature for 1 0 mm. The cells were then transferred to a flask con-

taming 15 ml RPMI 1640 with 10% FBS and allowed to recover. After 48h, cells were treated with 50 lU/mI of IFN-a2a, and the expression of

transfected DNA was assayed at various time intervals.CAT and �3-gal Assays. Cells resuspended in 220 �.tI ofTE [50 m� Tris

(pH 7.8) and 1 mM EDTA] were lysad by three rounds offreeze-thaw(5 mmon dry ice, followed by 1 mm at 37#{176}C)and microfuged at 12,000 rpm for1 0 mm at 4#{176}C.The supernatant obtained was the total cell lysate. An

aliquot of the cell lysate was heat inactivated and used for CAT assay,while the remaining nonheated lysate was used to determine the p-gal

activity. For CAT assay by the scintillation method, the heated cell extractwas mixed with 25 �I of 1 M Tris#{149}Cl(pH 7.8), 50 �d of 5 rn� chloroarn-phenicol, 0.5 �Ci of 3H-Iabeled acetyl-C0A substrate (ICN Radiochemi-

cals, Costa Mesa, CA) in a total volume of 200 �I. Appropriate positive(purified CAT enzyme; Sigma) and negative control (no extracts) were

used in the assay. The above reaction mixture was overlayed with 3 ml ofwater-immiscible scintillation fluid and then incubated at 37EC. Theamount of radioactivity was determined by scintillation counting at differ-

ant time intervals (0.5, 1 , 2, 4, 8, and 24 h). The data represented as

dprn/106 cells/h were obtained from the linear portion of the curve. Forp-gal assays, the nonheated cell lysates were mixed with 500 �I ofphosphate buffer(60 mM Na2PO4, 40 m� NaH2PO4, 10 mp�i KCI, and 1 mp�i

MgCI�), 3.6 �I of p-mercaptoethanol, 50 pJ of 48.6 mg/mI stock chloro-phenol red-f3-D-galactopyranoside (Boahringer Mannheim, Indianapolis,IN) to a total volume of 650 p.1. The reaction mixture was incubated at

37#{176}C,and the colorirnetric reaction was determined at intervals (every 15mm) by measuring the absorbance at 570 nrn. The data represented asA57�/10 cells/h were obtained from the linear portion of the curve. TheCAT:j3-gal activity ratio was calculated to correct for transfection effi-ciency, and the CAT:p-gal ratio for the negative control was subtracted

from the values to obtain the corrected CAT expression.

AcknowledgmentsWe would like to thank Dr. Richard Ashmun for his help with the flow

cytomatry analysis, Elisha Jenkins for CAT assays, and Frank Harwood for

the computer-generated images using ADOBE Photoshop 2.5 software.

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