Retinoic Acid Induces Myogenin Synthesis and Myogenic Differentiation in the Rat Rhabdomyosarcoma Cell Line BA-Han-IC

Two clonal rat rhabdomyosarcoma cell lines BA-Han-IB and BA-Han-IC with different capacities for myogenic differentiation have been examined for the expression of muscle regulatory basic helix-loophelix (bHLH) proteins of the MyoD family. Whereas cells of the BA-Han-IC subpopulation constitutively expressed MyoD1 and could be induced to differentiate with retinoic acid (RA), BA-Han-IB cells did not express any of the myogenic control factors and appeared to be largely differentiation-defective. Upon induction with RA, BA-Han-IC cells expressed also myogenin, in contrast to BA-Han-IB cells which never activated any of the genes encoding muscle bHLH factors. The onset of myogenin transcription in BA-Han1C cells required de novo protein synthesis and DNA replication suggesting that RA probably did not act directly on the myogenin gene. Although MyoD1 was expressed in proliferating BA-Han-IC myoblasts, muscle-specific reporter genes were not activated indicating that MyoD was biologically inactive. However, transfections with plasmid expressing additional MyoD1 protein resulted in the transactivation of muscle genes even in the absence of RA. mRNA encoding the negative regulatory HLH protein Id was expressed in proliferating BA-Han-IC cells and disappeared later after RA induction which suggested that it may be involved in the regulation of MyoD1 activity. The myogenic differentiation of malignant rhabdomyosarcoma cells strictly correlated with the activation of the myogenin gene. In fact, stable transfections of BA-Han-IC cells with myogenin expressing plasmids resulted in spontaneous differentiation. Taken together, our results suggest that the transformed and undifferentiated phenotype of BA-Han-IC rhabdomyosarcoma cells is associated with the inactivation of the myogenic factor MyoD1 as well as lack of myogenin expression. RA alleviates the inhibition of myogenic differentiation, probably by activating MyoD protein and myogenin gene transcription. BA-Han-IB cells did not respond to RA and the differentiated phenotype could not be restored by overexpression of MyoD1 or myogenin. T RANSFORMATION of normal cells to a malignant phenotype is believed to involve multistep mechanisms which alter regulatory processes controlling growth and differentiation. In many tumors individual cells can be found that spontaneously undergo partial or complete differentiation or can be induced to differentiate by various agents (Pierce, 1974). These observations have indicated that not all cells in a given tumor are necessarily in the same state of transformation and some can be reverted to a benign phenotype. Induction of differentiation has actually been proposed as an alternative approach to conventional therapy of cancer (Metcalf, 1983; Spremulli and Dexter, 1984; Sartorelli, 1985; Sachs, 1987). To investigate factors and conditions which may influence the balance between the transformed state and the differentiated phenotype, we have used clonal cell lines isolated from a dimethylbenzanthraceneinduced rat rhabdomyosarcoma which were shown to exert different capacities to differentiate in vitro (Gerharz et al., 1988). In contrast to normal myogenic cells in which differentiation can be induced by the mere removal of serum components, only a minute proportion of the rhabdomyosarcoma cen line BA-Han-IC forms myotubes under these conditions. The majority of cells requires retinoic acid (RA) ~ or a variety of other inducing agents for effective differentiation (Gabbert et al., 1988; Gerharz et al., 1989). The BAHan-lB cell line isolated from the same tumor can not be triggered to differentiate to the complete muscle phenotype by inducing agents, although it appears to be of skeletal muscle origin as judged by its morphology and the expression of desmin and muscle creatine kinase (Gerharz et al., 1988, 1989). In the absence of inducers both cell lines appear as mononuclear, rapidly dividing rhabdomyoblasts. The development of muscle cells has become a preferred model system to study molecular mechanisms underlying cellular differentiation. A group of myogenic control proteins encoded by the MyoD gene family has recently been identified (for review see Olson, 1990; Emerson, 1990; 1. Abbreviations used in this paper: bHLH, basic-helix-loop-helix; MHC, myosin heavy chain; RA, retinoic acid; RAR, retinoic acid receptor. 9 The Rockefeller University Press, 0021-9525/92/08/877/11 $2.00 The Journal of Cell Biology, Volume 118, Number 4, August 1992 877-887 877 on July 8, 2017 jcb.rress.org D ow nladed fom Weintraub et al., 1991). Four distinct cDNAs, MyoD1 (Davis et al., 1987; Braun et al., 1989a), myogenin (Wright et al., 1989; Edmondson and Olson, 1989; Braun et al., 1989a), Myf-5 ~rann et al., 1989b), and MRF4/herculin/Myf-6 (Rhodes and Konieczny, 1989; Miner and Wold, 1990; Braun et al., 1990) have been isolated and shown to possess the capacity to convert 101"1/2 fibroblasts and a variety of other nonmuscle cells to the myogenic lineage. All of these proteins contain a highly conserved basic region adjacent to a sequence motif that is believed to form two amphipathic helices which are connected by a short intervening loop structure. The basic-helix-loop-helix (bHLH) domain is responsible for heterooligomerization and sequencespecific DNA binding to a DNA sequence referred to as E-box (Davis et al., 1990; Murre et al., 1989a,b). This E-box motif consisting of the highly degenerate DNA consensus sequence CANNTG was found to be present in control elements of many muscle-specific genes where it mediates transcriptionai activation by the interaction with the muscle-specific regulatory bHLH proteins (reviewed in O1son, 1990; Emerson, 1990). The biochemical properties of these proteins and their exclusive expression in skeletal muscle and embryonic muscle progenitor cells suggest that their primary biological activity may be to activate the transcription of typical muscle genes. To investigate the role of the myogenic control factors in the differentiation-competent rhatxlomyosarcoma cell line BA-Han-IC and in the differentiation-refractory counterpart BA-Han-IB, we analyzed the expression and activity of the various members of the MyoD family. We were particularly interested in the effects that RA acting as a differentiation inducing agent may exert on the activity of the myogenic factors and their genes. Here, we report that MyoD1 is constitutively expressed in the inducible cell line BA-Han-IC but not in the uninducible line BA-Han-IB. Administration of RA results in a delayed transcriptional activation of the myogenin genc in BA-Han-IC but not in BA-Han-IB cells. Activation of myogenin genc expression is dependent on protein and DNA synthesis suggesting that RA may act rather indirectly. Our results provide evidence that the block of differentiation in the rhabdomyosarcoma cell line BA-Han-IC may be dependent on the lack of myogenin expression. However, a mechanism that inhibits the transcription activating function of MyoD1 may also play an important role for the inability of these cells to differentiate in the absence of the inducer RA. Both events may in fact be related. Materials and Methods Cell Culture and DNA Transfections The isolation and characterization of the rat rhabdomyosarcoma cell lines BA-Han-IB and BA-Han-IC have been described previously (Gerharz et ai., 1988, 1989). Cells were cultured in DME supplemented with 10% FCS, penicillin, and streptomycin. Ctflture media and sera were purchased from Gibco Laboratory (Eggenstein, Germany). To induce differentiation, RA (scrva Inc.) was added to a final concentration of 1 ~M from a 5 mM stock solution prepared in 95 % ethanol. Medium was routinely chan~od after 3 d. Rat L6 and mouse C2C12 celis (Yaffe and Saxel, 1977) were obtained from the American Type Culture Collection (Rockville, MD) and were cultured as described elsewhere (Braun et al., 1992; Sniminen et ai., 1991). Rhabdomyosarcoma cells were stably transfected with 1 ~g of supercoiled pSV2-neo plasmid conferring geneticin (G418) resistance and 20/zg of pEMSV-Myf4 by use of the calcium-phosphate precipitation method. G418-resistant colonies were selected in medium containing 400 ~g/ml G418 (Geneticin; Gibco Laboratories, Grand Island, NY). Transient transfections were performed as described previously (Braun et al., 1989c). CAT activity was determined by standard procedures (Gorman, 1985) two days after transfecfion. Transfection efliciencies were controlled by cotransfection of 5 #g RSV-Bgal plasmid and CAT activity was standardized according to ~-galactosidase activity obtained in the same cellular extracts. Reporter Plasmids and Expression Vectors Myf4L-CAT reporter containing 1.1 kb of the human Myf-4 gene promoter driving the CAT gene has been described previously (Saiminen et ai., 1991). MCK4R-CAT plasmid containing four oligomerized MyoDl-binding sites upstream of the thymidine kinase TATA-box linked to the CAT gene has been provided by A. Lassar (Harvard Medical School, Boston, MA) (Weintraub et ai., 1991). TK-CAT reporter plasmid has been obtained by G. Schlitz (German Cancer Research Center, Heidelberg, Germany). MLCCAT plasmid containing the muscle-specific enhancer of the rat myosin light chain 1/3 gene and the MLC1 core promoter linked to the CAT gene has been published previously ~senthal et al., 1990). The expression vector pEMSV-Myf4 has been described (Braun et ai., 1989a). Isolation of RNA and Northern Blot Analysis RNA was isolated from tissue culture cells by the guanidinium method (Chomzynski and Sacchi, 1987). 5-10 • 106 cells yielded between 200 and 300 ~g of total RNA. Glyoxylation, agarose gel electrophoresis, RNA transfer, and hybridization conditions have been described previously (Braun et al., 1989b). Briefly, 25 ~g of total RNA was separated on gels, transferred onto PALL Biodyne Membrane (Portsmouth, UK), and hybridized with 1-3 • 106 cpm/ml of radioactively labeled hybridization probe (sp act 1-2 • 10 a cpm/~g obtained through random priming with [32p] dCTP) (3,000 Ci/mmol). Filters were hybridized in 50% formamide, 5x Denhardi's solution, 5 • SSC (1• SSC contains 0.15 M sodium chloride and 0.015 M sodium citrate), 50 mM sodium phosphate, 0.1% SDS at 42~ for 18 h. Filters were washed in 0.1% SSC, 0.1% SDS at 55~ for 30 vain in several steps. The following probes were used for hybridization: (a) the mouse MyoDl probe was obtained as a 800-bp HpalI/EcoRI fragment from the cloned eDNA (Davis et ai., 1987) representing the 3' noncoding region; (b) the myogenin probe was prepared as a 700-bp fragment from the rat eDNA as described previously (Sassoon et at., 1989); (c) the GAPDH probe was a 1,100-bp segment of the mouse glyceraldehyde phosphate d e h y ~ eDNA: (d) the Id eDNA probe has been provided by H. Weintraub (Fred Hutchinson Cancer Center, Seattle, WA) and has been described (Benezra et ai., 1990); and (e) mouse Myf-5 and Myf-6 probes have been described previously (OR et ai., 1991; Bober et aL, 1991). Immunohistochemicai Staining of Culture Cells Cells were seeded on microscopic slides and cultured under the described conditions: ceils were fixed in methanol for 5 min followed by acetone treatment at -20~ for 10 s, and air dried. The mAb against rat myogenin, kindly provided by W. Wright (University of Texas, Dallas, TX) was applied for 30 rain at room temperature in a moist chamber. Slides were rinsed in PBS solution and incubated with the antimouse IgG second antibody using the avi~me-biotin complex and peroxidase staining for detection (Vectastain Kit; ABC, Burlingame, CA).