Effects of Phorbol 12-Myristate 13-Acetate on the Differentiation Program of Embryonic Chick Skeletal Myoblasts1

The effects of phorbol 12-myristate 13-acetate (PMA) on three aspects of myogenesis have been analyzed: (a) fusion of mononucleated myogenic cells to form myotubes; (b) synthesis and accumulation of two muscle-specific proteins; and (c) DNA syn thesis. Using autoradiography combined with immunofluorescent localization of muscle-specific light meromyosin and the musclespecific intermediate filament protein desmin, we have found that embryonic chick myogenic cells cultured in the presence of PMA (50 nw) initiate the synthesis of both desmin and muscle-specific light meromyosin and, by these criteria, partially differentiate. These cells differ from normal definitive postmitotic myoblasts, however, since they (a) do not fuse; (b) do not assemble normal myofibrils; and (c) incorporate [3H]thymidine. PMA does not appear to induce DNA synthesis in postmitotic myoblasts, but it apparently permits cells to initiate expression of muscle-specific proteins while preventing complete withdrawal from the cell cycle. Inhibition of fusion by PMA has been reported, but contin ued incorporation of [3H]thymidine in nuclei of cells expressing muscle-specific proteins is a previously undescribed effect of PMA. This effect is not achieved by 4-«-phorbol-12,13-didecanoate, a nonpromoting phorbol ester, and may be relevant to the action of PMA as a tumor promoter. INTRODUCTION The tumor promoter PMA5 has a wide variety of effects, including either the inhibition or stimulation of cell differentiation (for reviews, see Refs. 12, 28, 52, and 54). With respect to differentiation, the effect of PMA differs depending on cell type (13, 34, 43), species (21, 41, 45, 46), and in what compartment of its lineage the cell is (11, 35, 44). One of the first systems in which PMA was shown to reversibly inhibit different aspects of differentiation was in vitro myogenesis (8-10). PMA has been shown to (a) reversibly block the fusion of postmitotic myoblasts into multinucleated myotubes, (b) degrade striated myofibrils into amino acids, and (c) inhibit the striking increase in total Ca2+ which occurs as normal myotubes mature. Yeoh and Brighton (55) reported that, while PMA inhibited fusion, the cultures did accumulate small amounts of muscle-specific M-CK. However, 'This work was supported by NIH Grants T32-HD07152, HL-18708, POI CÕIS! 94, and HL-15835 (to the Pennsylvania Muscle Institute), and the Muscular Dystrophy Association. 2 Present address: The M.S. Hershey Medical Center of the Pennsylvania State University, Hershey, Pa. 17033. 3 Insurance Medical Scientist Scholarship Fund trainee sponsored by the Na tionwide Life Insurance Co. 4To whom requests for reprints should be addressed. 5The abbreviations used are: PMA, phorbol 12-myristate 13-acetate; M-CK, muscle-specific creatine kinase; dThd, thymidine; light meromyosin, muscle-specific light meromyosin; desmin, muscle-specific intermediate filament protein; 4-a-PDD, 4-n-phorbol-12,13-didecanoate; EGTA, eythyleneglycol bis(aminoethylether)-W,A/'tetraacetic acid; BrdUrd, bromodeoxyuridine. Received August 26, 1982; accepted March 8, 1983. this study did not determine whether the M-CK was synthesized by the definitive postmitotic myoblasts present at the time of plating or by the progeny of cells that replicated in vitro in the presence of PMA. Using [3H]dThd autoradiography combined with immunoflu orescent localization of light meromyosin and desmin, we have analyzed the effects of PMA on 3 aspects of myogenesis: (a) fusion into myotubes; (o) synthesis of muscle-specific proteins; and (c) synthesis of DNA. We have found that myogenic cells cultured in PMA initiate the synthesis of both desmin and light meromyosin and, by these criteria, differentiate partially. How ever, these cells differ considerably from normal definitive post mitotic myoblasts in that (a) they do not fuse to form myotubes; (b) they fail to assemble striated myofibrils; and (c) they incor porate [3H]dThd, and some enter mitosis. The fate of these atypical PMA-treated myogenic cells is unknown. MATERIALS AND METHODS Cell Cultures. Myogenic cultures were established as described pre viously (5) but with minor modifications. Briefly, breast muscle from 12day-old chick embryos was dissociated in Ca2+ and Mg2+-free salt solution [CMF 0.137 M NaCI-2.7 HIM KCI:1.5 mw NaPO42:6 mw NaHCO3:5.5 HIMglucose (pH 7.4)] containing 0.25% trypsin (Grand Island Biological Co., Grand Island, N. Y.). Following centrifugaron and resuspension in standard culture medium, the cells were filtered through lens tissue, and a dilution was counted in a hemocytometer. Cells were plated at a density of 3 x 105 cells/1.5 ml/35-mm tissue culture dish which was previously coated with rat tail collagen. Cells were also plated in 100mm tissue culture dishes to be used for subculturing. Culture medium consisted of Eagle's minimum essential medium containing 10% horse serum, 3% chick embryo extract, 0.2 mw glutamine, penicillin (50 units/ ml), streptomycin (50 ng/m\), and Fungizone (2.5 ^g/ml) (all obtained from Grand Island Biological Co., except embryo extract). The medium in all cultures was replaced daily. Cells were subcultured using a 1:10 dilution of trypsin:EDTA (final concentrations: trypsin, 0.05% and EDTA, 0.02%; Grand Island Biological Co.) in CMF for 2 to 4 min at 37°. PMA was obtained from Dr. Peter Borchert (Eden Prairie, Minn.). Stock PMA was prepared by dissolving the agent in absolute ethanol and obtaining an absorption spectrum to calculate the concentration of phorbol compound in solution (we are grateful to Dr. T. G. O'Brien, Wistar Institute, Philadelphia, Pa., for providing the equipment and as sistance needed for this procedure). PMA stock was stored in the dark at -20°, and fresh PMA medium at a concentration of 50 nw (30 ng/ml) was prepared daily. Nonpromoting phorbol ester 4-a-PDD stock solution was a gift from Dr. T. G. O'Brien. EGTA was obtained from Sigma Chemical Co. (St. Louis, Mo.), and a 40 HIM stock was prepared as described (40) with minor modifications. BrdUrd (Sigma) stock was prepared by dissolving the analogue in dimethyl sulfoxide to give a 20mg/ml solution. This was diluted 1:20 with a balanced salt solution (0.137 M NaCI:2.7 mM KCI:1.0 mM CaCI2:1.0 mw MgCI2:1.5 rnw NaPO42:6 mw NaHC03:5.5 mM glucose, pH 7.4) to give a 1-mg/ml working stock which was passed through a 0.22-iim filter and stored frozen at -20°. Antibodies. Preparation and characterization of antibodies against light meromyosin and desmin have been described (3, 10, 16, 17). In 2780 CANCER RESEARCH VOL. 43 on April 15, 2017. © 1983 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from cultures derived from embryonic chick skeletal muscle, both of these proteins are present in definitive postmitotic mononucleated myoblasts and myotubes and are thus markers of cells in the terminal compartment of the myogenic lineage (23, 24, 29). Immunofluorescent Microscopy. Cells were processed for immunofluorescent microscopy as described elsewhere (49) with only minor modifications. The DMA-binding fluorochrome, bisbenzimide [Hoechst No. 33258 (49)], was used as a nuclear counterstain. Cells were exam ined using either Zeiss or Leitz epifluorescent microscopes with appro priate filters for either fluorescein:bisbenzimide or rhodamine fluores cence. Immunofluorescent Autoradiography. Autoradiography was com bined with the Immunofluorescent technique to determine if cells binding muscle-specific antibody incorporated [3H]dThd into their DNA. Cultures were established as described above but at a density of 5 x 105 cells/ 1.5 ml/dish. Cells were exposed to [3H]dThd (0.1 to 0.5 /iCi/ml) (specific activity, 50 Ci/mmol; New England Nuclear, Boston, Mass.) for varying periods of time as indicated for each experiment. Cultures which were not sacrificed immediately were maintained in medium containing 0.32 rriM unlabeled dThd to prevent reuse of isotope (6). After immunofluorescent processing, the cultures were coated with a 1:1 dilution of NTB2 emulsion (Kodak, Rochester, N. Y.) at 45°and air-dried for 30 to 90 min. Cultures were stored in light-tight boxes and exposed for approximately 1 day at 4°and for the remaining 1 to 6 days at either 4°or -70°. Autoradiographs were developed using D-19 developer (Kodak) for 2 min, rinsed with deionized water, fixed, washed for 5 to 15 min with deionized water, and mounted as described above. A cell with greater than 30 to 40 grains overlying its nucleus was scored as labeled positively; frequently, most of the nucleus was obscured by grains.