Expression of Acetylcholine Receptor a-Subunit mRNA during Differentiation of the BC3H1 Muscle Cell Line*

The accumulation of translatable acetylcholine re- ceptor a-subunit mRNA was examined in the BC3H1 muscle cell line in response to serum and cell growth. Relative amounts of a-subunit mRNA were quantitated during differentiation by cell-free translation and immunoprecipitation with an a-subunit-specific mono- clonal antibody. Logarithmically growing cells do not possess cell surface acetylcholine receptors; however, a significant amount of a-subunit mRNA is detectable in cells under these conditions. Furthermore, a-subunit is synthesized in growing undifferentiated cells at a rate similar to that of differentiated cultures. Follow- ing growth arrest of BC3H1 cells, surface receptors are induced to levels greater than 100-fold above that of growing cells. The relative level of translatable a- subunit mRNA in differentiated cells, however, is only approximately 4-fold greater than in growing cultures. Induction of a-subunit mRNA appears to be reversible since reinitiation of growth in quiescent differentiated BC3H1 cells results in a reduction in relative abun- dance of this mRNA species to levels comparable to that of undifferentiated cells and the concomitant loss of surface receptors. These results indicate that receptor expression during differentiation is regulated both post-translationally and at the leveI of receptor subunit mRNA accumulation.

tions of Ca2+ and reduced levels of growth factors (mitogens), mononucleated myoblasts have been shown to withdraw from the cell cycle and differentiate (2,3). Similar results have also been obtained by Olson et al. (4), who reported that creatine phosphokinase expression in the BC3H1 nonfusing muscle cell line is initiated following growth-arrest in the presence of media containing low levels of serum. Differentiation in mononucleated muscle cells, whether permanent cell lines or myoblasts, appears to be at least partially reversible since reexposure of uncommitted differentiated cells to high levels of serum results in the arrest of further induction of musclespecific proteins and re-entry of cells into the cell cycle (4-6).
These studies indicate that myogenesis is intimately coupled to the withdrawal of muscle cells from the cell cycle and that serum components influence differentiation, at least in part, as a consequence of their mitogenic effect. Little information is available concerning the role of serum components and cell growth in the regulation of expression of muscle-specific mRNA sequences.
Among the proteins whose level of expression is regulated during muscle cell differentiation is the ACh' receptor. This multisubunit integral membrane glycoprotein is a pentamer composed of 4 different subunits in a molar stoichiometry of aZ, 6, y, 6, and mediates synaptic transmission at the vertebrate neuromuscular junction (7, 8). The four different subunits have been shown to be synthesized on membrane-bound polysomes as preproteins (9). Several recent reports have described the molecular cloning of mRNA sequences for receptor subunits from Torpedo (10)(11)(12)(13)(14)(15) and mouse (16). The ability to assay ACh receptor subunit mRNAs by in vitro translation and through the use of purified cDNA probes will permit a detailed examination of the molecular mechanisms involved in the regulation of receptor expression during myogenic differentiation and synaptogenesis.
Studies with the BCBHl mouse clonal muscle-like cell line have demonstrated that expression of the acetylcholine receptor (and other muscle-specific proteins) is intimately coupled to the state of growth of the cells (17)(18)(19)(20). Logarithmically growing BC3H1 cells do not possess cell surface ACh receptors (17); however, following cessation of cell division, ACh receptors accumulate to very high levels. We reported previously that addition of high concentrations of serum to quiescent differentiated BC,H1 cells resulted in the rapid loss of surface ACh receptors. The disappearance of receptors was attributed primarily to inefficient assembly of receptor subunits in addition to an apparent defect in the transport of newly assembled receptors to the cell surface (19,20). Following addition of serum to quiescent cells, the rate of a-subunit synthesis was not significantly reduced and in many experiments was actually increased compared to quiescent cultures.
In the present study, the effects of serum and cell division on the accumulation of functional ACh receptor a-subunit mRNA were examined. The results demonstrate that logarithmically growing cells contain functional a-subunit mRNA which is translated in vivo at a rate similar to that of differentiated cells. Cessation of cell division, by exposure to low levels of serum, results in a 4-fold increase in the relative levels of a-subunit mRNA and greater than a 100-fold induction in the level of surface ACh receptors. Reinitiation of cell division in quiescent BC3H1 cells arrests the accumulation of translatable ACh receptor a-subunit mRNA and causes the loss of surface ACh receptors within 24 to 48 h. Under these conditions, the level of a-subunit mRNA is decreased to a level similar to that in undifferentiated cells. These data indicate that during muscle cell differentiation ACh receptor expression is regulated only partially at the level of transcription (or accumulation) of translatable a-subunit mRNA. Reentry of differentiated cells into the cell cycle not only inhibits the post-translational processing of ACh receptors (20) but also prevents the further accumulation of translatable receptor subunit mRNA.

MATERIALS AND METHODS
Cell Culture-The clonal mouse muscle-like cell line, BC3H1 (18), was grown as described previously (4). Fetal calf serum was obtained from K.C. Biologicals and media were obtained from the Washington University Basic Cancer Center. Cell numbers were determined using a Coulter counter.
Labeling of cellular proteins with [%]methionine and preparation of cell extracts was performed as described (20).
Assay for Acetylcholine Receptor-Previously published procedures were used to measure the binding of '251-a-bungarotoxin to cell cultures as described (20).
Preparation of Total Cellular RNA-Total cellular RNA was extracted from BC3H1 cells grown on 10-cm Falcon tissue culture dishes according to the 8 M guanidine HC1 procedure of Cox as modified by Deeley et al. (21,22). Details of the RNA isolation procedure have been described previously (4). 20-25 pg of total cellular RNA/106 cells could generally be obtained from quiescent cultures in 1% fetal calf serum. Cells growing in 20% serum gave about a 25% greater yield.
Cell-free Protein Synthesis-Total cellular RNA was translated in 30-pl translation reactions in a micrococcal nuclease-treated rabbit reticulocyte lysate translation system (Bethesda Research Laboratories) containing [35S]methionine a t 1.65 mCi/ml (>IO00 Ci/mmol, Amersham Corp.) as described previously (23,24). Follwing incubation for 60 min at 30 "C, reactions were terminated by addition of 200 pl of NaCI/Pi containing 1% (w/v) Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 5 mM EDTA,and 0.1-0.2 unit/ml of bovine lung aprotinin (Sigma). Total [=S]methionine incorporation was determined by applying a 5-pl sample of the total translation reaction to a 3-cm disk of Whatman filter paper followed by boiling for 10 min in 10% trichloroacetic acid and washing in 95% ethanol. Filters were counted in a scintillation spectrometer.
Immunoprecipitation-Immunoprecipitation of nascent a-subunit from cell-free translations and of a-subunit from cell extracts was performed using mAb61 (an a-subunit-specific monoclonal antibody) exactly as described (20, 24).
Electrophoresis-NaDodSO4-polyacryIamide gel electrophoresis was performed according to Laemmli (25) as described previously (23). Following electrophoresis, gels were processed for fluorography as described (23), and the films were analyzed by densitometry. Peak height was found to be proportional to peak area and, therefore, was employed as a measurement of band intensity.

RESULTS
Cell Growth and Acetylcholine Receptor Induction-BCsH1 cells grow logarithmically with a doubling time of approximately 18 h in medium containing 20% fetal calf serum (17). Transfer of subconfluent cultures to medium containing 1% fetal calf serum results in the arrest of cell division within approximately 24 h (Fig. 1A). Following growth arrest, ACh receptors appear on the cell surface and continue to increase to a maximum level after about 5 days (Fig. 1, B and C). As described previously (20), exposure of subconfluent differentiated cultures to 20% serum results in the rapid loss of surface ACh receptors followed by reinitiation of cell division. In the experiment presented in Fig. 1, the number of surface abungarotoxin-binding sites decreased approximately 85% within 24 h of serum stimulation (compare day 5 (1% serum) with day 6 (20% serum)). The culture dishes used in these experiments contain approximately 1 x lo7 cells/dish at confluent cell densities; therefore, at the time of growth arrest in 1% serum the cells are relatively sparse (9 X lo5 cells/dish). By day 8 in 20% serum, areas of confluent cells can be observed. These regions of high cell density may contribute to the slight reinduction of a-subunit mRNA observed on day 8 in serum-stimulated cultures (see below).
Cell-free Translations and Immunoprecipitations-In order to determine whether exposure of differentiated cells to 20% serum influenced the accumulation of mRNA coding for the

T I M E ( d a y s )
FIG. 1. Effect of serum on cell growth and acetylcholine receptor expression. BC3Hl cells were plated on 10-cm Falcon dishes a t 1.5 X lo5 cells/dish in Dulbecco's minimal essential medium (Grand Island Biologicals) containing 20% fetal calf serum, 0.1 mg/ml of glutamine, 100 units/ml of penicillin, 100 pg/ml of streptomycin. On day 1, designated by the arrow, cultures were transferred to medium containing 1% fetal calf serum. On day 5, half of the cultures were transferred to medium containing 20% fetal calf serum subunits of the ACh receptor, total cellular RNA was extracted from BC,,H1 cells and translated in a micrococcal nuclease-treated rabbit reticulocyte lysate system. The primary translation product of 0-subunit mRNA was then immunoprecipitated from total translation reactions with mAb61 (an n-subunit-specific monoclonal antibody prepared against NaDodS04-denatured purified Electrophorus electricus ACh receptor) (26)(27)(28)(29) and analyzed by NaDodS0,-polyacrylamide gel electrophoresis (Fig. 2). As described previously, the in uitro translation product of n-subunit mRNA demonstrated an apparent molecular weight of 41,000 daltons which is approximately 2,000 daltons larger than the nonglycosylated tu-subunit in uiuo, due to the presence of a signal sequence (24). The identity of the a-subunit has been confirmed previously by peptide mapping (24).
When the results from the cell-free translations are com-pared with the data in Fig. 1, a discrepancy is observed between the induction of translatable tu-subunit mRNA and surface ACh receptors. During differentiation, the level of surface receptors increases by over 100-fold. In contrast, the relative abundance of translatable n-subunit mRNA increases only about 3-to 4-fold (Fig. 2, compare lanes 2-6, 8, and 10 and Fig. 3). These findings indicate that ACh receptor induction is regulated only partially at the level of accumulation of translatable n-subunit mRNA and suggest the possibility of some form of post-transcriptional control in the regulation of receptor expression.
As reported previously (4, 24), other differences in cell-free translation products can be observed between RNA samples isolated from cultures at different stages of differentiation ( Fig. 2 A ) . For 30-pI translation reactions were immunoprecipitated with mAhG1 as described under "Materials and Methods." Immunoprecipitates were denatured in NaDodSOI sample buffer, and polypeptides were separated on 10% polyacrylamide gels followed by fluorography (exposure time = 3 days for total translation products and 6 days for immunoprecipitations). Each lane represents the results obtained from translation of 2 pg of RNA from each culture condition. A, total cell-free translation products. Lanes: I , day 1 (20% serum); 2, day 3 (1% serum); 3, day 4 (1% serum); 4, day 5 (1% serum); 5 , day 6 (1% serum); 6, day 6 (205 serum); 7, day 7 (1% serum); 8, day 7 (20% serum); 9, day 8 (1% serum); IO, day 8 (20% serum); 11, endogenous translation without added RNA. B, cy-subunit immunoprecipitated from translation reactions with mAbG1 (anti-c~) (or nonimmune serum where indicated). Lanes: I . day 1 (20% serum immunoprecipitated with nonimmune serum); 2, day 1 (20%); 3, day 3 (1% serum); 4, day 4 (1% serum); 5, day 5 (1% serum); 6, day 6 (1% serum); 7, day G (20% serum); 8, day 7 (1% serum); 9, day 7 (20% serum); IO, day 8 (1% serum); 11, day 8 (20% serum); 12, day 7 (1% serum immunoprecipitated with nonimmune serum). The relative decrease in concentration of wsubunit mRNA following addition of 2 0 4 serum can be observed by comparing lane 5 in B, which represents cu-subunit mRNA at the time of addition of 20% serum to differentiated cultures, with lanes 7, 9, and 11, which represent trsubunit mRNA following exposure to 20% serum for 24, 48, and 72 h, respectively. The relative amounts of tysubunit mRNA during each day in 1 and 20% serum can be directly compared with the data in Fig. 1  FIG. 3. Quantitation of a-subunit mRNA during differentiation. Relative amounts of a-subunit mRNA correspond to the culture conditions described in Fig. 1. For quantitation of a-subunit mRNA, the gel in Fig. 2B and gels from 2 additional sets of translations of the same RNA samples (gels not shown) were scanned with a Joyce-Loebl densitometer, and the relative peak height of a-subunit bands was quantitated. Values represent the average of 3 sets of translations and are expressed as relative units of a-subunit per lo6 cpm incorporated into trichloroacetic acid-precipitable radioactivity in translations containing 2 pg of total cellular RNA extracted from cells under the specified culture conditions. 0, cultures in 1% fetal calf serum; 0, 20% fetal calf serum. It was necessary to normalize cysubunit to [35C]methionine incorporation due to minor differences in the efficiency of translation of the different RNA samples. be more abundant in growing cells, while other mRNAs such as those directing the synthesis of polypeptides with apparent M , -35,000, 40,000, and 200,000 are more prevalent in differentiated cultures. The identities of these developmentally regulated mRNA species remain to be determined. Note that a large number of proteins are nonspecifically immunoprecipitated from the translation reactions with mAb61; however, the a-subunit is among the most abundant. The nonspecific background bands in the gel are also precipitated with nonimmune serum (Fig. 2B, lunes 1 and 12). This background is not unexpected considering that a-subunit mRNA constitutes less than 0.1% of total mRNA in maximally differentiated BC3H1 cells.
Assay by cell-free translation of RNA from cells which were transferred from 1 to 20% serum, followed by immunoprecipitation of the protein product of a-subunit mRNA, indicates that re-entry of quiescent cells into the cell cycle is associated with a decrease in a-subunit mRNA of 3-to 4-fold, resulting in a basal a-subunit mRNA level characteristic of undifferentiated cells. At t.he time of addition of 20% serum to differentiated cultures, a-subunit mRNA represented approximately 205 relative units of translatable mRNA based upon densitometry of immunoprecipitable a-subunit from translation reactions (Fig. 2B, lune 5, Fig. 3, and Table I). The fraction of total translatable mRNA that is represented by asubunit mRNA decreased to 133 and 106 relative units following exposure of cultures to 20% serum for 24 and 48 h, respectively (Fig. 2B, compare lane 5 with lanes 7 and 9, Fig.  3, and Table I). A slight increase in the relative abundance of a-subunit mRNA was observed after 3 days growth in 20% serum (Fig. 2B, lane 11). This may be due to reinduction of this mRNA since these cultures are beginning to reach high cell density. A minor species which comigrates with a-subunit was immunoprecipitated with nonimmune serum from a translation of mRNA extracted from cells on day 7 in 1% serum (Fig. 2B, lune 12). We are uncertain as to whether this is actually a-subunit or a nonspecific contaminant. The relative intensity of the peak corresponds to 10 relative units on the scale in Fig. 3. The intensity of this band has not been subtracted from the intensities of a-subunit bands immunoprecipitated with mAb61. Because the translation efficiencies of the different RNAs were not absolutely identical for all samples, a-subunit mRNA is expressed in Fig. 3 as relative units which are equivalent to the relative peak heights of the a-subunit bands in the gel in Fig. 2 and gels from 2 additional sets of translations (gels not shown) divided by the amount of radioactivity incorporated into total protein in each translation reaction. The differences in translation efficiency of the different RNA samples are due to variations between RNA samples rather than differences in the translation reactions. Similar values for the relative abundance of a-subunit mRNA are obtained if the a-subunit intensities are normalized to one of the nonreceptor polypeptides such as actin.
The apparent reduction in the concentration of a-subunit mRNA following reinitiation of cell division can probably be attributed to at least two factors. 1) The steady state level of a given mRNA species is governed by the relative rates of transcription and degradation. Therefore, if as appears to be the case, addition of 20% serum prevents further accumulation of a-subunit mRNA, then pre-existing a-subunit mRNA will decay with time and thus comprise a smaller fraction of total cellular RNA/culture. 2 ) Addition of 20% serum to quiescent cultures results in reinitiation of cell division which is accompanied by an increase in total RNA content/culture. The transition of cells from the resting to the growing state has been reported to result in a rapid and significant increase in mRNA content/cell (30). Thus, under these conditions the amount of a-subunit mRNA will appear to decrease as a fraction of total cellular RNA due to dilution by the newly transcribed RNA from cells as they re-entered the cell cycle. The actual reduction in abundance of a-subunit mRNA/ culture is, therefore, not as dramatic as indicated from cellfree translation assays of an equivalent amount of total RNA per translation from each culture condition. Currently, we are unable to examine the effects of growth-arrest and reinitiation of cell division on the p, y, or 6 ACh receptor subunit mRNAs due to the high degree of protease sensitivity of these subunits and the lack of sufficiently characterized antisera which recognize the primary translation products of the individual subunit mRNAs from mouse muscle.
Rates of a-Subunit Synthesis in Vioo-Because of the presence of a-subunit mRNA in undifferentiated cultures which lacked ACh receptors, we decided to determine whether or not this mRNA species was translated in these cells or whether some form of translational control might explain the apparent discrepancy between receptor induction and a-subunit mRNA accumulation. Relative rates of a-subunit synthesis were measured in vivo by a 5-min pulse with [35S] methionine followed by immunoprecipitation of labeled 01subunits from cell extracts with mAb61. The rate of total cellular protein synthesis, as measured by incorporation of ["S]methionine into trichloroacetic acid-insoluble material, was approximately 2-to %fold greater in undifferentiated growing cells compared to quiescent differentiated cultures (Table I, compare log phase (20% serum) and day 5 (1% serum)). The cell densities of the growing cultures and the differentiated cultures (day 5, 1% serum) were equivalent in this experiment (1.5 x lo6 cellsldish). The rates of a-subunit synthesis/dish under the two conditions can, therefore, be directly compared as rates of a-subunit synthesis/cell. Surprisingly, the rate of a-subunit synthesis/culture in growing cultures was approximately 60% that of differentiated cultures; however, because of the greater rate of total protein synthesis in growing cells, a-subunit comprised a smaller fraction of total proteins synthesized in growing compared to quiescent cultures ( Fig. 4 and Table I) (compare the rate of a-subunit synthesis/dish with the relative rate of a-subunit synthesis). The relative rate of a-subunit synthesis represents the rate of cy-subunit synthesisldish divided by the total [3sS] with rates of a-subunit synthesis in vivo Culture conditions correspond to those in Fig. 1. Rates of a-subunit synthesis were determined by densitometry of the fluorograms of the gels in Fig. 4 and the relative peak height of the n-subunit hands were quantitated. ["S]Methionine incorporation represents trichloroacetic acid-precipitable radioactivity incorporated into cells during a 5-min laheling period. Relative abundance of e-subunit mRNA corresponds to the data in Fig. 3. Relative rates of a-subunit synthesis are equivalent to <?-subunit synthesis/dish divided by ["S]methionine incorporation/dish. The cell densities were equivalent for log phase (20%) and day 5 (1%) cultures (1.5 X IO6 cells/dish); therefore, the rates of cr-subunit synthesis under the two conditions can he directly compared as rates of a-subunit synthesis/cell. Rates of [RSS]methionine incorporation/dish were approximately 2-to 3-fold greater in cultures exposed to 20% serum. Therefore, cr-subunit synthesis/dish was greater compared to cultures in 1% serum, despite the lower relative abundance of cr-subunit mRNA.  Fig. 1. 10cm culture dishes were pulse-labeled in 1 5 ml of medium containing 20 P M methionine, 1% (day 5) or 20% (day 5 and log phase) dialyzed fetal calf serum, and 150 pCi/ml of [:"'S]methionine for 5 min. At the end of the laheling period, cells were washed with NaCl/P,, scraped, and collected hy centrifugation at 2000 rpm for 10 min. Pellets were then extracted and immunoprecipitated with mAb6l (anti-tu subunit) as descrihed under "Materials and Methods." Immunoprecipitates were solubilized and radioactive polypeptides were analyzed by 10rE NaDodS0,-polyacrvlamide gel electrophoresis. Gels were treated for fluorographv as descrihed and exposed for 6 days. Lanes: I, log phase cultures in W E serum, 1 5 x 10" cells/dish; 2, day 5 (1% serum, 1.5 X IO6 cells/dish): 3 . day 6 (Ir; serum).
Nunit mRNA during Differentiation methionine incorporation/dish. As reported previously (20), exposure of differentiated cultures to 20% serum for 24 h resulted in a similar increase in the rate of total protein synthesis (Table I, compare day 5 (1% serum) and day 6 (20% serum)). The rate of a-subunit synthesis/dish increased by 50% following reinitiation of growth in differentiated cells despite the disappearance of ACh receptors during this same period. These findings indicate that a-subunit mRNA is translated in undifferentiated BCBHl cells and suggests the possibility of post-translational control in the regulation of ACh receptor expression.

DISCUSSION
Previously we reported that the surface expression of ACh receptors in the BCsHl muscle cell line was inhibited following addition of high concentrations of serum to quiescent differentiated cells (20). In the present study, the role of serum and cell growth in the regulation of expression of functional ACh receptor a-subunit mRNA was examined. Particular attention was focused upon the control of accumulation of this mRNA species during differentiation of quiescent cells and in cultures which, once differentiated, were stimulated to re-enter the cell cycle.
The results indicate that ACh receptor expression is regulated during differentiation of B G H 1 cells only partially at the level of accumulation of translatable a-subunit mRNA. Growing cells, which lack receptors, contain translatable asubunit mRNA, the relative abundance of which increases approximately 3to 4-fold following cessation of cell division. This change in the level of mRNA is insufficient to account for the greater than 100-fold increase in the level of surface ACh receptors in differentiated compared to growing cells and suggests the possibility of some form of post-translational regulation of receptor expression. Using the C, fusing muscle cell line originally described by Yaffe and Saxe1 (31), a similar disparity beteen the induction of a-subunit mRNA and ACh receptors has also been observed., Previously, Sebbane et al. (24) reported a 10-to 12-fold induction of translatable a-subunit mRNA during differentiation of BC,Hl cells at confluent densities. The lower level of induction reported here may reflect the fact that cells used for these studies were differentiated at low cell densities in low concentrations of serum. If, for example, extensive cellcell contacts are required for maximum a-subunit mRNA induction, then the cells used in these studies may not be fully differentiated. In both cases, however, there is a clear discrepancy between the level of induction of translatable asubunit mRNA and surface receptors.
Induction of a-subunit mRNA appears to be reversible since conditions which reinitiate cell division in differentiated BCsHl cultures result in the arrest of further accumulation of this mRNA species and the deinduction of a-subunit mRNA to levels characteristic of growing cells. I t is unclear at the present time as to whether the arrest of accumulation of tu-subunit mRNA is obligatorily coupled to the reinitiation of cell division or whether these cellular responses can be dissociated by various culture conditions.
The rate of tu-subunit synthesis/culture was previously shown to remain approximately constant and in many cases to increase, following exposure of differentiated cultures to high concentrations of serum (20). The degree to which asubunit synthesis increased depended upon the extent of stimulation of total protein synthesis in the presence of serum. Considered in light of the present study, the increased rate of

Regulation of A C h Receptor cr-Subunit mRNA during Differentiation
a-subunit synthesis/culture is not due to an increase in asubunit mRNA content/culture but is due to the enhanced rate of total protein synthesis/culture. Thus, despite the reduction in translatable a-subunit mRNA in cells following serum stimulation, the increased rate of total protein synthesis under these conditions results in a net increase in the rate of a-subunit synthesis/culture. Similar increases in total protein synthesis have been reported previously following addition of serum to quiescent fibroblasts (32,33). The increased rate was attributed to an enhanced rate of initiation of translation. The finding that a-subunit is synthesized in cells which have never been differentiated indicates that this protein is constitutively expressed by muscle cells, independent of the degree of differentiation, and is apparently not limiting for assembly of a multisubunit receptor complex. Receptor expression during differentiation, therefore, may be regulated post-translationally, possibly at the level of subunit assembly and/or transport to the surface as we previously reported for differentiated cells which were stimulated to reinitiate growth (20). We are currently investigating these possibilities in an effort to determine whether the lack of receptor expression in growing cells and in differentiated cells which have been stimulated with serum is regulated by common mechanisms.
It is also possible that the mRNA encoding the p, y, and/or 6 subunit is precisely regulated during differentiation of BC3H1 cells and that the accumulation of this mRNA governs the expression of the ACh receptor. Previously, we reported that the inhibitory effects of serum on receptor expression appear to be initiated within a few hours following serum stimulation (20). A rate-limiting subunit mRNA, therefore, would be predicted to exhibit a very short half-life under these conditions in order for the subunit which it encodes to be limiting for receptor assembly within this time period. Examination of the short term effects of actinomycin D on receptor expression in differentiated cells could provide indirect evidence to support this model. Direct examination of the relative levels of the p, 7 , and 6 subunit mRNAs, however, will require the availability of subunit-specific antibodies or cDNAs.
The molecular mechanisms involved in the regulation of muscle-specific mRNA accumulation during myogenic differentiation have been the subject of considerable controversy. A role for translational control of protein synthesis in muscle cell differentiation was suggested in early studies by Dym et al. (34) and others (35,36) who demonstrated the presence of myosin heavy chain mRNA in translationally inactive mRNP particles prior to myoblast fusion and the subsequent transfer of this mRNA species to polysomes at the time of fusion. Similarly, Yaffe and Dym (37,38) reported the presence of muscle-specific mRNAs prior to fusion as evidenced by the ability of cultured rat muscle cells to synthesize these proteins following treatment with actinomycin D at the time of fusion.
Transcriptional regulation of muscle gene expression has been suggested in more recent studies by Hastings and Emerson (39) and others (40, 41) who have shown through the use of purified cDNA probes that the accumulation of mRNAs coding for the proteins of the thick and thin filaments of the contractile apparatus is coordinated with myoblast fusion.
The coordination between the growth state of muscle cells and the expression of differentiation-specific mRNAs has been previously reported for other muscle-specific RNA sequences. Nguyen et al. found that the transcription of myosin heavy chain mRNA was obligatorily coupled to the withdrawal of L6E9 myoblasts from the cell division cycle. Furthermore, under nonfusing conditions the accumulation of this mRNA species was reversible during the early stages of differentiation by reinitiation of cell division (42). Accumulation of translatable creatine phosphokinase mRNA in BC3H1 cells has also been shown to be dependent on the withdrawal of cells from the cell cycle. Conversely, re-entry of quiescent differentiated cells into the cell division cycle was found to result in the arrest of accumulation of creatine phosphokinase mRNA in a similar manner to that described for a-subunit mRNA in the present study (4). The low level of induction (3-to 4-fold) of a-subunit mRNA during differentiation of BC3H1 cells, in addition to the presence and functional translation of this mRNA species in undifferentiated cells, however, clearly differs from the characteristics of induction of other musclespecific proteins.
Whether or not the accumulation of a-subunit mRNA and creatine phosphokinase mRNA is regulated during differentiation of the BC3H1 cell line at the level of transcription or post-transcriptionally, by stabilization or processing of RNA transcripts, for example, will be the subject of future studies. It also remains to be determined whether or not receptor regulation in the developing muscle fiber in vivo takes place by mechanisms similar to those described in the present study.