Regulation of Human Chorionic Gonadotropin (hCG) Secretion by Serum and Dibutyryl Cyclic AMP in Malignant Trophoblast Cells in Vitro ROBERT O. HUSSA, MICHAEL T. STORY, AND ROLAND A. PATTILLO Department of Gynecology and Obstetrics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226 was prevented by Actinomycin D. When cells were preincubated for 2 days in the absence of serum but with dbcAMP plus theophylline, subsequent addition of serum resulted in supra-additive stimulation of hCG secretion within a single day; the continued presence of dbcAMP plus theophylline was not necessary for this stimulation by serum. These findings suggested that dbcAMP activates a slow process requiring RNA synthesis, resulting in increased hCG secretion. Serum appears to protect the hCG synthesizing system from degradation. (J Clin Endocrinol Metab 40: 401, 1975)
ABSTRACT. Human chorionic gonadotropin (hCG) secretion was studied in human malignant trophoblast cells (Jar line) in continuous culture. Radioimmunoassayable hCG in the cells and in the daily culture fluid was increased for at least 3 days by 10% newborn calf serum and by 1 mM N^O2'dibutyryl cyclic adenosine 3',5'-monophosphoric acid (dbcAMP) plus 1 mM theophylline. Incubation of the cells in the presence of serum, dbcAMP and theophylline resulted in additive stimulation of hCG secretion after 1 day. After 2 or 3 days, a supra-additive stimulation was observed, which
S
EVERAL agents have recently been reported to stimulate hCG1 secretion. Handwerger et al. (1) observed increased levels of hCG in the culture fluid after incubating human term placenta in vitro for 1 day in the presence of either 1 mM dbcAMP (which mimics the actions of cyclic AMP) or 1 mM theophylline (which increases cyclic AMP levels by inhibition of cyclic nucleotide phosphodiesterase). Synergism was observed when both agents were added together. In the same study, neither agent affected hPL 1 secretion. Stimulation of hCG secretion for at least 3 days by dbcAMP and by theophylline was subsequently confirmed in cultures of malignant trophoblast cells in this laboratory (2). Stimulation of hCG secretion by Act D has also been reported (3). When 10~8M Act D was added to cultures of BeWo trophoblast cells (incubated in the absence of serum), the cells contained and secreted
more hCG than did control cultures. The increase was sustained for at least 3 days in spite of severe cell injury and greater than 90% inhibition of RNA and DNA synthesis. The stimulation was relatively specific for Act D, since other agents toxic to the trophoblast inhibited hCG secretion. Increased hCG levels, as measured by bioassay and radioimmunoassay, were also observed in the serum of a patient with choriocarcinoma following administration of Act D (3). The results suggested that Act D acts at the post-transcriptional level to stimulate hCG production. The objective of the present investigation was to provide further insight into the regulation of hCG secretion in human trophoblast cells. The study describes the stimulation of hCG secretion by serum, and extends previous reports (1-3) on the effects of dbcAMP and Act D in the trophoblast.
Received April 18, 1974. 1 Abbreviations: hCG, human chorionic gonadotropin; hPL, human placental lactogen; dbcAMP, N6,02'-dibutyryl cyclic adenosine-3',5'-monophosphoric acid; TCA, trichloroacetic acid; Act D, Actinomycin D.
Materials and Methods The Jar trophoblast cell line (4), maintained in continuous culture as previously described (5), was employed in this series of experiments. To assure comparable cell populations in all
401
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JCE & M • 1975 Vol 40 • No 3
HUSSA, STORY AND PATTILLO
flasks of each experiment, cells from 8-10 confluent flasks were pooled at the time of subculture and dispensed in equal quantities into 24-30 flasks. Following subculture, all cells were maintained for 1 day on medium containing 10% newborn calf serum. The medium, minus serum in some cases, was replenished daily. Four days following subculture, the medium was supplemented with the following agents, alone or in combination: 10% newborn calf serum, 1 mM dbcAMP (Sigma), 1 mM theophylline (Sigma) and 10"8M Act D (Cosmegen, Merck Sharp & Dohme). Incubations were continued for 3 days. The daily culture fluids and in 1 experiment, the cells, were frozen for later analysis of hCG by double antibody radioimmunoassay (2,3). Results were expressed as IU Second International Reference Preparation for hCG. The reference standard was kindly supplied by Dr. Derek Bangham, World Health Organization. Highly purified hCG for iodination (CR-115) was kindly provided by the National Institute of Child Health and Human Development and the National Institute of Arthritis Metabolic and Digestive Diseases, National Institutes of Health. At the termination of the
FIG. 1. Stimulation of hCG secretion by serum, and by dbcAMP plus theophylline. Jar cells were maintained 86OJ for 3 days on serumfree medium. Cultures then received medium supple40. mented with 10% newborn calf serum (stippled bars), 1 mM dbcAMP plus 1 mM theophylline (cross hatched bars), or the com2 bination of all 3 DAYS agents (solid bars). Control cultures (open bars) received serum-free medium. The culture fluid was exchanged daily. Vertical lines represent the SEM of triplicate cultures. Arrows indicate the arithmetic sum of the hCG levels in cultures incubated with serum and in the cultures incubated with dbcAMP plus theophylline. Values differing significantly from serum-free controls are indicated (*P < 0.05, \P < 0.001, t test).
incubation, the cells were assayed for incorporation of 2-14C-thymidine (59 mCi/mmol, New England Nuclear) and 4,5-3H-leucine (41 Ci/ mmol, New England Nuclear) into TCA-precipitable material, and for protein content (6).
Results
The stimulation of hCG secretion by serum, by dbcAMP plus theophylline, and by all 3 agents in combination is shown in Fig. 1. Addition of 10% newborn calf serum to the medium enhanced hCG secretion by 1.8, 6, and 11 times after 1, 2, and 3 days of incubation, respectively, compared to control cultures that received no serum. Increasing the serum content to 20 or 30% gave no further increase in hormone production. The stimulatory effect of 1 mM dbcAMP plus 1 mM theophylline was similar to the stimulatory effect of serum. When serum, dbcAMP, and theophylline were all present during incubation, the stimulation of hCG secretion was additive after 1 day, but increasingly supra-additive after 2 or 3 days of treatment; after 3 days the hCG levels were 230 times greater than in control cultures receiving none of the 3 agents. In a comparable experiment, the levels of hCG in the cell sheet were measured after 2 and 3 days of stimulation (Table 1). Addition of serum, dbcAMP plus theophylline, or all 3 agents to the culture fluid for 2 days resulted in increases of 2, 3.6, and 23 times, respectively, in the amount of hCG in the cell sheet; these increases were significant (P < 0.01). After 3 days of incubation with serum, the level of hCG in the cell sheet was 5.5 times greater than in cells incubated with serum-free medium (P < 0.01), while the level in cells incubated with serum, dbcAMP, and theophylline was 48 times greater (P < 0.01). By contrast, the amount of hormone in cells incubated with dbcAMP plus theophylline in serum-free medium was no greater than in untreated cells, even though the culture fluid contained 10 times more hormone than did the medium of serum-free con-
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403
CONTROL OF hCG SECRETION
trols. This probably reflected, at least in part, the toxicity of dbcAMP and theophylline in the absence of serum; in the experiment of Table 1, the cell sheet of serumfree controls contained more than 10 times more protein than did cells incubated with dbcAMP plus theophylline for 3 days (P < 0.001). The degree of this cellular injury, however, varied considerably between experiments (Table 2). Although serum, dbcAMP, and theophylline all stimulated hCG secretion, these agents had opposing effects on cellular protein content and on incorporation of 2-14Cthymidine or 4,5-3H-leucine into TCA-precipitable material (Table 2). Addition of serum stimulated the incorporation of 4,5-3H-leucine (P < 0.05) and of 2-14C-thymidine into TCA-precipitable material, and increased the protein content of the cells. On the other hand, addition of dbcAMP to the serum-free medium tended to decrease all 3 parameters after 3 days of incubation; these effects were more pronounced when theophylline was also present. When compared to the effects of serum, dbcAMP and dbcAMP plus theophylline significantly decreased all 3 growth parameters. When added along with dbcAMP or dbcAMP plus theophylline, serum prevented the tendency of dbcAMP to decrease the 3 parameters, and largely prevented the inhibitory effects of dbcAMP plus theophylline.
TABLE 1. Levels of hCG (IU/culture) in Jar trophoblast cells after 2 or 3 days of incubation with 10% newborn calf serum, 1 mM dbcAMP, and 1 mM theophylline Days
Additions to c
medium
2
3
None dbcAMP, theophylline Serum Serum, dbcAMP, theophylline
0.457 ±0.063tff
0.330 ± 0.113ft
0.915 ± 0.005** ft 1.65 ±0.11***
0.160 ±o.oiott 1.81 ± 0.25**
10.6
16.0
± 1.0*** ft
± 3.3** t
Culture fluids were replenished daily. The mean ± SEM of 3 cultures is given. ****** Differs significantly (**P < 0.01, ***P < 0.001, t test) from cultures receiving no additions to serum-free medium.
t . t t . t t t Differs significantly (fP < 0.05, f fF < 0.01, 11 fP < 0.001) from cultures incubated with medium containing 10% newborn calf serum.
The data of Fig. 1 suggested a lag of approximately 1 day before the stimulation of hCG secretion by any agent became pronounced. Such observations, however, did not explain whether the lag resulted from the action of dbcAMP, of serum, or of both agents. To help resolve this question, cells were preincubated for 2 days in serum-free medium containing 1 mM dbcAMP and 1 mM theophylline. Serum was then added, either in the presence or absence of dbcAMP and theophylline (Fig. 2). When added along with dbcAMP and theophylline at the start of the experiment, serum stimulated the cells to secrete 7.4 ± 0.84 IU of hCG in 1 day.
TABLE 2. Effect of 10% newborn calf serum, 1 mM dbcAMP, and 1 mM theophylline on protein content and incorporation of 2-14C-thymidine and 4,5-3H-leucine into TCA-precipitable material in Jar cells Incorporation Additions to serum-free medium None Serum dbcAMP dbcAMP, theophylline Serum, dbcAMP Serum, dbcAMP, theophylline
Protein (mg/culture) 3.58 4.68 3.00 1.89 5.02 3.23
± ± ± ± ± ±
0.64 0.35 0.08** 0.46** 0.36 0.40
thymidine (cpm/culture x 10~3) 4.03 5.72 1.87 1.38 6.46 4.95
± ± ± ± ± ±
0.97 0.68 0.09** 0.12** 0.74 0.55
leucine (cpm/culture x 10"4) 1.46 ± 2.56 ± 1.15 ± 0.63 ± 2.56 ± 2.00 ±
0.34* 0.07 0.02*** 0.08*** 1.14 0.10*
Cultures were incubated for 3 days with the agents. The mean ± SEM of 3 cultures is given. Other conditions as in Fig. 1. ****** Differs significantly (*P < 0.05, **P < 0.01, ***P < 0.001) from cultures incubated with medium containing 10% newborn calf serum.
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404
HUSSA, STORY AND PATTILLO
7O
6Q -dbc-l
"£ 5O. & 3 40J •
*
+ dbc+T
-
o
JCE & M • 1975 Vol 40 • No 3
than 1 day was observed before the stimulation of hCG secretion became pronounced (Fig. 3). To determine if the stimulation of hCG secretion by dbcAMP and theophylline was dependent on RNA synthesis, experiments were performed in which 10~8M Act D (which inhibits RNA synthesis by more than 90% in the trophoblast) was added to the culture fluid. Incubation of the trophoblast cells for 3 days in the presence of
2 3OH
o o
120, 20.
too
80
FIG. 2. Effects of dbcAMP plus theophylline, and of serum, on the rate of hCG secretion by Jar trophoblast cells. Serum was omitted from the culture fluid for 3 days prior to the start of the experiment. Cultures were then incubated in the presence (closed circles) or absence (open circles) of 1 mM dbcAMP (dbc) plus 1 mM theophylline (T), and in the presence (solid lines) or absence (dashed lines) of 10% newborn calf serum; culture fluids were exchanged daily. Vertical arrow indicates the addition of serum to cultures preincubated with serum-free medium containing dbcAMP plus theophylline. The mean ± SEM of triplicate cultures is given.
However, when added to cultures preincubated for 2 days with dbcAMP plus theophylline, serum stimulated the cells to secrete 47 to 56 IU of hCG in 1 day. In preincubated cells, the continued presence of dbcAMP and theophylline was. not required for manifestation of the serum effect (Fig. 2). This observation suggested that the stimulation of hCG secretion by dbcAMP required a lag of at least 1 day, but that the serum stimulation was expressed in less than a day. Furthermore, when cells, maintained continuously in the presence of serum, were incubated with dbcAMP and theophylline, a lag of more
5 60
O O 40
FIG. 3. Effect of Act D on the stimulation of hCG secretion by dbcAMP plus theophylline. Jar cells were subcultured and maintained for 3 days on x). medium containing 10% newborn calf serum (x On the following day (Day O), the culture fluid was supplemented with either 10~8M Act D (A A), 1 mM dbcAMP plus 1 mM theophylline (• •), or 1 mM dbcAMP plus 1 mM theophylline plus 10~8M Act D (A A). On Day 1, 10"8M Act D was included in the daily culture fluid of some cultures that had been maintained in the presence of 1 mM dbcAMP plus 1 mM theophylline (vertical arrow) (• •). Each point represents the mean of closely agreeing determinations on triplicate cultures. Other details were as described in Fig. 1.
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CONTROL OF hCG SECRETION 10~8M Act D resulted in little change in the daily rate of hCG secretion (Fig. 3). The stimulation of hCG secretion by dbcAMP plus theophylline, however, was essentially prevented by Act D when the drug was added at the start of the experiment. Furthermore, when added after 1 day of incubation with dbcAMP and theophylline, Act D was still effective in inhibiting the stimulation of hCG. Discussion The present observations demonstrate that dbcAMP and serum act differently to increase the level of radioimmunoassayable hCG in the culture fluid of trophoblast cells. Growth of the trophoblast cells was stimulated by serum but retarded by dbcAMP and theophylline (Table 2). Serum also stimulates cell proliferation (as measured by cell enumeration) in trophoblast cultures (7). These opposing effects of serum and dbcAMP on cell growth have been reported for many cell types (8-10). This observation in the trophoblast suggested that the stimulation of hCG secretion caused by dbcAMP is not simply related to cell growth. The increased secretion of hCG caused by serum or by dbcAMP plus theophylline probably involves synthesis of the hormone as well as release (Table 1, Fig. 1). Furthermore, the enhancement of hCG secretion by dbcAMP plus theophylline appears to be a slow process (Fig. 1) requiring RNA synthesis, since Act D largely prevented the supra-additive response to serum and dbcAMP plus theophylline (Fig. 3). Preliminary studies in this laboratory with a-amanitin, an inhibitor of mammalian nuclear Mn2+-ammonium sulfateactivated RNA polymerase of extranucleolar origin (11), support this interpretation. At
405
a concentration of 1 /ag/ml, a-amanitin prevented the stimulation of hCG secretion by dbcAMP plus theophylline, in the presence or absence of serum. Once the process of hCG secretion has been stimulated, it is relatively stable; the continuous presence of dbcAMP was not required for subsequent stimulation by serum (Fig. 2). The present investigation suggests that serum protects the hCG synthesizing system from degradation. However, our data do not exclude a possible stimulation by serum of transcription or translation. Acknowledgments The authors are grateful to Mrs. Martha Rinke and Mrs. Pilar Shalaby, and to Miss Anna Ruckert, Mr James Kurtz, and Mrs. Jimmie Martin for expert technical assistance. This work was supported in part by Grant No. RO1-CA14232-01A1, National Institutes of Health, U.S. Public Health Service.
References 1. Handwerger, S., J. Barrett, L. Tyrey, and D. Schomberg, J Clin Endocrinol Metab 36: 1268, 1973. 2. Hussa, R. O., M. T. Story, and R. A. Pattillo, J Clin Endocrinol Metab 38: 338, 1974. 3. , R. A. Pattillo, E. Delfs, and R. F. Mattingly, Obstet Gynecol 42: 651, 1973. 4. Pattillo, R. A., A. Ruckert, R. Hussa, R. Bernstein, and E. Delfs, In Vitro 6: 398, 1971 (Abstract). 5. and G. O. Gey, Cancer Res 28: 1231, . 1968. 6. Hussa, R. O., R. A. Pattillo, J. C. Garancis, A. C. F. Ruckert, and R. F. Mattingly, J Nat Cancer Inst 45: 1119, 1970. 7. Story, M. T., R. O. Hussa, and R. A. Pattillo, J Clin Endocrinol Metab 39: 877, 1974. 8. Abell, C. W., and T. M. Monahan, J Cell Biol 59: 549, 1973. 9. Rozengurt, E., and A. B. Pardee, J Cell Physiol 80: 273, 1972. 10. Bombik, B. M., and M. M. Burger, Exp Cell Res 80: 88, 1973. 11. Goldberg, I. H. and P. A. Friedman, Ann Rev Biochem 40: 775, 1971.
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ABSTRACT. Human chorionic gonadotropin (hCG) secretion was studied in human malignant trophoblast cells (Jar line) in continuous culture. Radioimmunoassayable hCG in the cells and in the daily culture fluid was increased for at least 3 days by 10% newborn calf serum and by 1 mM N^O2'dibutyryl cyclic adenosine 3',5'-monophosphoric acid (dbcAMP) plus 1 mM theophylline. Incubation of the cells in the presence of serum, dbcAMP and theophylline resulted in additive stimulation of hCG secretion after 1 day. After 2 or 3 days, a supra-additive stimulation was observed, which
S
EVERAL agents have recently been reported to stimulate hCG1 secretion. Handwerger et al. (1) observed increased levels of hCG in the culture fluid after incubating human term placenta in vitro for 1 day in the presence of either 1 mM dbcAMP (which mimics the actions of cyclic AMP) or 1 mM theophylline (which increases cyclic AMP levels by inhibition of cyclic nucleotide phosphodiesterase). Synergism was observed when both agents were added together. In the same study, neither agent affected hPL 1 secretion. Stimulation of hCG secretion for at least 3 days by dbcAMP and by theophylline was subsequently confirmed in cultures of malignant trophoblast cells in this laboratory (2). Stimulation of hCG secretion by Act D has also been reported (3). When 10~8M Act D was added to cultures of BeWo trophoblast cells (incubated in the absence of serum), the cells contained and secreted
more hCG than did control cultures. The increase was sustained for at least 3 days in spite of severe cell injury and greater than 90% inhibition of RNA and DNA synthesis. The stimulation was relatively specific for Act D, since other agents toxic to the trophoblast inhibited hCG secretion. Increased hCG levels, as measured by bioassay and radioimmunoassay, were also observed in the serum of a patient with choriocarcinoma following administration of Act D (3). The results suggested that Act D acts at the post-transcriptional level to stimulate hCG production. The objective of the present investigation was to provide further insight into the regulation of hCG secretion in human trophoblast cells. The study describes the stimulation of hCG secretion by serum, and extends previous reports (1-3) on the effects of dbcAMP and Act D in the trophoblast.
Received April 18, 1974. 1 Abbreviations: hCG, human chorionic gonadotropin; hPL, human placental lactogen; dbcAMP, N6,02'-dibutyryl cyclic adenosine-3',5'-monophosphoric acid; TCA, trichloroacetic acid; Act D, Actinomycin D.
Materials and Methods The Jar trophoblast cell line (4), maintained in continuous culture as previously described (5), was employed in this series of experiments. To assure comparable cell populations in all
401
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402
JCE & M • 1975 Vol 40 • No 3
HUSSA, STORY AND PATTILLO
flasks of each experiment, cells from 8-10 confluent flasks were pooled at the time of subculture and dispensed in equal quantities into 24-30 flasks. Following subculture, all cells were maintained for 1 day on medium containing 10% newborn calf serum. The medium, minus serum in some cases, was replenished daily. Four days following subculture, the medium was supplemented with the following agents, alone or in combination: 10% newborn calf serum, 1 mM dbcAMP (Sigma), 1 mM theophylline (Sigma) and 10"8M Act D (Cosmegen, Merck Sharp & Dohme). Incubations were continued for 3 days. The daily culture fluids and in 1 experiment, the cells, were frozen for later analysis of hCG by double antibody radioimmunoassay (2,3). Results were expressed as IU Second International Reference Preparation for hCG. The reference standard was kindly supplied by Dr. Derek Bangham, World Health Organization. Highly purified hCG for iodination (CR-115) was kindly provided by the National Institute of Child Health and Human Development and the National Institute of Arthritis Metabolic and Digestive Diseases, National Institutes of Health. At the termination of the
FIG. 1. Stimulation of hCG secretion by serum, and by dbcAMP plus theophylline. Jar cells were maintained 86OJ for 3 days on serumfree medium. Cultures then received medium supple40. mented with 10% newborn calf serum (stippled bars), 1 mM dbcAMP plus 1 mM theophylline (cross hatched bars), or the com2 bination of all 3 DAYS agents (solid bars). Control cultures (open bars) received serum-free medium. The culture fluid was exchanged daily. Vertical lines represent the SEM of triplicate cultures. Arrows indicate the arithmetic sum of the hCG levels in cultures incubated with serum and in the cultures incubated with dbcAMP plus theophylline. Values differing significantly from serum-free controls are indicated (*P < 0.05, \P < 0.001, t test).
incubation, the cells were assayed for incorporation of 2-14C-thymidine (59 mCi/mmol, New England Nuclear) and 4,5-3H-leucine (41 Ci/ mmol, New England Nuclear) into TCA-precipitable material, and for protein content (6).
Results
The stimulation of hCG secretion by serum, by dbcAMP plus theophylline, and by all 3 agents in combination is shown in Fig. 1. Addition of 10% newborn calf serum to the medium enhanced hCG secretion by 1.8, 6, and 11 times after 1, 2, and 3 days of incubation, respectively, compared to control cultures that received no serum. Increasing the serum content to 20 or 30% gave no further increase in hormone production. The stimulatory effect of 1 mM dbcAMP plus 1 mM theophylline was similar to the stimulatory effect of serum. When serum, dbcAMP, and theophylline were all present during incubation, the stimulation of hCG secretion was additive after 1 day, but increasingly supra-additive after 2 or 3 days of treatment; after 3 days the hCG levels were 230 times greater than in control cultures receiving none of the 3 agents. In a comparable experiment, the levels of hCG in the cell sheet were measured after 2 and 3 days of stimulation (Table 1). Addition of serum, dbcAMP plus theophylline, or all 3 agents to the culture fluid for 2 days resulted in increases of 2, 3.6, and 23 times, respectively, in the amount of hCG in the cell sheet; these increases were significant (P < 0.01). After 3 days of incubation with serum, the level of hCG in the cell sheet was 5.5 times greater than in cells incubated with serum-free medium (P < 0.01), while the level in cells incubated with serum, dbcAMP, and theophylline was 48 times greater (P < 0.01). By contrast, the amount of hormone in cells incubated with dbcAMP plus theophylline in serum-free medium was no greater than in untreated cells, even though the culture fluid contained 10 times more hormone than did the medium of serum-free con-
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403
CONTROL OF hCG SECRETION
trols. This probably reflected, at least in part, the toxicity of dbcAMP and theophylline in the absence of serum; in the experiment of Table 1, the cell sheet of serumfree controls contained more than 10 times more protein than did cells incubated with dbcAMP plus theophylline for 3 days (P < 0.001). The degree of this cellular injury, however, varied considerably between experiments (Table 2). Although serum, dbcAMP, and theophylline all stimulated hCG secretion, these agents had opposing effects on cellular protein content and on incorporation of 2-14Cthymidine or 4,5-3H-leucine into TCA-precipitable material (Table 2). Addition of serum stimulated the incorporation of 4,5-3H-leucine (P < 0.05) and of 2-14C-thymidine into TCA-precipitable material, and increased the protein content of the cells. On the other hand, addition of dbcAMP to the serum-free medium tended to decrease all 3 parameters after 3 days of incubation; these effects were more pronounced when theophylline was also present. When compared to the effects of serum, dbcAMP and dbcAMP plus theophylline significantly decreased all 3 growth parameters. When added along with dbcAMP or dbcAMP plus theophylline, serum prevented the tendency of dbcAMP to decrease the 3 parameters, and largely prevented the inhibitory effects of dbcAMP plus theophylline.
TABLE 1. Levels of hCG (IU/culture) in Jar trophoblast cells after 2 or 3 days of incubation with 10% newborn calf serum, 1 mM dbcAMP, and 1 mM theophylline Days
Additions to c
medium
2
3
None dbcAMP, theophylline Serum Serum, dbcAMP, theophylline
0.457 ±0.063tff
0.330 ± 0.113ft
0.915 ± 0.005** ft 1.65 ±0.11***
0.160 ±o.oiott 1.81 ± 0.25**
10.6
16.0
± 1.0*** ft
± 3.3** t
Culture fluids were replenished daily. The mean ± SEM of 3 cultures is given. ****** Differs significantly (**P < 0.01, ***P < 0.001, t test) from cultures receiving no additions to serum-free medium.
t . t t . t t t Differs significantly (fP < 0.05, f fF < 0.01, 11 fP < 0.001) from cultures incubated with medium containing 10% newborn calf serum.
The data of Fig. 1 suggested a lag of approximately 1 day before the stimulation of hCG secretion by any agent became pronounced. Such observations, however, did not explain whether the lag resulted from the action of dbcAMP, of serum, or of both agents. To help resolve this question, cells were preincubated for 2 days in serum-free medium containing 1 mM dbcAMP and 1 mM theophylline. Serum was then added, either in the presence or absence of dbcAMP and theophylline (Fig. 2). When added along with dbcAMP and theophylline at the start of the experiment, serum stimulated the cells to secrete 7.4 ± 0.84 IU of hCG in 1 day.
TABLE 2. Effect of 10% newborn calf serum, 1 mM dbcAMP, and 1 mM theophylline on protein content and incorporation of 2-14C-thymidine and 4,5-3H-leucine into TCA-precipitable material in Jar cells Incorporation Additions to serum-free medium None Serum dbcAMP dbcAMP, theophylline Serum, dbcAMP Serum, dbcAMP, theophylline
Protein (mg/culture) 3.58 4.68 3.00 1.89 5.02 3.23
± ± ± ± ± ±
0.64 0.35 0.08** 0.46** 0.36 0.40
thymidine (cpm/culture x 10~3) 4.03 5.72 1.87 1.38 6.46 4.95
± ± ± ± ± ±
0.97 0.68 0.09** 0.12** 0.74 0.55
leucine (cpm/culture x 10"4) 1.46 ± 2.56 ± 1.15 ± 0.63 ± 2.56 ± 2.00 ±
0.34* 0.07 0.02*** 0.08*** 1.14 0.10*
Cultures were incubated for 3 days with the agents. The mean ± SEM of 3 cultures is given. Other conditions as in Fig. 1. ****** Differs significantly (*P < 0.05, **P < 0.01, ***P < 0.001) from cultures incubated with medium containing 10% newborn calf serum.
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404
HUSSA, STORY AND PATTILLO
7O
6Q -dbc-l
"£ 5O. & 3 40J •
*
+ dbc+T
-
o
JCE & M • 1975 Vol 40 • No 3
than 1 day was observed before the stimulation of hCG secretion became pronounced (Fig. 3). To determine if the stimulation of hCG secretion by dbcAMP and theophylline was dependent on RNA synthesis, experiments were performed in which 10~8M Act D (which inhibits RNA synthesis by more than 90% in the trophoblast) was added to the culture fluid. Incubation of the trophoblast cells for 3 days in the presence of
2 3OH
o o
120, 20.
too
80
FIG. 2. Effects of dbcAMP plus theophylline, and of serum, on the rate of hCG secretion by Jar trophoblast cells. Serum was omitted from the culture fluid for 3 days prior to the start of the experiment. Cultures were then incubated in the presence (closed circles) or absence (open circles) of 1 mM dbcAMP (dbc) plus 1 mM theophylline (T), and in the presence (solid lines) or absence (dashed lines) of 10% newborn calf serum; culture fluids were exchanged daily. Vertical arrow indicates the addition of serum to cultures preincubated with serum-free medium containing dbcAMP plus theophylline. The mean ± SEM of triplicate cultures is given.
However, when added to cultures preincubated for 2 days with dbcAMP plus theophylline, serum stimulated the cells to secrete 47 to 56 IU of hCG in 1 day. In preincubated cells, the continued presence of dbcAMP and theophylline was. not required for manifestation of the serum effect (Fig. 2). This observation suggested that the stimulation of hCG secretion by dbcAMP required a lag of at least 1 day, but that the serum stimulation was expressed in less than a day. Furthermore, when cells, maintained continuously in the presence of serum, were incubated with dbcAMP and theophylline, a lag of more
5 60
O O 40
FIG. 3. Effect of Act D on the stimulation of hCG secretion by dbcAMP plus theophylline. Jar cells were subcultured and maintained for 3 days on x). medium containing 10% newborn calf serum (x On the following day (Day O), the culture fluid was supplemented with either 10~8M Act D (A A), 1 mM dbcAMP plus 1 mM theophylline (• •), or 1 mM dbcAMP plus 1 mM theophylline plus 10~8M Act D (A A). On Day 1, 10"8M Act D was included in the daily culture fluid of some cultures that had been maintained in the presence of 1 mM dbcAMP plus 1 mM theophylline (vertical arrow) (• •). Each point represents the mean of closely agreeing determinations on triplicate cultures. Other details were as described in Fig. 1.
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CONTROL OF hCG SECRETION 10~8M Act D resulted in little change in the daily rate of hCG secretion (Fig. 3). The stimulation of hCG secretion by dbcAMP plus theophylline, however, was essentially prevented by Act D when the drug was added at the start of the experiment. Furthermore, when added after 1 day of incubation with dbcAMP and theophylline, Act D was still effective in inhibiting the stimulation of hCG. Discussion The present observations demonstrate that dbcAMP and serum act differently to increase the level of radioimmunoassayable hCG in the culture fluid of trophoblast cells. Growth of the trophoblast cells was stimulated by serum but retarded by dbcAMP and theophylline (Table 2). Serum also stimulates cell proliferation (as measured by cell enumeration) in trophoblast cultures (7). These opposing effects of serum and dbcAMP on cell growth have been reported for many cell types (8-10). This observation in the trophoblast suggested that the stimulation of hCG secretion caused by dbcAMP is not simply related to cell growth. The increased secretion of hCG caused by serum or by dbcAMP plus theophylline probably involves synthesis of the hormone as well as release (Table 1, Fig. 1). Furthermore, the enhancement of hCG secretion by dbcAMP plus theophylline appears to be a slow process (Fig. 1) requiring RNA synthesis, since Act D largely prevented the supra-additive response to serum and dbcAMP plus theophylline (Fig. 3). Preliminary studies in this laboratory with a-amanitin, an inhibitor of mammalian nuclear Mn2+-ammonium sulfateactivated RNA polymerase of extranucleolar origin (11), support this interpretation. At
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a concentration of 1 /ag/ml, a-amanitin prevented the stimulation of hCG secretion by dbcAMP plus theophylline, in the presence or absence of serum. Once the process of hCG secretion has been stimulated, it is relatively stable; the continuous presence of dbcAMP was not required for subsequent stimulation by serum (Fig. 2). The present investigation suggests that serum protects the hCG synthesizing system from degradation. However, our data do not exclude a possible stimulation by serum of transcription or translation. Acknowledgments The authors are grateful to Mrs. Martha Rinke and Mrs. Pilar Shalaby, and to Miss Anna Ruckert, Mr James Kurtz, and Mrs. Jimmie Martin for expert technical assistance. This work was supported in part by Grant No. RO1-CA14232-01A1, National Institutes of Health, U.S. Public Health Service.
References 1. Handwerger, S., J. Barrett, L. Tyrey, and D. Schomberg, J Clin Endocrinol Metab 36: 1268, 1973. 2. Hussa, R. O., M. T. Story, and R. A. Pattillo, J Clin Endocrinol Metab 38: 338, 1974. 3. , R. A. Pattillo, E. Delfs, and R. F. Mattingly, Obstet Gynecol 42: 651, 1973. 4. Pattillo, R. A., A. Ruckert, R. Hussa, R. Bernstein, and E. Delfs, In Vitro 6: 398, 1971 (Abstract). 5. and G. O. Gey, Cancer Res 28: 1231, . 1968. 6. Hussa, R. O., R. A. Pattillo, J. C. Garancis, A. C. F. Ruckert, and R. F. Mattingly, J Nat Cancer Inst 45: 1119, 1970. 7. Story, M. T., R. O. Hussa, and R. A. Pattillo, J Clin Endocrinol Metab 39: 877, 1974. 8. Abell, C. W., and T. M. Monahan, J Cell Biol 59: 549, 1973. 9. Rozengurt, E., and A. B. Pardee, J Cell Physiol 80: 273, 1972. 10. Bombik, B. M., and M. M. Burger, Exp Cell Res 80: 88, 1973. 11. Goldberg, I. H. and P. A. Friedman, Ann Rev Biochem 40: 775, 1971.
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