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Cycloheximide (actidione) inhibition of protein synthesis and the uterine response to estrogen
ARCHIVES
OF
BIOCHEMISTRY
AND
Cycloheximide and
the Department
517-520
106,
(Actidione) the
J. GORSKI From
BIOPHYSICS
Uterine AND
of Physiology
(1964)
Inhibition
of Protein
Response
to Estrogen’
SISTER
M. CLAUDINE
and
Biophysics,
Received
October
University
Synthesis
AXMAF of Illinois,
Urbana,
Illinois
17, 1963
Cycloheximide (actidione) was shown to be a potent inhibitor of protein synthesis and the estrogen response in the immature rat uterus. Increases in uterine wet weight and cytidine-H3 incorporation into RNA associated with the four hour response to estrogen were blocked by actidione administration. Increases in orthophosphate-Pa and acetate-Cl4 incorporation into lipid and cytidine-H3 incorporation into RNA at 2 hours after estrogen were also blocked by actidione. Protein synthesis in the uterus was not significantly increased by estrogen at 2 hours and was blocked to 10% of controls by a single injection of 200 pg. of actidione into immature female rats.
Studies by Mueller, Gorski, and Aizawa have indicated that when puromycin blocks protein synthesis in the rat uterus, increased biosynthetic activity due to estrogen is blocked to control levels (1). More recently Noteboom and Gorski (2) have used puromycin in studies which suggest that estrogen regulates the synthesis of a limited number of proteins possibly by a mechanism similar to induction of enzyme synthesis in microorganisms. The above conclusions depend to a great extent on the assumption that puromycin’s only direct effect on the rat uterus is to inhibit protein synthesis. The question has been raised, therefore, as to whether puromycin might be inhibiting the response to estrogen by some other mechanism. Observations that liver glycogen is depleted (3) and O2 uptake into Earles L cells and ascites cells is decreased (4) by puromycin have stimulated interest in this question. Recently, Young et al. (5) reported that cycloheximide (actidione) and its acetoxy derivative inhibit protein synthesis without 1 Supported by a grant (A-6327) from the National Institute of Arthritis and Metabolic Diseases. 2 NSF Post-Doctoral Research Trainee; present address: Sacred Heart College, Witchita, Kansas. 517
affecting ribonucleic acid (RNA) and phospholipid synthesis. Previously, Bradley and co-workers (6, 7) had shown that actidione blocked induced enzyme synthesis and antibody formation. Arnow et al. in the studies mentioned above on puromycin effects on O2 uptake observed that actidione did not affect O2uptake (4). This study was undertaken to further test the hypothesis that the synthesis of a limited number of proteins is essential to the uterine response to estrogen. It will be shown that actidione blocks protein synthesis in the rat uterus and at the same time blocks the increase in biosynthetic activity in the uterus caused by estrogen administration. EXPERIMENTAL
PROCEDURES
Immature female rats, 22-25 days old and weighing 40-50 g., were obtained from Holtzman Rat Company, Madison, Wisconsin. Estradiol-17p (Cal. Biochem., 5 pg./rat) was administered intraperitoneally in 0.5 ml. of a 1% ethanol in 0.15 M NaCl solution. Actidione (Upjohn Co.) was dissolved in 0.5 ml. of 0.15 M NaCl and injected intraperitoneally. Protein synthesis was measured by the incorporation of glycine-2-C” (New England Nuclear) into protein as described previously (2). RNA synthesis was measured by incorporation of cyti-
518
GORSKI
AND
200 pg. of actidione were administered 30 minutes prior to estrogen administration. Table I shows the effects of actidione and 4-hour estrogen treatment on glycine incorporation into protein, uterine wet weight, and cytidine-H3 incorporation into RXA. Protein synthesis is obviously inhibited whereas no effect of actidione alone can be seen on cytidine incorporation into RNA or on wet weight of uterus. It is apparent that the estrogen response is blocked in both instances. A count of an aliquot of the crude acid soluble fraction showed that actidione caused approximately a 50 % increase in acid soluble carbon-14 counts. There was little effect of actidione or estrogen on acid soluble tritium. Table II summarizes the results of three experiments testing the effect of actidione on 2-hour estrogen-treated animals. This time period is of importance as Noteboom and Gorski reported that over-all protein synthesis is not affected by estrogen until after this time (2). Protein synthesis as measured in vivo by the incorporation of glycine-2-Cl4 into protein was inhibited to less than 10 70 of controls by actidione. Estrogen treated rats showed a slight increase in incorporation of glycine-2-Cl4 but this was not significant by the Student Ttest (p > 0.10). The estrogen response was significant at the 0.05 level or lower in all other experiments at this time period. In vivo incorporation of cytidine-H3 into RNA at 2 hours is increased by estrogen and this increase is inhibited by actidione (Table II). In this experiment as well as another at 2 hours, actidione itself stimulated cytidine
dine-H3 (Schwara Bioresearch) into the acidinsoluble, defatted residue described previously (2). RNA was hydrolyzed by incubating the above residue with 25 pg. ribonuclease (Worthington) in 0.05 M tris buffer for 1 hour at 37°C. The samples were chilled in an ice bath, made up to 0.5 N HCl, and the soluble oligonucleotides separated by centrifugation. The ultraviolet absorption of the supernate was determined in a recording Beckman DB spectrophotometer and the quantity of RNA calculated using values for yeast RNA (8). Aliquots were evaporated to dryness and counted in a scintillation counter. Data are expressed as counts per minute (cpm) per 10 pg. RNA. Phospholipids were determined as described previousIy (9) after in vitro incubation of uteri with 20 PC. orthophosphate-P” in 2.0 ml. of Eagles tissue culture medium Hela (Difco) at 37°C. for 1 hour under an atmosphere of 95% O2 and 57, COZ. Data are expressed as cpm per fitmole of lipid phosphate. Acetate-2-Cr4 incorporation into lipid was determined by the same procedure as above except 11~. acetate-2.Cl4 (New England Nuclear) was the isotope used. An aliquot of the total lipid fraction was counted in a scintillation counter and data are expressed as cpm per uterus. RESULTS In 100,
separate
experiments,
dosages
of
AXMAN
50,
200, and 500 pg. of actidione per 50 g. rat resulted in inhibition of amino acid incorporation into uterine protein to 36, 25, 10, and 4% of controls respectively at 4 hours after treatment. The treated animals showed a previously described syndrome of lethargy and diarrhea (5). No deaths occurred in the 4-hour experimental period even with a dose of 2 mg. per rat, but four of five rats died within 20 hours after a 200 pg. dose. In the experiments to be reported here, TABLE EFFECT
OF ACTIDIONE
ON
~-HOUR
I ESTROGEN
RESPONSE
Each group represents the mean of six animals. Animals were injected with 200 pg. actidione or saline 30 minutes before estradiol injections. Five pg. estradiol or control solutions were injected 4 hours prior to killing. Cytidine-H3 (1 PC.) and glycine-2-04 (1 PC.) were injected 1 hour prior to killing. Glycine-2-C” into kP~/W.)
Control Actidione Estradiol Estradiol 0 Values
are
+
96.5 11.0 162.5 10.5
actidione
average
values
f
standard
f f f f error.
protein
21.2’ 3.0 26.5 1.4
Uterine
wet weight
26.5 30.2 43.7 30.5
f f f f
2.3 2.5 2.8 2.3
(mg.)
Cytidine-Ha (cpm/lo
72 66 137 81
into RNA pg. RNA)
f f f f
9.1 18.6 17.6 11.4
ACTIDIONE
EFFECTS
ON
ESTROGEN
TABLE EFFECT
OF ACTIDIONE
519
ACTION
II
ON ~-HOUR
ESTROGEN
RESPONSE
Data reported here are from three separate experiments. Data on the incorporation of cytidine-H3 into RNA and P32 into lipid were from one group of rats. Actidione (200 pg.) or saline injected 30 minutes before estrogen. Estradiol (5 pg.) injected 2 hours before killing. Glycine-2-Cia (2 PC.) or cytidine-H3 (2 PC.) injected 1 hour prior to killing. Incorporation of isotope into lipid was carried out in vitro as described under experimental procedures. Uteri from rats injected with cytidine-H3 were divided with one horn being used for RNA determination and the other horn incubated with P32 for phospholipid assay. Glycine-?-Cl4 into protein (cpm/mg. protem)
Control Sctidione Estradiol Estradiol a Values
249 22 308 21
+ actidione are average
values
f
+ * f f
Cytidine-Hs (cpm/lO
23.8” 4.2 32.0 1.8
standard
125 156 290 168
into RNA KS. RNA)
f 3.6 rt 13.9 I!L 12.4 + 18.4
Pa* into (cpm/timole
4156 6370 9413 6903
lipid PO4)
rt + f zt
368 442 317 529
Acetate-2474 into (cpm/uterus)
2040 1760 3709 2330
lipid
f 119 & 125 rt 295 f 229
error.
incorporation into RNA. The combination of estrogen plus actidione results in incorporation rates very close to that of the actidione group in all cases. A similar situation occurs in orthophosphate-P32 incorporation into lipid. In this case uteri were removed from the animals and one horn from each animal incubated for 1 hour with orthophosphate-Pa3 and the incorporation into the total lipid fraction determined. Again actidione has a marked effect itself on l? incorporation into lipid, but. the effect of estrogen is blocked. When a similar experiment was carried out with in ZGO Pa2 administration, the actidione effect was eyual to the estrogen effect. The combination of estrogen and actidione was the same as actidione alone and suggests that estrogen is not working in the presence of actidione. The last experiment in Table II shows the effects of actidione and 2-hour estrogen treatment on in v&o acetate-2-Cl4 incorporation into lipid. Aizawa and Mueller (10) had shown that estrogen markedly increases this pathway, and these results confirm their observations. Actidione again blocks the increase due to estrogen to control levels. Actidione had a small inhibitory effect on acetate incorporation into lipid in this experiment. This inhibitory effect also showed up in acetate-2-Cl4 incorporation into RNA. No estrogen effect on this latter pathway was noted.
DISCUSSIOK
Actidione has been shown to be a potent in vivo inhibitor of protein synthesis in the rat uterus as has been demonstrated previously in other tissues (5). It shows a pattern of responses very similar to that of puromycin (1, 2). Its effects on protein synthesis, increased incorporation of P32 into lipid and RNA in the uterus, and its toxic syndrome of diarrhea and hypotension are also common to puromycin. Actidione has the advantages of effectively inhibiting protein synthesis over at least a 4-hour period (no longer time periods studied) with a single injection and of being relatively inexpensive. The differences in structures of actidione and puromycin as well as some known differences in effects on oxygen utilization (4) are in contrast to the similarity of their effects on the estrogen response in this study. As the principal effect of these compounds is an inhibition of protein synthesis, we feel these results confirm previous reports that the response of the rat uterus to estrogen is dependent on protein synthesis. It is still possible that both compounds are blocking the estrogen response by some mechanism other than protein synthesis. The probability of this being the case is definitely lessened by these observations that two chemically different protein synthesis inhibitors produce the same effect. The recent work of Ui and Mueller (11) demonstrating that actinomycin D blocks
520
GORSKI
AND
the estrogen response is also in accord with the hypothesis that protein synthesis is a necessary step in estrogen action. Their report would suggest that the synthesis of RNA is an essential prerequisite for the estrogen-dependent protein synthesis discussed here. The data presented here again point out that although the early estrogen responses are dependent on protein synthesis, this is not a random effect on over-all protein synthesis. As reported in previous work (2) and confirmed here, no significant effect on overall protein synthesis occurs within the first 2 hours after estrogen administration. At the same time, in this and previous studies, RNA and lipid synthesis are markedly stimulated by estrogen. The actual number of proteins that need to be synthesized is still not known but could be a rather limited number. A recent study in this laboratory suggests that much of the estrogen effect on RNA synthesis may be due to activation of RNA polymerase (12). This activation, in turn, is dependent on protein synthesis. Whether other early estrogen responses are regulated in a similar manner is not known. If they are, it is possible that the actidione or puromycin sensitive step in the response to estrogen is the synthesis of only a very small number of proteins.
AXMAN ACKNOWLEDGMENT The authors greatly Rex Mann and Paul Company in obtaining able technical assistance and Mrs. Nira Jeanne edged.
appreciate the help of Drs. O’Connell of the Upjohn samples of actidione. The of Mrs. Sylvin Rittschof Nelson is also acknowl-
REFERENCES 1. MUELLER, G. C., GORSKI, J., AND AIZAWA, Y., Proc. Natl. Acad. Sci. 47, 164 (1961). 2. NOTEBOOM, W., AND GORSKI, J., Proc. Natl. ilcad. Sci. 60, 250 (1963). 3. HOFFERT, J., GORSKI, J., MUELLER, G. C., AND BOUTWELL, R. K., Arch. Biochem. Biophys. 97, 134 (1962). 4. ARNOW, P., BRINDLE, S. A., GIUFFRE, N. A., AND PERLMAN, D., Antimicrobial Agents and Chemotherapy-I%Z, 731 (1963). 5. YOUNG, C. W., ROBINSON, P. F., AND SACKTOR, B., Biochem. Pharmacol. 12, 855 (1963). 6. BRADLEY, S. G., Nature 194, 315 (1962). 7. COONEY, W. J., AND BRADLEY, S. G., Antimicrobial Agents and Chemotherapy-f 961, 1 (1963). 8. DE DEKEN-GRENSON, M., AND DE DEKEN, R. G., Biochem. Biophys. Acta 31, 195 (1959). 9. GORSKI, J., AND NICOLETTE, J., Arch. Biochem. Biophys.. 103, 418 (1963) 10. AIZAWA, Y., AND MUELLER, G. C., J. Biol. Chem. 236, 381 (1961). 11. UI, H., AND MUELLER, G. C., Proc. N&Z. Acad. Sci. 60, 256 (1963). 12. GORSKI, J., J. Biol. Chem. 239, 889 (1964).
OF
BIOCHEMISTRY
AND
Cycloheximide and
the Department
517-520
106,
(Actidione) the
J. GORSKI From
BIOPHYSICS
Uterine AND
of Physiology
(1964)
Inhibition
of Protein
Response
to Estrogen’
SISTER
M. CLAUDINE
and
Biophysics,
Received
October
University
Synthesis
AXMAF of Illinois,
Urbana,
Illinois
17, 1963
Cycloheximide (actidione) was shown to be a potent inhibitor of protein synthesis and the estrogen response in the immature rat uterus. Increases in uterine wet weight and cytidine-H3 incorporation into RNA associated with the four hour response to estrogen were blocked by actidione administration. Increases in orthophosphate-Pa and acetate-Cl4 incorporation into lipid and cytidine-H3 incorporation into RNA at 2 hours after estrogen were also blocked by actidione. Protein synthesis in the uterus was not significantly increased by estrogen at 2 hours and was blocked to 10% of controls by a single injection of 200 pg. of actidione into immature female rats.
Studies by Mueller, Gorski, and Aizawa have indicated that when puromycin blocks protein synthesis in the rat uterus, increased biosynthetic activity due to estrogen is blocked to control levels (1). More recently Noteboom and Gorski (2) have used puromycin in studies which suggest that estrogen regulates the synthesis of a limited number of proteins possibly by a mechanism similar to induction of enzyme synthesis in microorganisms. The above conclusions depend to a great extent on the assumption that puromycin’s only direct effect on the rat uterus is to inhibit protein synthesis. The question has been raised, therefore, as to whether puromycin might be inhibiting the response to estrogen by some other mechanism. Observations that liver glycogen is depleted (3) and O2 uptake into Earles L cells and ascites cells is decreased (4) by puromycin have stimulated interest in this question. Recently, Young et al. (5) reported that cycloheximide (actidione) and its acetoxy derivative inhibit protein synthesis without 1 Supported by a grant (A-6327) from the National Institute of Arthritis and Metabolic Diseases. 2 NSF Post-Doctoral Research Trainee; present address: Sacred Heart College, Witchita, Kansas. 517
affecting ribonucleic acid (RNA) and phospholipid synthesis. Previously, Bradley and co-workers (6, 7) had shown that actidione blocked induced enzyme synthesis and antibody formation. Arnow et al. in the studies mentioned above on puromycin effects on O2 uptake observed that actidione did not affect O2uptake (4). This study was undertaken to further test the hypothesis that the synthesis of a limited number of proteins is essential to the uterine response to estrogen. It will be shown that actidione blocks protein synthesis in the rat uterus and at the same time blocks the increase in biosynthetic activity in the uterus caused by estrogen administration. EXPERIMENTAL
PROCEDURES
Immature female rats, 22-25 days old and weighing 40-50 g., were obtained from Holtzman Rat Company, Madison, Wisconsin. Estradiol-17p (Cal. Biochem., 5 pg./rat) was administered intraperitoneally in 0.5 ml. of a 1% ethanol in 0.15 M NaCl solution. Actidione (Upjohn Co.) was dissolved in 0.5 ml. of 0.15 M NaCl and injected intraperitoneally. Protein synthesis was measured by the incorporation of glycine-2-C” (New England Nuclear) into protein as described previously (2). RNA synthesis was measured by incorporation of cyti-
518
GORSKI
AND
200 pg. of actidione were administered 30 minutes prior to estrogen administration. Table I shows the effects of actidione and 4-hour estrogen treatment on glycine incorporation into protein, uterine wet weight, and cytidine-H3 incorporation into RXA. Protein synthesis is obviously inhibited whereas no effect of actidione alone can be seen on cytidine incorporation into RNA or on wet weight of uterus. It is apparent that the estrogen response is blocked in both instances. A count of an aliquot of the crude acid soluble fraction showed that actidione caused approximately a 50 % increase in acid soluble carbon-14 counts. There was little effect of actidione or estrogen on acid soluble tritium. Table II summarizes the results of three experiments testing the effect of actidione on 2-hour estrogen-treated animals. This time period is of importance as Noteboom and Gorski reported that over-all protein synthesis is not affected by estrogen until after this time (2). Protein synthesis as measured in vivo by the incorporation of glycine-2-Cl4 into protein was inhibited to less than 10 70 of controls by actidione. Estrogen treated rats showed a slight increase in incorporation of glycine-2-Cl4 but this was not significant by the Student Ttest (p > 0.10). The estrogen response was significant at the 0.05 level or lower in all other experiments at this time period. In vivo incorporation of cytidine-H3 into RNA at 2 hours is increased by estrogen and this increase is inhibited by actidione (Table II). In this experiment as well as another at 2 hours, actidione itself stimulated cytidine
dine-H3 (Schwara Bioresearch) into the acidinsoluble, defatted residue described previously (2). RNA was hydrolyzed by incubating the above residue with 25 pg. ribonuclease (Worthington) in 0.05 M tris buffer for 1 hour at 37°C. The samples were chilled in an ice bath, made up to 0.5 N HCl, and the soluble oligonucleotides separated by centrifugation. The ultraviolet absorption of the supernate was determined in a recording Beckman DB spectrophotometer and the quantity of RNA calculated using values for yeast RNA (8). Aliquots were evaporated to dryness and counted in a scintillation counter. Data are expressed as counts per minute (cpm) per 10 pg. RNA. Phospholipids were determined as described previousIy (9) after in vitro incubation of uteri with 20 PC. orthophosphate-P” in 2.0 ml. of Eagles tissue culture medium Hela (Difco) at 37°C. for 1 hour under an atmosphere of 95% O2 and 57, COZ. Data are expressed as cpm per fitmole of lipid phosphate. Acetate-2-Cr4 incorporation into lipid was determined by the same procedure as above except 11~. acetate-2.Cl4 (New England Nuclear) was the isotope used. An aliquot of the total lipid fraction was counted in a scintillation counter and data are expressed as cpm per uterus. RESULTS In 100,
separate
experiments,
dosages
of
AXMAN
50,
200, and 500 pg. of actidione per 50 g. rat resulted in inhibition of amino acid incorporation into uterine protein to 36, 25, 10, and 4% of controls respectively at 4 hours after treatment. The treated animals showed a previously described syndrome of lethargy and diarrhea (5). No deaths occurred in the 4-hour experimental period even with a dose of 2 mg. per rat, but four of five rats died within 20 hours after a 200 pg. dose. In the experiments to be reported here, TABLE EFFECT
OF ACTIDIONE
ON
~-HOUR
I ESTROGEN
RESPONSE
Each group represents the mean of six animals. Animals were injected with 200 pg. actidione or saline 30 minutes before estradiol injections. Five pg. estradiol or control solutions were injected 4 hours prior to killing. Cytidine-H3 (1 PC.) and glycine-2-04 (1 PC.) were injected 1 hour prior to killing. Glycine-2-C” into kP~/W.)
Control Actidione Estradiol Estradiol 0 Values
are
+
96.5 11.0 162.5 10.5
actidione
average
values
f
standard
f f f f error.
protein
21.2’ 3.0 26.5 1.4
Uterine
wet weight
26.5 30.2 43.7 30.5
f f f f
2.3 2.5 2.8 2.3
(mg.)
Cytidine-Ha (cpm/lo
72 66 137 81
into RNA pg. RNA)
f f f f
9.1 18.6 17.6 11.4
ACTIDIONE
EFFECTS
ON
ESTROGEN
TABLE EFFECT
OF ACTIDIONE
519
ACTION
II
ON ~-HOUR
ESTROGEN
RESPONSE
Data reported here are from three separate experiments. Data on the incorporation of cytidine-H3 into RNA and P32 into lipid were from one group of rats. Actidione (200 pg.) or saline injected 30 minutes before estrogen. Estradiol (5 pg.) injected 2 hours before killing. Glycine-2-Cia (2 PC.) or cytidine-H3 (2 PC.) injected 1 hour prior to killing. Incorporation of isotope into lipid was carried out in vitro as described under experimental procedures. Uteri from rats injected with cytidine-H3 were divided with one horn being used for RNA determination and the other horn incubated with P32 for phospholipid assay. Glycine-?-Cl4 into protein (cpm/mg. protem)
Control Sctidione Estradiol Estradiol a Values
249 22 308 21
+ actidione are average
values
f
+ * f f
Cytidine-Hs (cpm/lO
23.8” 4.2 32.0 1.8
standard
125 156 290 168
into RNA KS. RNA)
f 3.6 rt 13.9 I!L 12.4 + 18.4
Pa* into (cpm/timole
4156 6370 9413 6903
lipid PO4)
rt + f zt
368 442 317 529
Acetate-2474 into (cpm/uterus)
2040 1760 3709 2330
lipid
f 119 & 125 rt 295 f 229
error.
incorporation into RNA. The combination of estrogen plus actidione results in incorporation rates very close to that of the actidione group in all cases. A similar situation occurs in orthophosphate-P32 incorporation into lipid. In this case uteri were removed from the animals and one horn from each animal incubated for 1 hour with orthophosphate-Pa3 and the incorporation into the total lipid fraction determined. Again actidione has a marked effect itself on l? incorporation into lipid, but. the effect of estrogen is blocked. When a similar experiment was carried out with in ZGO Pa2 administration, the actidione effect was eyual to the estrogen effect. The combination of estrogen and actidione was the same as actidione alone and suggests that estrogen is not working in the presence of actidione. The last experiment in Table II shows the effects of actidione and 2-hour estrogen treatment on in v&o acetate-2-Cl4 incorporation into lipid. Aizawa and Mueller (10) had shown that estrogen markedly increases this pathway, and these results confirm their observations. Actidione again blocks the increase due to estrogen to control levels. Actidione had a small inhibitory effect on acetate incorporation into lipid in this experiment. This inhibitory effect also showed up in acetate-2-Cl4 incorporation into RNA. No estrogen effect on this latter pathway was noted.
DISCUSSIOK
Actidione has been shown to be a potent in vivo inhibitor of protein synthesis in the rat uterus as has been demonstrated previously in other tissues (5). It shows a pattern of responses very similar to that of puromycin (1, 2). Its effects on protein synthesis, increased incorporation of P32 into lipid and RNA in the uterus, and its toxic syndrome of diarrhea and hypotension are also common to puromycin. Actidione has the advantages of effectively inhibiting protein synthesis over at least a 4-hour period (no longer time periods studied) with a single injection and of being relatively inexpensive. The differences in structures of actidione and puromycin as well as some known differences in effects on oxygen utilization (4) are in contrast to the similarity of their effects on the estrogen response in this study. As the principal effect of these compounds is an inhibition of protein synthesis, we feel these results confirm previous reports that the response of the rat uterus to estrogen is dependent on protein synthesis. It is still possible that both compounds are blocking the estrogen response by some mechanism other than protein synthesis. The probability of this being the case is definitely lessened by these observations that two chemically different protein synthesis inhibitors produce the same effect. The recent work of Ui and Mueller (11) demonstrating that actinomycin D blocks
520
GORSKI
AND
the estrogen response is also in accord with the hypothesis that protein synthesis is a necessary step in estrogen action. Their report would suggest that the synthesis of RNA is an essential prerequisite for the estrogen-dependent protein synthesis discussed here. The data presented here again point out that although the early estrogen responses are dependent on protein synthesis, this is not a random effect on over-all protein synthesis. As reported in previous work (2) and confirmed here, no significant effect on overall protein synthesis occurs within the first 2 hours after estrogen administration. At the same time, in this and previous studies, RNA and lipid synthesis are markedly stimulated by estrogen. The actual number of proteins that need to be synthesized is still not known but could be a rather limited number. A recent study in this laboratory suggests that much of the estrogen effect on RNA synthesis may be due to activation of RNA polymerase (12). This activation, in turn, is dependent on protein synthesis. Whether other early estrogen responses are regulated in a similar manner is not known. If they are, it is possible that the actidione or puromycin sensitive step in the response to estrogen is the synthesis of only a very small number of proteins.
AXMAN ACKNOWLEDGMENT The authors greatly Rex Mann and Paul Company in obtaining able technical assistance and Mrs. Nira Jeanne edged.
appreciate the help of Drs. O’Connell of the Upjohn samples of actidione. The of Mrs. Sylvin Rittschof Nelson is also acknowl-
REFERENCES 1. MUELLER, G. C., GORSKI, J., AND AIZAWA, Y., Proc. Natl. Acad. Sci. 47, 164 (1961). 2. NOTEBOOM, W., AND GORSKI, J., Proc. Natl. ilcad. Sci. 60, 250 (1963). 3. HOFFERT, J., GORSKI, J., MUELLER, G. C., AND BOUTWELL, R. K., Arch. Biochem. Biophys. 97, 134 (1962). 4. ARNOW, P., BRINDLE, S. A., GIUFFRE, N. A., AND PERLMAN, D., Antimicrobial Agents and Chemotherapy-I%Z, 731 (1963). 5. YOUNG, C. W., ROBINSON, P. F., AND SACKTOR, B., Biochem. Pharmacol. 12, 855 (1963). 6. BRADLEY, S. G., Nature 194, 315 (1962). 7. COONEY, W. J., AND BRADLEY, S. G., Antimicrobial Agents and Chemotherapy-f 961, 1 (1963). 8. DE DEKEN-GRENSON, M., AND DE DEKEN, R. G., Biochem. Biophys. Acta 31, 195 (1959). 9. GORSKI, J., AND NICOLETTE, J., Arch. Biochem. Biophys.. 103, 418 (1963) 10. AIZAWA, Y., AND MUELLER, G. C., J. Biol. Chem. 236, 381 (1961). 11. UI, H., AND MUELLER, G. C., Proc. N&Z. Acad. Sci. 60, 256 (1963). 12. GORSKI, J., J. Biol. Chem. 239, 889 (1964).