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Regulation of human interferon production
VIROLOGY
70, 542-544
(1976)
SHORT Regulation II. Inhibition
The Rockefeller
of Human
of Interferon
PRAVINKUMAR
COMMUNICATIONS Interferon
Messenger RNA Synthesis ribofuranosylbenzimidazole
B. SEHGAL,
IGOR TAMM,
Production by 5,6-Dichloro-l-p-o-
AND
JAN
VILCEK’
University, New York, New York 10021; and ‘Department of Microbiology, Uniuerstiy School of Medicine, New York, New York 10016
New York
Accepted December 9, 1975 Polyinosinic:polycytidylic RNA in a strain of diploid n-ribofuranosylbenzi6idazole
acid (poly(I:C))-induced synthesis of interferon messenger human fibroblasts (FS-4) can be inhibited by 5,6-dichloro-l-P(DRB).
We have reported previously that 5,6dichloro-1-p-n-ribofuranosylbenzimidazole (DRB), a reversible inhibitor of nuclear heterogeneous RNA synthesis (l-5), can enhance (superinduce) polyinosinic:polycytidylic acid (poly(I:C)-induced interferon production in a strain of diploid human fibroblasts (FS-4) (6, 7). In the experiments reported earlier (6, 7), it appeared as if the synthesis of interferon messenger RNA (mRNA) might be, at least operationally, relatively resistant to inhibition by DRB. It was therefore necessary to determine whether the synthesis of interferon mRNA was indeed resistant to DRB inhibition or whether the marked superinducing effect of DRB masked inhibition of interferon mRNA synthesis. That the latter might be the case was suggested by our earlier observations that interferon yield in the presence of DRB and cycloheximide was lower than in the presence of cycloheximide alone (Fig. 1 of Ref. 6) and that the rate of interferon production in the presence of DRB was lower than in untreated control cultures for the first 3 hr after poly(I:C)induction (Fig. 3 of ref. 6). In the present report we describe a drug regimen which effectively eliminated interferon superinduction as an experimental variable and aliowed us to show clearly 542 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
that the synthesis of interferon mRNA is sensitive to inhibition by DRB. The cell cultures and media used, the procedure for interferon induction, the assay for interferon, and the measurement of the rate of cellular RNA synthesis have been described before (6-9). The beginning of poly(I:C)treatment (100 kg/ml in Eagle’s minimum essential medium for 1 hr at 37”) is designated as “0 hr” in all experiments. Poly(I:C) was obtained from the Antiviral Substances Program, Infectious Disease Branch, National Institute of Allergy and Infectious Diseases, Bethesda, Md. The drug regimen described in Fig. 1 has been developed on the basis of the following observations. Transcription of interferon mRNA is confined to the first 23 hr from the beginning of poly(I:C)induction (7). We have also observed that when cultures in the shutoff phase of interferon production (6, 7) are treated with DRB or actinomycin D, the rate of interferon production continues to decline for a further 3-4 hr and then reaches a plateau at a lower level (10). These observations indicate that the post-transcriptional control element responsible for the shutoff of interferon production (whose inhibition leads to superinduction), in addition to being operative during the first hour of
SHORT
poly(I:C)induction (6, 7), has a lifetime of 3-4 hr at 37” and inhibits interferon production in an irreversible manner (10). Hence, on the basis of the translational repressor model (II ) as a working hypothesis, it was decided to induce cells in the presence of cycloheximide (50 pg/ml, Polysciences, Inc., Warrington, Pa.) and to commence the inhibition of RNA synthesis using actinomycin D (5 pg/ml, Merck, Sharp and Dohme, Rahway, N.J.) 3.5 hr prior to removal of cycloheximide. It has been reported that cycloheximide does not inhibit the transcription of interferom mRNA (11). Further, the presence of cycloheximide from 0 to 6 hr would prevent the synthesis of the putative repressor protein even though repressor mRNA may have been synthesized. The 3.5-hr overlap between the beginning of actinomycin D treatment and the end of cycloheximide exposure would allow repressor mRNA synthesized during the first 2.5 hr to decay without undergoing translation. This drug regimen would maximize interferon superinduction and hence eliminate it as an experimental variable. Interferon yields obtained between 6 and 24 hr would therefore be proportional to the amount of interferon mRNA synthesized in the first 2.5 hr of poly(I:C)induction. Treatment of such cells with DRB at varying concentrations during the first 2.5 hr should permit the detection of an inhibition of interferon mRNA synthesis by DRB. The results of a typical experiment are CYCLOHEXIMIDE I 3RB
I ACTINOMYCIN D
543
COMMUNICATIONS
INTERFERON YIELD _ _ _ _ _ _ _ _ _ _ _ _ _ _ _,
FIG. 1. Drug regimen used to detect inhibition of interferon messenger RNA synthesis by DRB. Cultures in 60-mm dishes were induced with poly(I:C), 100 pg/ml, 2 ml/dish in Eagle’s medium for 1 hr beginning at 0 hr (solid bar). Cultures were treated with cycloheximide (50 pglml) from 0 to 6 hr and actinomycin D (5 Kg/ml) from 2.5 to 3.5 hr. DRB was present at varying concentrations, in duplicate cultures, between 0 and 2.5 hr. The cultures were washed four times with warm PBS at 1, 3.5, and 6 hr, and interferon yield from 6 to 24 hr was determined in 2 ml of inhibitor-free maintenance medium.
;i; 2 8
60
+
20
0 1001
t
601
-4
P
+ALL-UL 5 25 IO 5,6-Dlchloro-I-P-o-rtbofuranosylbenzlmldazole
20
30 40 60 (DREi),,M
FIG. 2. Inhibition of interferon production by DRB. Poly(I:C)-induced cultures were treated with DRB at varying concentrations using the drug regimen described in Fig. 1. (A) n - --W, Interferon yields expressed as percentage of the yield from DRB-free controls (20,480 ref. units/ml); -, rate of RNA synthesis (from Ref. 6). (B) Normalized doseeffect curves for the inhibition of interferon production and of RNA synthesis derived from data in (A).
presented in Fig. 2. Duplicate cultures in 60-mm dishes were treated with DRB at varying concentrations during the first 2.5 hr of the drug regimen indicated in Fig. 1. The cultures were washed four times with warm phosphate buffered saline at 1, 3.5, and 6 hr. Interferon produced from 6 to 24 hr was measured and expressed as ref. units/ml in terms of the 69/19 reference standard for human interferon (obtained from the Antiviral Substances Program, Infectious Disease Branch, National Institute of Allergy and Infectious Diseases, Bethesda, Md.). Figure 2A shows that DRB at concentrations of 5 fl or higher inhibited interferon yields. DRB at 50 fl caused 94% inhibition of interferon production. The solid line in Fig. 2A depicts the rate of RNA synthesis in the presence of varying concentrations of DRB as reported in an earlier publication (6). Figure 2B shows that the normalized dose-response curves of the effects of DRB on interferon yield and on RNA synthesis, constructed as described earlier (6, 8, 9), are closely
544
SHORT COMMUNICATIONS
similar. This provides additional evidence that the inhibition of interferon yield by DRB is the result of the inhibition of RNA synthesis. Our findings provide the first indirect evidence that DRB can inhibit the synthesis of a messenger RNA. ACKNOWLEDGMENTS We thank Dr. Arthur F. Wagner of the Merck, Sharp and Dohme Research Laboratories, Rahway, N.J., for a gift of DRB. This investigation was supported in part by Research Grant Nos. CA-18608 and AI-07057 from the United States Public Health Service. REFERENCES 1. EGYHAZI, E., DANEHOLT, B., EDSTROM, J.-E., LAMBERT, B., and RINGBORG, U., J. Cell Biol.
47, 516-520 (1970). 2. EGYHAZI, E., J. Mol. Biol. 84, 173-183 (1974). 3. EGYHAZI, E., Prm. Nat. Acad. Sci. USA 72,
947-950 (1975). 4. TAMM, I., HAND, R., and CALIGUIRI, L. A., J. Cell Biol., in press (1976). 5. GRANICK, D., J. Cell Biol. 65, 398-417 (1975). 6. SEHGAL, P. B., TAMM, I., and VItiek, J., Science 190, 282-284 (1975). 7. SEHGAL, P. B., TAMM, I., and VILCEK, J., Virology, 70, 532-541 (1976). 8. SEHGAL, P. B., TAMM, I., and VILEEK, J., J. Exp. Med. 142, 1283-1300 (1975). 9. SEHGAL, P. B., TAMM, I., and Vilcek, J., Virology, 70, 256-259 (1976). 10. SEHGAL, P. B., and TAMM, I., Proc. Nat. Acad. Sci. USA, in press (1976). 11. NG, M. H., and VILCEK, J., Adu. Prot. Chem. 26,
173-241 (1972).
70, 542-544
(1976)
SHORT Regulation II. Inhibition
The Rockefeller
of Human
of Interferon
PRAVINKUMAR
COMMUNICATIONS Interferon
Messenger RNA Synthesis ribofuranosylbenzimidazole
B. SEHGAL,
IGOR TAMM,
Production by 5,6-Dichloro-l-p-o-
AND
JAN
VILCEK’
University, New York, New York 10021; and ‘Department of Microbiology, Uniuerstiy School of Medicine, New York, New York 10016
New York
Accepted December 9, 1975 Polyinosinic:polycytidylic RNA in a strain of diploid n-ribofuranosylbenzi6idazole
acid (poly(I:C))-induced synthesis of interferon messenger human fibroblasts (FS-4) can be inhibited by 5,6-dichloro-l-P(DRB).
We have reported previously that 5,6dichloro-1-p-n-ribofuranosylbenzimidazole (DRB), a reversible inhibitor of nuclear heterogeneous RNA synthesis (l-5), can enhance (superinduce) polyinosinic:polycytidylic acid (poly(I:C)-induced interferon production in a strain of diploid human fibroblasts (FS-4) (6, 7). In the experiments reported earlier (6, 7), it appeared as if the synthesis of interferon messenger RNA (mRNA) might be, at least operationally, relatively resistant to inhibition by DRB. It was therefore necessary to determine whether the synthesis of interferon mRNA was indeed resistant to DRB inhibition or whether the marked superinducing effect of DRB masked inhibition of interferon mRNA synthesis. That the latter might be the case was suggested by our earlier observations that interferon yield in the presence of DRB and cycloheximide was lower than in the presence of cycloheximide alone (Fig. 1 of Ref. 6) and that the rate of interferon production in the presence of DRB was lower than in untreated control cultures for the first 3 hr after poly(I:C)induction (Fig. 3 of ref. 6). In the present report we describe a drug regimen which effectively eliminated interferon superinduction as an experimental variable and aliowed us to show clearly 542 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
that the synthesis of interferon mRNA is sensitive to inhibition by DRB. The cell cultures and media used, the procedure for interferon induction, the assay for interferon, and the measurement of the rate of cellular RNA synthesis have been described before (6-9). The beginning of poly(I:C)treatment (100 kg/ml in Eagle’s minimum essential medium for 1 hr at 37”) is designated as “0 hr” in all experiments. Poly(I:C) was obtained from the Antiviral Substances Program, Infectious Disease Branch, National Institute of Allergy and Infectious Diseases, Bethesda, Md. The drug regimen described in Fig. 1 has been developed on the basis of the following observations. Transcription of interferon mRNA is confined to the first 23 hr from the beginning of poly(I:C)induction (7). We have also observed that when cultures in the shutoff phase of interferon production (6, 7) are treated with DRB or actinomycin D, the rate of interferon production continues to decline for a further 3-4 hr and then reaches a plateau at a lower level (10). These observations indicate that the post-transcriptional control element responsible for the shutoff of interferon production (whose inhibition leads to superinduction), in addition to being operative during the first hour of
SHORT
poly(I:C)induction (6, 7), has a lifetime of 3-4 hr at 37” and inhibits interferon production in an irreversible manner (10). Hence, on the basis of the translational repressor model (II ) as a working hypothesis, it was decided to induce cells in the presence of cycloheximide (50 pg/ml, Polysciences, Inc., Warrington, Pa.) and to commence the inhibition of RNA synthesis using actinomycin D (5 pg/ml, Merck, Sharp and Dohme, Rahway, N.J.) 3.5 hr prior to removal of cycloheximide. It has been reported that cycloheximide does not inhibit the transcription of interferom mRNA (11). Further, the presence of cycloheximide from 0 to 6 hr would prevent the synthesis of the putative repressor protein even though repressor mRNA may have been synthesized. The 3.5-hr overlap between the beginning of actinomycin D treatment and the end of cycloheximide exposure would allow repressor mRNA synthesized during the first 2.5 hr to decay without undergoing translation. This drug regimen would maximize interferon superinduction and hence eliminate it as an experimental variable. Interferon yields obtained between 6 and 24 hr would therefore be proportional to the amount of interferon mRNA synthesized in the first 2.5 hr of poly(I:C)induction. Treatment of such cells with DRB at varying concentrations during the first 2.5 hr should permit the detection of an inhibition of interferon mRNA synthesis by DRB. The results of a typical experiment are CYCLOHEXIMIDE I 3RB
I ACTINOMYCIN D
543
COMMUNICATIONS
INTERFERON YIELD _ _ _ _ _ _ _ _ _ _ _ _ _ _ _,
FIG. 1. Drug regimen used to detect inhibition of interferon messenger RNA synthesis by DRB. Cultures in 60-mm dishes were induced with poly(I:C), 100 pg/ml, 2 ml/dish in Eagle’s medium for 1 hr beginning at 0 hr (solid bar). Cultures were treated with cycloheximide (50 pglml) from 0 to 6 hr and actinomycin D (5 Kg/ml) from 2.5 to 3.5 hr. DRB was present at varying concentrations, in duplicate cultures, between 0 and 2.5 hr. The cultures were washed four times with warm PBS at 1, 3.5, and 6 hr, and interferon yield from 6 to 24 hr was determined in 2 ml of inhibitor-free maintenance medium.
;i; 2 8
60
+
20
0 1001
t
601
-4
P
+ALL-UL 5 25 IO 5,6-Dlchloro-I-P-o-rtbofuranosylbenzlmldazole
20
30 40 60 (DREi),,M
FIG. 2. Inhibition of interferon production by DRB. Poly(I:C)-induced cultures were treated with DRB at varying concentrations using the drug regimen described in Fig. 1. (A) n - --W, Interferon yields expressed as percentage of the yield from DRB-free controls (20,480 ref. units/ml); -, rate of RNA synthesis (from Ref. 6). (B) Normalized doseeffect curves for the inhibition of interferon production and of RNA synthesis derived from data in (A).
presented in Fig. 2. Duplicate cultures in 60-mm dishes were treated with DRB at varying concentrations during the first 2.5 hr of the drug regimen indicated in Fig. 1. The cultures were washed four times with warm phosphate buffered saline at 1, 3.5, and 6 hr. Interferon produced from 6 to 24 hr was measured and expressed as ref. units/ml in terms of the 69/19 reference standard for human interferon (obtained from the Antiviral Substances Program, Infectious Disease Branch, National Institute of Allergy and Infectious Diseases, Bethesda, Md.). Figure 2A shows that DRB at concentrations of 5 fl or higher inhibited interferon yields. DRB at 50 fl caused 94% inhibition of interferon production. The solid line in Fig. 2A depicts the rate of RNA synthesis in the presence of varying concentrations of DRB as reported in an earlier publication (6). Figure 2B shows that the normalized dose-response curves of the effects of DRB on interferon yield and on RNA synthesis, constructed as described earlier (6, 8, 9), are closely
544
SHORT COMMUNICATIONS
similar. This provides additional evidence that the inhibition of interferon yield by DRB is the result of the inhibition of RNA synthesis. Our findings provide the first indirect evidence that DRB can inhibit the synthesis of a messenger RNA. ACKNOWLEDGMENTS We thank Dr. Arthur F. Wagner of the Merck, Sharp and Dohme Research Laboratories, Rahway, N.J., for a gift of DRB. This investigation was supported in part by Research Grant Nos. CA-18608 and AI-07057 from the United States Public Health Service. REFERENCES 1. EGYHAZI, E., DANEHOLT, B., EDSTROM, J.-E., LAMBERT, B., and RINGBORG, U., J. Cell Biol.
47, 516-520 (1970). 2. EGYHAZI, E., J. Mol. Biol. 84, 173-183 (1974). 3. EGYHAZI, E., Prm. Nat. Acad. Sci. USA 72,
947-950 (1975). 4. TAMM, I., HAND, R., and CALIGUIRI, L. A., J. Cell Biol., in press (1976). 5. GRANICK, D., J. Cell Biol. 65, 398-417 (1975). 6. SEHGAL, P. B., TAMM, I., and VItiek, J., Science 190, 282-284 (1975). 7. SEHGAL, P. B., TAMM, I., and VILCEK, J., Virology, 70, 532-541 (1976). 8. SEHGAL, P. B., TAMM, I., and VILEEK, J., J. Exp. Med. 142, 1283-1300 (1975). 9. SEHGAL, P. B., TAMM, I., and Vilcek, J., Virology, 70, 256-259 (1976). 10. SEHGAL, P. B., and TAMM, I., Proc. Nat. Acad. Sci. USA, in press (1976). 11. NG, M. H., and VILCEK, J., Adu. Prot. Chem. 26,
173-241 (1972).