Superinduction of human fibroblast interferon production: Further evidence for increased stability of interferon mRNA

VIKOI.OGY

89, 186-198

(1978)

Superinduction Evidence PRAVINKUMAR

of Human Fibroblast Interferon Production: for Increased Stability of Interferon mRNA B. SEHGAL, The Rockefeller

DOUGLAS

S. LYLES,

AND

IGOR

Further

TAMM

Uniuersity, Nru~ York, Nwr York 10021 Accepted Mny 2, 1978

Cytoplnsmw polyadenylated interferon mRNA, synthesized in human diploid fibroblasts (FS-4 strain) largely within the first ,3 hr of exposure to poly(I) .poly(C), is approximately 850-900 nucleotides in size (12 S) as determined by sedimentation of dimethybulfoxide denatured mRNA through a sucrose gradient and translation of RNA in each gradient fraction by microinjection into oocytes of Xenopus Zaeu~s. Newly synthesized cytoplasmic interferon mRNA has a poly(A) length >lOO nucleotides as determined by step elution from a poly(lJ)-Sepharose column with varying concentrations of formamide and subsequent translation of each eluted fraction. There is a pool of translatable nuclear polyadenylated interferon mRNA molecules which also sediment at approximately 12 S. No poly(A)lacking interferon mRNA is detectable. One hour after the beginning of poly(I)-poly(C) induction the concentration of translatable interferon mRNA in the nuclear pool is higher than that in the cytoplasmic pool. Interferon mRNA in the nuclear pool peaks at 2 hr and is undetectable by 4-5 hr while in the cytoplasmic pool it peaks between 2 and 3 hr and is barely detectable by 6 hr. The rate of interferon secretion in a culture of FS-4 cells peaks between 2.5 and 3.5 hr after the beginning of polytI).poly(C) induction and secretion is shut off by 6-8 hr. The concentration of cytoplasmic, phenol-extractable, interferon mRNA declines with a half-life of 0.5 to 1.0 hr during the shutoff phase, as does the rate of interferon secretion. The rapid shutoff of interferon production which occurs between 3 and 8 hr after induction is prevented when FS-4 cultures are induced and maintained in 5.6dichloro-I-/-n-rihofuranosylhenzimidazolr (I)RR, 40 /r&f), a selective and reversible inhihitor of hnRNA and mRNA synthesis. This leads to an approximately IO-fold increase in the cumulative interferon yield in the first 24 hr of induction. In the presence of DRB, both the corn-ntration of phenol-extractable, translatable interferon mRNA and the rate of interferon secretion in induced cultures decline with virtually identical half-lives of approximately 6-8 hr A theoretical analysis of the available data indicates that the increased functional stability of interferon mRNA is the major factor in interferon superinduction by DRB. The increased stability possibly results from an inhibition by DRB of the synthesis of a rapidly turning over repressor RNA involved in the inactivation or degradation of interferon mRNA. Since the poly(A) of interferon mRNA continues to shorten in the presence of L)RB. it is unlikely that increased stahility of interferon mRNA is the result of an inhibition of poly(A) metabolism synthesis of a rapidly turning over RNA, which leads to an inhibition in the translation of poly(1). poly(C)-induced interferon mRNA (Vilcek et al., 1969; Tan et al., 1970; Sehgal et al., 1975a, 197Gb; Sehgal and Tamm, 1976; Tamm and Sehgal, 1978). The key experimental basis for this hypothesis is the observation that induced FS-4 cultures appropriately exposed to a wide range of inhibitors of RNA or protein synthesis show a paradoxical enhancement of interferon production (Sehgal et al., 1975b; Vil-

Indirect, inhibitor-based experiments carried out over the last few years suggest, that the translation of interferon mRNA in poly(1) poly(C)-induced diploid human fibroblasts is regulated by a repressor mechanism which inactivates or degrades interferon mRNA (reviewed in Vilcek et al., 1976; Sehgal et al., 1977; Tamm and Sehgal, 1978). The poly( I) poly( C) -induced repressor mechanism is thought to involve the 186 0042.6X22/78/0891-0162$02.00/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any furm reserved

STABILITY

OF

HLJMAN

FIHKORLAS1‘

cek et al., 1976; Sehgal et al., 1977; Tamm and Sehgal, 1978). In our studies we have analyzed the enhancing effect of 5,6-dichloro-l-[%n-ribofuranosylbenzimidazole (DRB), a selective and reversible inhibitor of hnRNA and mRNA synthesis (Tamm and Sehgal, 1978)) on poly(1). poly(C)-induced interferon production by FS-4 cells (Sehgal et al., 1975a; Sehgal and Tamm, 1976; Tamm and Sehgal, 1978). These studies have led to a view of interferon synthesis depicted in Fig. 1. The rapid shutoff of the rate of interferon production by 6-8 hr is ascribed not to sirnple decay of interferon mRNA subsequent to the termination of transcription but to a repressor mechanism which inactivates or degrades interferon mRNA. When cultures are induced and maintained in DRB at a moderate concentration (30-40 I*M), interferon production continues for up to 4 days leading to a large (lo-20-fold) increase in cumulative interferon yield (Sehgal et al., 1975a, 1976b). This observation suggests that interferon mRNA is relatively stable provided cellular RNA synthesis has been at least partially inhibited. Additional indirect experiments suggest that the shutoff of interferon production is unlikely to be the result of simple competition by newly synthesized cellular mRNA but is likely to be due to a repressor mechanism which has a life-time of 3-4 hr at 37” (Sehgal and Tamm, 1976). Recently we have obtained direct biochemical evidence for the increased stability of interferon mRNA in DRB-treated cells (Sehgal et al., 1977). We assayed hu-

FIG. 1. Induction poly(I)

-poly(C)

of in cultures

interferon of FS-4

production cells.

by

IN’I’EKFKKON

187

mRNA

man fibroblast interferon mRNA in induced cultures by translation of microinjetted mRNA in oocytes of Xenopus Laevis into biologically active interferon (Reynolds et al., 1975). The oocyte assay is a sensitive, quantitative and reliable assay for interferon mRNA (Cavalieri et al., 1977a; Sehgal et al., 1977). We demonstrated the disappearance of phenol-extractable, translatable interferon mRNA from induced FS4 cells during the shutoff phase and a persistence of such RNA in DRB-treated cells (Sehgal et nl., 1977). In the present communication we provide a detailed description of these phenomena. We have used the oocyte assay to characterize nuclear and cytoplasmic interferon mRNA molecules, to determine the kinetics of their appearance during induction and their rapid decay during the shutoff phase as well as to demonstrate a quantitative correlation between the concentration of translatable interferon mRNA and the rate of interferon secret,ion when FS-4 cultures are induced and maintained for an extended period of time in the continuous presence of DRB (40 fl. The data presented are best interpreted within the framework of the McAuslan-Tomkins repressor hypothesis (McAuslan, 1963; Carren et al., 1964; Tomkins et al., 1972). MATERIALS

AND

M~:1’HOI~S

The procedures used for growing FS-4 cells in 150-mm Falcon petri dishes, induction of interferon with poly(1). poly(C) (P.L. Biochemicals, 20 pg/ml), harvesting cells by trypsinization, cell fractionation, ext,raction of nucleic acid from whole cells as well as from the cytoplasmic and nuclear fractions, poly(U)-Sepharose chromatography, denaturation of RNA by dimethylsulfoxide and its analysis by sucrose gradient centrifugation, and the assay of interferon mRNA by translation of microin.jetted mRN*A in X. laevis oocytes have all been described in earlier publications (Sehgal et al., 1975a, b; 1976d; 1977; Derman and Darnell, 1974; Derman et al., 1976). Oocytes used in most of this investigation were obtained by operating on female X. laerks kept submerged in an ice bath. A small piece of the ovary was excised without sacrificing the animal. Typically the translation assay consisted of

188

SEHGAL,

LYLES.

IO oocytes each microinjected with 100-150 nl of RNA solution, incubated for 36-48 hr at 23”, and subsequently homogenized in 0.2 ml of modified Barth’s medium that had been used for the incubation. Interferon was assayed on FS-4 cells using vesicular stomatitis virus (Indiana strain) by a modification of the semi-micro method initially developed by Armstrong (1971) (Have11 and Vilcek, 1972). Interferon titers are expressed in terms of the 69/19 reference standard for human interferon. The procedure used for step eiution of polyadenylated RNA bound to a poly(U)Sepharose column was developed by S. Sawicki, M. Wilson, and J. E. Darnell (personal communication). Briefly, RNA was bound to a 2-cm column of poly(U)-Sepharose in a Pasteur pipette in buffer containing 0.4 M NaCl, 0.01 M EDTA, 0.01 M Tris (pH 7.4), and 0.2% sodium dodecyl sulfate (SDS) (0.4 h4 NETS). The column was washed with 10 ml each of 0.4 M NETS and ETS (buffer containing no NaCl). For each step elution 2 ml of the formamide solution in ETS was passed through the column followed by 1 ml of an ETS rinse. The eluted RNA was adjusted to 0.2 M LiCl and ethanol precipitated after addition of carrier RNA. RESULTS

Human fibroblast interferon mRNA injected into X. laeuis oocytes is efficiently translated in the next 24-48 hr into biologically active interferon which is antigenitally indistinguishable from human fibroblast interferon (Reynolds et al., 1975; Cavalieri et al., 1977a; Sehgal et al., 1977). The oocyte product comigrates with authentic human fibroblast interferon in SDS-polyacrylamide gels with an apparent molecular weight of 20,000 (Cavalieri et al., 1977a). Microinjection of as little as 1 ng of polyadenylated RNA from induced FS-4 cells into each of 15 oocytes (followed by incubation at 23” for 36-48 hr and homogenization in 0.3 ml of modified Barth’s medium) leads to detectable interferon synthesis. There is a linear increase in the amount of interferon synthesized with increasing amounts of injected mRNA in the range of 1 to 20 ng RNA per oocyte. At

ANI)

‘I’AMM

saturation (40-80 ng RNA per oocyte), 10 or 15 injected oocytes homogenized in 0.2 or 0.3 ml medium produce 192-256 reference units of interferon per ml. The main source of error in this assay is the two-fold variation in the antiviral assay for interferon. However the biological assay for interferon constitutes an extremely sensitive procedure than can detect picogram amounts of interferon in a crude cellular homogenate.

Characterization

of Interferon

mRNA

Our initial objective was to characterize the interferon mRNA molecule induced in human fibroblasts by poly(1) poly( C). Earlier reports have indicated that human fibroblast interferon mRNA is polyadenylated (Reynolds et al., 1975; Pestka et al., 1975; Cavalieri et al., 1977a; Sehgal et al., 1977) and that a nuclear as well as a cytoplasmic pool of translatable interferon mRNA molecules is present in induced cells (Sehgal et al., 1977). Figure 2 describes the sedimentation through a sucrose gradient of dimethylsulfoxide-denatured nuclear and cytoplasmic polyadenylated RNA from FS-4 cells that had been induced and maintained in cycloheximide (50 pg/ml) for 4 hr. Cultures were treated with cycloheximide in an attempt to prevent possible degradation of interferon mRNA during the shutoff phase and hence to obtain the maximum yield of interferon mRNA (Reynolds et al., 1975; Pestka et aZ., 1975; Sehgal et al., 1977). RNA in each gradient fraction was precipitated with ethanol and assayed for interferon mRNA by translation in oocytes. It is apparent that both nuclear and cytoplasmic interferon mRNA species sediment at approximately 12 S. The poly(A) length of interferon mRNA was estimated by step elution of interferon mRNA bound to poly(U)-Sepharose with formamide at varying concentrations, using a procedure developed recently by S. Sawicki, M. Wilson, and J. E. Darnell (personal communication). Typically, elution of a poly(U)-Sepharose column with 20,25,27.5, 30, or 50% (v/v) formamide at room temperature leads to the removal of poly(A)containing RNA with poly(A) lengths equivalent to approximately 55, 75-95, 125,

STABII,I’I’Y

OF

HllMAN

FIBKOBLASI

FIc. 2. Srdimentatiun analysis of nuclear (0) and cytoplasmic (0) interferon mRNA molecules. t14 FS-4 cultures in 150 mm Falcon petri dishes were induced with poly(I) polytC) (XI /Lg/ml) and maintained continuously in the presence of cycloheximide (50 pg/ml). Four hours later the cells were harvested by trypsinization. allowed to swell for 15 min in one-thirdstrength reticulocytr standard buffer, broken by Dounce homogenization and the cytoplasmic as well as the detergent (half-strength)-washed nuclear fractions ohtained (Penman. 1969; Sehgal et ctl., 1977). RNA was phenol extracted and passed through a pol?;(LJ)-Sepharose column to select polyadenylatrd KNA which wac then ethanol precipitated. The nuclear and cytoplasrnic~ polyadenylatetl KNA preparations were dissolid in %O 1~1 and lot) ~1, respectively, of inject ion buffer (88 mJ4 NaCI, 0.01 ;W ‘l’ris, pH 7.4) (Gurdon, 1974; Sehgal et (I/.. 1957) to give solutions containing O.‘L? g/p1 and 0.5 &WI of KNA, respectively. Ten mic roliters of each of the two preparations were mixed with Z(H) ~1 of dimethvlsulfoxide, denatured at 65” for 2 min (Derman and Darnell, 1X4), and then centrifugrd through a IS-JO’r sucrose gradient in 0.05 M NETS in an SW?7 rotor at %,(I00 rpm for Z-3 hr. KNA in eac,h fraction was ethanol precipitated after addition of carrier KNA, dissolved in 5 pl injection buffer, and approximately half of each fraction was assayed for interferon mKNA by translation in oocytes. The hrokel, line indicates sedimentation of marker 4 S and 1X S KNA in a separate gradient.

INTEKFE:KON

The extracted RNA was bound to poly(U)Sepharose columns in 0.4 M NaCl. The columns were washed thoroughly to remove unbound nucleic acid and the bound RNA step-eluted with formamide at different concentrations. RNA in each fraction was ethanol precipitated and the amount of interferon mRNA measured by translation in oocytes. Figure 3 shows that the poly(A) length of nuclear and cytoplasmic interferon mRNA molecules at 3 hr is similar. The 6.25-hr cytoplasmic sample shows some shortening of the poly{A). At 6.25 hr little or no interferon mRNA is detectable in the nucleus, a result that reflects the

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175, and >175 nucleotides, respectively. The procedure gives highly reliable results in simultaneous comparative evaluation of poly(A) lengths of several mRNA preparations. Examination of similar preparations in separate analyses reveals a procedural variation of at least one elution class, which may be due to the sensitivity of this procedure to fluctuations in ambient temperature (Sawicki, Wilson and Darnell, personal communication). Cytoplasmic and nuclear RNA was extracted from FS-4 cells induced and maint,ained in cycloheximide for 3 or 6.25 hr.

lH9

mKNA

25

275-

C: r- c m, 2 e

33 ‘j

5””

, ”

FIG. 3. PoIy(A) length distribution of nuclear and cytoplasmic interferon mKNA. Nuclear and cvtoplasmic KNA was extracted from FS-4 cultures (10 per group) which had been induced and maintained in cycloheximide for 3 or 6.25 hr. Poly(A) length of interferon mRNA wab assayed by stepelution of poly(U)-Sepharose hound mKNA using varying concentrations of formamide followed by translation ot half of each eluted KNA sample The total amount of interferon synthesized in oocytes by KNA described in panels A, B, and C was 90, 34. and 18 reference units/ml. I’olyadenylated interferon mRNA was not detectable in the nuclear fraction at 6.25 hr (~4 reference units/ml).

190

SEHGAL,

LYLES,

termination of transcription of interferon mRNA as outlined in Fig. 1 (also see Fig. 4). That the absence of translatable interferon mRNA in the shorter poly(A) classes is not due to the inability of oocytes to translate interferon mRNA with short poly(A) is shown in Fig. 6B in which translatable interferon mRNA with short poly(A) is depicted. We conclude that interferon mRNA, like other mammalian mRNA species, has a poly(A) >lOO nucleotides long. We have attempted to detect poly(A)lacking interferon mRNA (Table 1). All earlier experiments directed at this question have involved translation of the unbound fraction of whole cell nucleic acid after passage through an oligo(dT)-cellulose or a poly(U)-Sepharose column (Reynolds et al., 1975; Pestka et al., 1975; Thang et al., 1975). However, as pointed out earlier (Sehgal et al., 1977) such an unbound fraction is strongly inhibitory to the translation of interferon mRNA. We have observed that this inhibitory activity is located in the nuclear fraction (P. B. Sehgal, unpublished observations). Gentle fractionation of cells into a nuclear and a cytoplasmic fraction prevents the appearance of inhibitory material in cytoplasmic RNA preparations. Table 1 describes an attempt to detect interferon mRNA in the unbound fraction of cytoplasmic RNA in the preparation used in Fig. 3C. It can be seen that poly(A)lacking interferon mRNA was not detected in the cytoplasmic fraction 6.25 hr from the TABLE TEST FOR PoI,Y(A)-LACKING RNA Unbound RNA Unbound mRNA Interferon

assayed

fraction fraction

+

mRNA

alone

interferon

TAMM

start of induction. A similar result (not shown) was obtained using the cytoplasmic RNA preparation obtained at 3 hr and described in Fig. 3B. Kinetics of Accumulation mRNA During Normal Shutoff

of Interferon Induction and

We have attempted to correlate the kinetics of secretion of interferon in induced FS-4 cultures with the concentration of interferon mRNA in nuclear and cytoplasmie pools. Figure 4A indicates that secretion of interferon in poly(I) poly(C)-induced FS-4 cultures is detectable by 1-2 hr after the beginning of induction. The rate of secretion peaks between 2.5 and 3.5 hr and secretion falls below detectable levels by 6-8 hr (Fig. 4A; Fig. 3 of Sehgal et al. (1975a); and Fig. 4 of Sehgal et al. (1977)). Figure 4A also indicates that the cellular content

1 INTERFERON Interferon erence .-

of cytoplasmic

AND

mRNA” .~ titer (refunits/ml) <3 48 24

” Cytoplasmic RNA described in Fig. 3C was passed through a poly(U)-Sepharose column and the unbound RNA recovered, ethanol precipitated, and dissolved in 10 ~1 of distilled water. The mixing experiment was carried out by microinjecting into oocytes an equal volume of a mixture (2 ~1 + 2 ~1) of the unbound RNA and an mRNA preparation known to contain interferon mRNA.

FIG. 4. Interferon mRNA content during normal induction and shutoff. FS-4 cultures were induced with poly(1) poly(C) for 1 hr beginning at “0” hr. At hourly intervals cells were harvested from 8 cultures and fractionated into cytoplasm and detergent-washed nuclei. The amount of polyadenylated interferon mRNA was determined by poly(U)-Sepharose selection and subsequent translation of half of each preparation. One culture was subjected to hourly medium changes to determine the rate of interferon secretion. Panel A: (O- - -0) interferon secretion per hour; (M) interferon mRNA, nucleus + cytoplasm. Panel B: (M) interferon mRNA, nuclear pool; (0- - -0) interferon mRNA, cytoplasmic pool.

STABILITY

OF

HUMAN

FIBROBLAST

of phenol-extractable interferon mRNA reaches a maximum between 2 and 3 hr and decreases thereafter such that it is barely detectable by 5-6 hr (also see Fig. 4 of Sehgal et al. (1977)). Figure 4B describes the nuclear and cytoplasmic pools. At 1 hr there is a greater amount of interferon mRNA in the nuclear pool than in the cytoplasmic pool. The nuclear pool reproducibly reaches a maximum at 2 hr and decreases to below detectable levels by 4-5 hr. A similar decrease in the nuclear pool also occurs in the presence of cycloheximide or DRB (not shown). These data further support the conclusion that transcription of interferon mRNA terminates by 3-4 hr of induction in the absence or the presence of cycloheximide or DRB. The cytoplasmic pool of phenol-extractable interferon mRNA reaches a maximum between 2 and 3 hr and decreases rapidly to barely detectable levels by 5 to 6 hr. Both the rate of secretion of interferon in culture and the amount of interferon mRNA in the cytoplasm decline with a similar half-life (0.5-1.0 hr). The data in Figure 4 provide an internally consistent picture of interferon synthesis. The key feature that needs emphasis is the disappearance of phenolextractable interferon mRNA during the shutoff phase. Furthermore, appropriate mRNA mixing experiments demonstrate that the reduced levels of interferon mRNA observed during the shutoff phase correctly reflect the levels of interferon mRNA (Sehgal et al., 1977). These data demonstrate that the rapid shutoff of interferon production is not the result of cellular mRNA displacing interferon mRNA from polysomes with survival of translatable interferon mRNA in the cytoplasm, but is the result of rapid inactivation or degradation of interferon mRNA. Several control experiments have shown that polyadenylated interferon mRNA extracted from detergent-washed nuclei (Figs. 2-4) does not represent cytoplasmic contamination. First, the maximum contamination of nuclear RNA with ribosomall8 S RNA is approximately 5% as estimated by the optical density profiles (260 nm) of nuclear and cytoplasmic RNA (unbound fraction) of the preparations described in Fig. 2 (nuclear to cytoplasmic interferon mRNA

INTERFERON

191

mRNA

ratio 1 to 5). Second, only 2% as much interferon mRNA is obtained in the nuclear fraction compared to the cytoplasmic at 6 hr after induction and continued maintenance in DRB (40 IIJI/I). Third, the poly(A) length of nuclear interferon mRNA is detectably larger than that of cytoplasmic molecules in the same cells (Fig. 3, A and B, and unpublished data). Decreased Inactivation Interferon mRNA tion

or Degradation of During Superinduc-

We have measured the half-life of interferon mRNA during superinduction by DRB and compared it to the decline in the rate of interferon secretion by DRBtreated, induced cultures. Since the nuclear interferon mRNA pool is completely depleted by 4-5 hr, cytoplasmic polyadenylated interferon mRNA was prepared by phenol extraction of whole cells at 5, 17, and 25 hr after FS-4 cells had been induced in and continuously exposed to DRB (40 m. One culture from the 25-hr group was subjected to repeated medium change in order to determine the rate of secretion of interferon. The results presented in Fig. 5

IO

20

3

FIG. 5. Interferon mRNA content of induced FS-4 cells in the continued presence of DRB (40 M. FS-4 cultures were induced with poly(I).poly(C) in the presence of DRB (40 a&f) and were continuously maintained in DRB. Cells were harvested at 5, 17, and 25 hr after the beginning of induction (3 cultures per group) and polyadenylated RNA selected from whole cell nucleic acid. Interferon mRNA was quantitated by translation of half of each preparation (0). One culture in the 25-hr group was subjected to repeated medium changes in order to determine the rate of interferon secretion (0).

192

SEHGAL,

LYLES,

confirm that DRB-treated. induced cultures secrete interferon for a prolonged period of time, and that DRB prevents the rapid loss of phenol-extractable interferon mRNA, which occurs in its absence. Furthermore, both the rate of interferon secretion and the concentration of interferon mRNA decrease with virtually identical half-lives (6-8 hr in this experiment). Table 2 summarizes the half-life data derived from Figs. 4 and 5. In the Appendix we TABLE HALF-LIFE Measurement

2

OF INTERFEKON

carried

out

mKNA

Estimate

based

on

during Rate of secretion in culture (hr)

mKNA concentration assayed in oocytes (hr)

0.5-0.7 (i-8

05 I .O (i-8

Normal shutoff DHB (40 m superinduc-

r DRB

AND

TAMM

demonstrate that the increase in cumulative interferon yield in the presence of DRB can be largely explained in terms of the observed increase in stability of interferon mRNA. Shortening During

of Poly(A) of Interferon DRB Superinduction

mRNA

Inhibition of poly(A) shortening is one possible mechanism by which the stability of interferon mRNA might be increased in the continued presence of DRB. We have evaluated this possibility by comparing the poly(A) length distribution of interferon mRNA in DRB-treated, induced cultures 6 hr after induction to that at 11 hr after induction. It is apparent from Fig. 6 that the poly(A) of interferon mRNA undergoes a dramatic reduction in length during continued incubation in the presence of DRB. A comparison of Figs. 3 and 6 indicates that the oocyte translation assay responds to interferon mRNA of all poly(A) length classes.

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t

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50

DISCUSSION

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20

25

275

Formamide,

30

50

%v/v

FIG. 6. PolytA) length distribution of interferon mRNA in the continued presence of DHB (40 m. The step-elution procedure described in Fig. 3 was used to assay the poly(A) length of interferon mHNA in RNA extracted from whole cells (10 cultures per group) that were induced and continuously maintained in DKB (40 t&f) for 6 or 11 hr. The total amount of interferon synthesized on translation of half of each eluted fraction was 264 and 168 reference units in oanels A and B. resnectivelv . ~.I

We reported previously that interferon mRNA induced by poly(1) poly(C) in FS-4 cells escapes rapid inactivation or degradation if the cultures are treated with DRB (Sehgal et al., 1977). We have now demonstrated that there is an excellent correlation between the rate of secretion of interferon in FS-4 cell cultures and the concentration of interferon mRNA in these cells both during normal induction and during superinduction in the presence of DRB. The paradoxical enhancement of interferon prodLction by DRB is largely due to an increase in the functional stability of interferon mRNA. It is likely that DRB interferes with the synthesis of RNA involved in the inactivation or degradation of interferon mRNA. Investigation of the regulation of interferon production at the molecular level was made possible by the recent development of the translation assays for interferon mRNA (Reynolds et al., 1975; Thang et al., 1975; Pestka et al., 1975; Cavalieri et al., 1977a; Sehgal et al., 1977). Microinjection of human fibroblast interferon mRNA into oocytes of X. laetis provides a highly sen-

S’I’AHII,I’I’Y

OF

H1iMAN

FIBROBLASl

sitive and reliable assay for interferon mRNA (Reynolds et nl., 1975; Cavalieri et al., 1977a; Sehgal et al., 1977). We have used this assay to delineate some of the events that underlie the induction and the subsequent shutoff of interferon production in human diploid fibroblasts exposed to poly(1) poly(C). Induction of FS-4 cultures with poly(1) poly(C) leads to the rapid appearance of polyadenylated interferon mRNA whereas uninduced FS-4 cells do not contain any polyadenylated RNA translatable into interferon (Reynolds et al., 1975; Pestka et al., 1975; Sehgal et al., 1977). Inhibitor-based evidence suggested that interferon mRNA is newly transcribed starting soon after the beginning of induction and that transcription of interferon mRNA is largely complete 3 hr after induction (Sehgal et al., 1976b). The results presented in Fig. 4 which show a rapid decline in the nuclear pool of interferon mRNA between 2 and 5 hr after induction are consistent with t.his interpretation. Furthermore, even when FS-4 cells are superinduced in the presence of DRB, transcription of interferon mRNA is largely terminated by 3 hr after the beginning of induction (Sehgal et nl., 1976b). Preliminary observations (P. B. Sehgal) have shown a rapid decline of interferon mRNA in the nuclear pool 3-6 hr after induct.ion in the presence of DRB or cycloheximide. Table 3 summarizes the physical characteristics of the mRNA for human fibroblast interferon. The estimate of 450-500 nucleotides in the coding region is based on the observation that deglycosvlated human fibroblast interferon migrates”in SDS-polyacrylamide gels as a polypeptide of molecular weight approximately 15,000-16,000 (Bose et al., 1976; Have11 et al., 1977). The assumption that 50-60 nucleotides may ‘I’AHIX

:j

(‘HARA(.‘~IIKI%A,TI~N OF HITMAN INTFJIFEKON mHNA Feat urc Size

(-12

I’oI~IAJ C’otiing Signal Noncodmg

S)

region sequence region

FIIIKOI~I.AST Nucleotides X50-W) >loo 4:50-5(W) 50-60 -2(X)

IN’I’FXFEKON

nrllNh

193

represent the N-terminal signal sequence is based on observations which indicate that human fibroblast interferon is a typical secretory glycoprotein (Have11 and Vilcek, 1975; Sehgal et al., 1975b; Falcoff et al.. 1976). The nucleotide assignment for the noncoding region was arrived at by subtraction. At the present time we do not know whether this molecule has a 5’.capped end. We have been unable t,o detect translatable poly(A)-lacking int,erferon mRNA in FS-4 cells using the oocyte assay. Furthermore, we have observed that polyadenylated and enzymatically deadenylated interferon mRNA are translated with equal efficiency and stability in oocytes (P. B. Sehgal, H. Soreq and I. Tamm, manuscript in preparation). The detection of a nuclear pool of translatable polyadenylated interferon mRNA molecules raises two obvious questions: 1) what is the relationship of these molecules to the primary transcript containing interferon mRNA sequences. and 2) do these molecules represent precursors to cytoplasmic interferon mRNA? At the present time it is difficult t,o relate the nuclear interferon mRNA molecules to their primary transcripts except to point out that in the globin system one of the polyadenylated nuclear precursors is of the same size as the cytoplasmic mRNA molecule, whereas the largmolecule lacks est. nuclear precursor poly(A) (Bastos and Aviv, 1977). It is therefore not surprising that both the nuclear and the cytoplasmic polyadenylated interferon mRNA species described in this report are of similar size (Fig. 2). The kinetic data presented in Fig. 4 suggest that, at least some of the nuclear molecules may be precursors to cytoplasmic -Interferon mRNA. Our previous observation that the shutoff of interferon production which occurs between 3 and 8 hr of induction is accompanied by a loss of phenol-extractable interferon mRNA from the induced cells (Sehgal et al., 1977) has recently been confirmed by Cavalieri et al. (1977b) and Greene et (11. (1978). In the present report we demonstrate that both the secretion of interferon in FS-4 cultures and the concentration of interferon mRNA decay with similar halflives of between 0.5 and 1.0 hr during the shutoff phase. This result confirms that the

194

SEHGAL.

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AND

TAMM

shutoff of interferon production is not a competition is not involved in this phenomconsequence of displacement of interferon enon (Sehgal and Tamm, 1976). Direct biomRNA from polysomes with survival of chemical experiments in which mRNA masked but translatable mRNA in the cypreparations extracted from cells during sutoplasm. perinduction and during shutoff were mixed It appears likely that the decay of interprior to translation in X. Zaevis oocytes also feron mRNA during the shutoff phase is indicate that mRNA competition is undue to a specific inactivating or degrading likely to play a significant role in the regumechanism. In the presence of DRB, interlation of human interferon production feron mRNA escapes inactivation or deg(Sehgal et al., 1977). radation and as a result synthesis of interThere is persuasive indirect evidence feron proceeds for a prolonged period, that the hypothetical repressor mechanism which results in a large paradoxical increase operative in the interferon system involves in interferon yield. Our data suggest that the synthesis of RNA (reviewed in Sehgal interferon superinduction can be largely ex- et al. (1977) and Tamm and Sehgal (1978)). plained in terms of the increased half-life of Evidence for the involvement of protein is interferon mRNA in the presence of DRB less convincing (Sehgal et al., 1976a). It (also see Appendix). takes a 20-fold higher concentration of cyAlthough Cavalieri et al. (1976b) are in cloheximide to cause half-maximal superagreement with these conclusions, it should induction than it does to cause half-maxibe noted that there is a large systematic mal inhibition of protein synthesis (Sehgal disagreement between their and our quan- et al., 1976a). Whatever the chemical natitative delineation of the kinetics of inter- ture of the molecules involved, it is clear feron mRNA accumulation during induc- that they are turned over rapidly (life-time tion, shutoff and superinduction in FS-4 3-4 hr at 37”, Sehgal and Tamm, 1976). cells. Cavalieri et al. (1977b) report that the There is also evidence that this mechanism half-life of the decline in the rate of inter- is induced by poly(1) poly(C) and is operferon secretion is substantially longer than ational within the first hour of induction the half-life of interferon mRNA in nor- (Sehgal et al., 1975a, 1976b). Our current mally induced cells as well as in cells super- working hypothesis is that the predominant of poly(1) induced with cycloheximide with or withmechanism of regulation out actinomycin D. It is possible that these poly(C)-induced interferon synthesis falls investigators did not recover and assay in- within the framework of the repressor hyterferon mRNA from FS-4 cells quantitapothesis proposed by McAuslan (1963) and by Tomkins and his colleagues (Garren et tively, since they used nitrocellulose filter al., 1964; Tomkins et al., 1972) for translabinding to select out polyadenylated RNA from whole cell nucleic acid. The data pre- tional regulation in mammalian cells. sented by Cavalieri et al. (1977b, p. 4416) APPENDIX: A THEORETICAL ANALYSIS OF indicate that up to 50% of polyadenylated INTERFERON SIJPERINDUCTION BY DRB RNA was lost during nitrocellulose filter binding compared to selection on oligo(dT)Is the observed increase in half-life of cellulose. That there is a good correlation interferon mRNA in the presence of DRB between the rate of secretion of interferon sufficient to explain the increase in cumuduring normal induction and the concentralative interferon yield? We have attempted tion of interferon mRNA has been inde- to evaluate this question by constructing a pendently confirmed by two other labora- mathematical model for the induction of tories (Greene et al., 1978; P. M. Pitha and interferon. The primary assumption in the N. B. K. Raj, personal communication). following analysis is that the rate of secrePitha and Raj (personal communication) tion of interferon in cell culture reflects the also ascribe superinduction largely to an concentration of interferon mRNA in these cells. We believe that the experimental data increased stability of interferon mRNA. The exact mechanism by which interpresented in this paper adequately validate feron production is shut off is unknown. this assumption (cf. Figs. 4 and 5). We also Indirect experiments suggest that mRNA assume that virtually every FS-4 cell in a

S1‘AHII,I’I’Y

OF

HUMAN

FIHHOH1,AS’I’

1N’I’E:HFEIION

195

mKNA

presence of DRB is inhibited to the same extent as synthesis of total mRNA (Sehgal et al., 1976c, d) then it would be expected that &,I’ = 0.2 S,,(‘ (where S,,,(’ is the synthesis rate in control cultures not treated with DRB and SnlU that in DRB-treated cultures). However, recent data suggest that there is a 3-4-fold higher rate of appearance of translatable interferon mRNA in the presence of DRB than would be expected. We estimate that S,,,‘) = -0.7 S’,,z(’ for 40 fl DRB (0.2 multiplied by 3-4; Sehgal and Tamm, manuscript in preparation). 2. The half-life of interferon mRNA in the presence of 40 pM DRB was found to be 6-8 hr, or approximately 7 hr, which gives k,” = 0.1 hrr’, where the superscript ClM ___ = S,, - k,,,M 111 denotes the presence of DRB. The decrease dt in the degradation rate could be explained by postulating an inhibition by DRB of the Solving this equation for M as a function of synthesis of a repressor which causes degt gives: radation of interferon mRNA. It should be mentioned that the effect of DRB on the M = ” (1 - em”!“‘), for t 5 f’ [aa] degradation rate of interferon mRNA is not ?,,I linearly related to the effect of DRB on and cellular mRNA synthesis, since 40 ,uJ~ DRB &f = M&“J“J, for t > t’ causes a 14-fold decrease in the degradation Pbl rate of interferon mRNA while causing only where Mt, is the level of mRNA at the time a 5-6-fold decrease in cellular mRNA synwhen synthesis is stopped (t’) and reflects thesis (i.e., -80% inhibition of mRNA synt,he maximum level obtained. For cells stimthesis, Sehgal et al., 1967c, d). ulated with poly(1) .poly(C) at t = 0, t’ has The ratio of the maximal rates of secrebeen determined to be approximately 3 tion of interferon in the absence and the hours (Sehgal et al., 1976b), and the halfpresence of DRB, which corresponds to the life of interferon mRNA is approximately increase in the peak concentration of inter0.5 h (Sehgal et al., 1975a, 1977; Sehgal and feron mRNA in the presence of DRB, can Tamm, 1976; Table 2) which gives be calculated from equation 2a. At t = 3 hours, ln2 0.693 k,,,=1.4hr-’ L=tl,L=tl, .

culture responds to poly(1) ‘poly(C). It is likely that this assumption is also valid (Kronenberg, 1977; Brown and Simon, 1977). Two further assumptions concerning the synthesis and degradation of interferon mRNA are necessary to define the concentration of mRNA, M, at any time, t. First, that interferon mRNA is synthesized at a constant rate, S,,, for a specified length of time, t’, subsequent to which its synthesis is terminated. Second, that interferon mRNA is subject to a decay process which follows the form of a first order reaction with a rate constant h,,. The rate of change in interferon mRNA concentration is then given by:

1

The assumpt,ions that S,,, and k,,, are constant from t = 0 to t = t’ introduce some error into these calculations. A description of the possible variations in these two parameters during induction may constitute future refinements of the model in equation 2. In general, equation 2a tends to overestimate M at early times prior to t’, particularly in the control curve. If induction is carried out in the presence of DRB, both the synthesis and the degradation of interferon mRNA are affected: 1. If interferon mRNA synthesis in the

[31 0.7 o.l s,“ =

I. C‘

(1 _. e-0.‘3) 2.6

This value is in excellent agreement with the observed 2-3-fold higher peak rate of interferon secretion in the presence of DRB (40 ELM) (cf. Figs. 4 and 5 and also see Sehgal et al., 1975a). Furthermore, the

196

SEHGAL.

LYLFCS.

AND

TAMM

model predicts that the peak rate of secretion should occur at a time = t’ plus the time it takes for newly synthesized interferon mRNA to exit into the cytoplasm, be translated into interferon and for the newly synthesized interferon to be secreted into the culture medium. The observed maximum in the rate of interferon secretion in the presence of DRB occurs approximately 4-5 hr after induction and thus correlates with the termination of interferon mRNA transcription at 3 hr (t’ = 3 hr) (Sehgal et al., 1976b, 1977, and unpublished data). The cumulative yield (I) of interferon is the integral of M over time:

shutoff, would by itself cause only a small (3-4-fold) increase in cumulative yield. On the other hand, a 14-fold increase in the half-life of interferon mRNA with no other variable altered would cause a 12-fold increase in cumulative interferon yield in the first 24 hr and is thus the major contributor to superinduction. The two factors together would produce a 40-50-fold increase. This situation may prevail under the combined protocol using cycloheximide from 0 to 6 hr and DRB from 3 to 24 hr (Sehgal et al., 1976b). With continuous DRB treatment from 0 hr and no cycloheximide, the inhibitory effect of DRB on interferon mRNA synt.hesis reduces the 40-50-fold stimulaI = KJMdt 141 tion to approximately 10. Equations 2 and where K is an arbitrary proportionality con- 5 suggest that if an increase in the duration stant. From equation 2: of transcription (increase in t') occurs in the presence of DRB, it must be small (tl hr) I srn t _ (1 - e-‘d) and is unlikely to play a significant role in for t 5 t’ [5a] z=h,, km superinduction by DRB. This conclusion agrees very closely with the experimental and observation that actinomycin D (5 ag/ml) can be added to DRB-containing, induced 15bl cultures at t = 3 hr without reducing subsequent interferon yields (Sehgal et al., 197613). It is clear from this analysis that superinduction of interferon production by DRB can be explained largely in terms of the increased functional stability of interfor t > t’. For t = 24 hr, with t’ = 3 hr, k,” = 1.4 feron mRNA. hr-‘, kmU = 0.1 hr~‘, S,,,’ = 0.7 S,,,’ and M,, ACKNOWLEDGMICNTS calculated according to equation 2a: We thank IJr. .I. E. I>arnell for helpful discussions

1

I sm t, _ (1- e-‘w’) K=E k,,, I

I I)

7 = 8.7. r’ This value is in agreement with the observed lo-fold increase in cumulative yield over 24 hr in the presence of DRB (cf. Figs. 4 and 5; see also Sehgal et al., 1975a, 197613). Equation 5 underestimates the increase in cumulative interferon yield because of a possible overestimation of M in control cultures at early times as indicated above in discussing equation 2. We can use equation 5 to assess the contribution of each of several variables to interferon superinduction. A 3-4-fold increase in the rate of appearance of interferon mRNA (3-4-fold increase in S,,,, Sehgal and Tamm, manuscript in preparation), with no alteration in the half-life during the

and for poly(lJ)-Sepharose and Drs. S. Sawicki, M. Wilson, and 1’. I’itha for sharing their unpublished data with us. We aiso thank I>rs. S. Koide and L. Burzio for providing us with X. lawns used in this investigation and Mrs. Erna Olhert for teaching us how to operate on and care for the animals. DHB was a gift from Ilr. Arthur F. Wagnrr, Merck Sharp & Dohme Research Laboratories. Hahway, New -Jersey. This investigation was supported by Research Grant CA- 1860X and Program Project Grant CA-IX213 awarded by the National Cancer Institute. P.B.S. is a postdoctoral fellow of the National Cancer Institute and 11.S.L. is a postdoctoral fellow of the National Institute of Allergy and Infectious 1)iseases.

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