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2-5A synthetase activity induced by interferon α, β, and γ in human cell lines differing in their sensitivity to the anticellular and antiviral activities of these interferons
VIROLOGY
117,425-424
(1982)
2-5A Synthetase Activity Induced by Interferon a, 0, and y in Human Ceil Lines Differing in Their Sensitivity to the Anticellular and Antiviral Activities of These lnterferons M. VERHAEGEN-LEWALLE,* T. KUWATA,t Z.-X. ZHANG,* K. CANTELL,O AND J. CONTENT*,l
E. DECLERCQ,$
*D&nartement de Virobgie, Institut Pasteur du Bmbant, Rue du Remorqueur 28, B-1040 Brussels, Belgium, ~Lkpartment of Mtkdkbgp School of Medicine, Chiba University, Chiba 280, Japan, $.Rega Institute for Medical Research, Katholkke Universiteit Leuven, B-2000 L.euven, Belgium, and &%ntral Public Health Luboratwq, Helsinki, Finland Received July 8, 1981; accepted October 28, 1981 Two human cell lines (HEC-1, IF’) which are resistant to the anticellular and/or antiviral action of HuIFN-a or -,9 have been tested with respect to the anticellular and antiviral activities of HuIFN-y. HEC-1 cells were totally resistant to the antiviral activity of the IFN--y, with either EMC or VSV as challenge virus. Per antiviral unit HuIFN--y exerted a much stronger inhibitory effect on the growth of the interferon-sensitive cell line RSa than did IFN-a. IF’ cells were more resistant to the anticellular effect of IFNy than the wild-type cells (RSa), and HEC-1 cells were totally resistant to the anticellular effect of HuIFN-y. 2-5A synthetase was present at a high basic level in untreated HEC1 cells, as previously reported. This activity was not influenced by either HuIFN-fi or y. Similar levels of 2-5A synthetase were induced by HuIFN--y in IF’ and RSa cells; however, this induction was 10 times lower than that obtained for HuIFN-8. A 2’-phosphodiesterase activity was detected in control extracts from RSa and was not induced by either HuIFN-jl or -7. There was no correlation between the amplitude of 2-5A synthetase induction and extent of anticellular effect of either HuIFN-r in different cell lines or of different interferons in a given cell line. Finally, HEC-1 represents the first example of a human cell line which is resistant to all three types of interferon (HuIFNa, -8, and -7).
important role in the cytostatic effect of IFN (for review see Content and Verhaegen-Lewalle, 1981). The evidence in favor of this hypothesis is as follows: (1) The artificial introduction of 2-5A oligonucleotides into permeabilized Daudi cells (Williams and Kerr, 1978) or other cells (Hovanessian et al., 1979; Resinger and Martin, 1980) or the mere incubation of cells with high concentrations of 2-5A oligonucleotide cores (e.g., 2-5A oligonucleotides produced from ATP by the 2-5A synthetase and treated with alkaline phosphatase to remove the 5’-triphosphate) (Kimchi et al., 1979b, 1981) leads to an inhibition of DNA, RNA, and protein synthesis in these cells, concomitantly with the activation of the 2-SA-dependent en-
INTRODUCTION
Interferons (IFN) could be considered as a family of natural pleiotropic mediators of various cell functions (for review see Gresser, 19’77, and Stewart, 1979). Most of these effects, i. e., the cytostatic activity, are still poorly understood in molecular terms, although they have been known for a long time (Paucker et al., 1962) and obtained with both crude and pure interferon (Gresser et al., 1979; Evinger et al., 1989). Recently, several investigators proposed that one of the -interferon-induced, double-stranded (ds) RNA activated enzymes (the 2-5A synthetase) could play an ‘To whom requests for reprints dressed.
should be ad-
425
0042~6g22/82/040425-10$02.00/0 Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
426
VERHAEGEN-LEWALLE
donuclease (Williams et al., 1979; Hovanessian et al., 1979). (2) IFN-treated L cells contain at least 40-fold more 2-5A trimers, tetramers, and trimer cores than untreated cells (Knight et al., 1980), which implies that in some conditions the interferon-induced 2-5A dynthetase can function in the absence of a known source of double-stranded RNA. (3) For murine lymphocyte, 3T3 and BSC-1 cells, the balance between 2-5A synthesis and degradation, as monitored by the cytoplasmic levels of 2-5A synthetase and 2-phosphodiesterase, respectively, strongly correlates with the cell growth rate (Kimchi et al., 1981). However, as recently shown for a human fibroblast cell subline [IF’, originally isolated by Kuwata et al. (1976)], the induction of 2-5A synthetase by interferon is not always correlated with anticellular activity (Vandenbussche et al., 1981). IF’ cells differ from the parental (RSa) cells only by their resistance to the anticellular effect of interferon. In these two cell lines IFN induces similar levels of 2-5A synthetase, and 2-5A similarly activates the BdA-dependent endonuclease. Thus, the induction of the 2-5A pathway may not be a sufficient or universal requirement for the establishment of the anticellular effect of IFN. A similar conclusion could be reached from the results reported by other investigators for other human cell lines, as summarized elsewhere (Vandenbussche et al., 1981; Content and Verhaegen-Lewalle, 1981). Immune IFN (IFN-7) although antigenically and chemically distinct, shares some of the pleiotropic activities of IFN-a and $3 (Epstein, 1979). More interestingly, IFN-r may possess a higher antitumor activity in wivo (Glasgow et al., 1978) and a higher cytostatic activity in vitro (Blalock et al., 1980; Rubin and Gupta, 1980) when compared to IFN-a or -,9 on the same antiviral unit basis. Ankel et al. (1980) have reported that MuIFN-y binds to different gangliosides than MuIFN-ar or -B and that murine leukemia (L1210R) cells, while resistant to the antiviral and anticellular effects of MuIFN-a or -8, are sen-
ET AL.
sitive to the anticellular or antiviral effects of MuIFN-7. Hovanessian et al. (1980b) further established that MuIFNy, like MuIFN-(Y and -& induces a dsRNAdependent 67K protein kinase, a 2-5A synthetase, and several new proteins of unknown function in L929 mouse cells. Finally, MuIFN-y was found to induce significant levels of 67K protein kinase and the 2-5A synthetase activity in both L121OS and L1210R cells (Hovanessian et al., 1980a). We have now explored the antiviral and anticellular activity of HuIFN-7 in two different human cell lines known for their resistance to HuIFN-a and -p: (i) IF’, which is resistant to the anticellular activity of HuIFN-(Y and -@ (Kuwata et al., 1976; Vandenbussche et al., 1981); and (ii) HE&l, a human uterine cervix adenocarcinema-derived cell line, which is resistant to the anticellular and antiviral actions of IFN-a and -/3 and constitutive for both the protein kinase and 2-5A synthetase activities (Verhaegen et al., 1980). We have also measured the amplitude of the 2-5A synthetase activity induced by HuIFN-7, in an attempt to correlate this enzymatic response to the cytostatic activity of interferon. MATERIALS
AND
METHODS
Materials. Human leukocyte interferon (HuIFN-a), human fibroblast interferon (HuIFN-B), and human immune interferon [“Helsinki” HuIFN-7 (produced by human leukocytes stimulated with staphylococcal enterotoxin A) and “Leuven” HuIFN-7 (produced by human splenocytes stimulated with staphylococcal enterotoxin A)] were partially purified preparations. Specific activities of the interferon preparations (HuIFN-(u, HuIFN-8, “Helsinki” HuIFN-7, and “Leuven” HuIFN-7) were 106.‘, 105.‘, 104.‘, and 103.6 U/mg protein, respectively. These values are only indicative since for HuIFNy no international standard exists and the cell-virus system used for our titrations (VSV cytopathogenicity in LR, a strain of trisomic-21 cells) may not have been optimal. Three HuIFN samples, HuIFN-a
2-5A
SYNTHETASE
(BY5 reference units/ml), HuIFN-/3 (lo’.’ reference units/ml), and HuIFN-y (102.’ laboratory units/ml) gave 104.‘, 103.2, and 102.5 U/ml when titered on trisomic-21 cells, as compared to l@.‘, 102.‘, and lo’.’ U/ml, when titered on RSa cells. Thus, although the IFN titers were higher on trisomic-21 than on RSa cells, the relative order of activity of the three HuIFN types remained the same. The “Leuven” HuIFNy had been partially purified on controlled-pore glass bead columns and was cross-checked with the “Helsinki” HuIFNy for anticellular activity and ability to induce 2-5A synthetase. Both HuIFN-r preparations proved equally effective in this regard. Fetal calf serum was obtained from Gibco and bacterial alkaline phosphatase was from Boehringer. [6-‘H]Thymidine (20 Ci/mmol) was purchased from New England Nuclear Corporation (Boston, Massachusetts) and [&“!P]ATP (200 Ci/mmol) from the Radiochemical Centre (Amersham, England). CeU cdtures. HEC-1 cells (Kuramoto et al., 1972), and RSa and IF’ clonal transformed human cells (Kuwata et al., 1976) were grown as monolayers in Eagle’s minimal essential medium (MEM) containing 10% (v/v) fetal calf serum at 37” in a humidified 5% CO2 atmosphere. Confluent cultures were treated with interferon for 20-24 hr, as indicated in the text. Inhibition of vimLs growth. The antiviral activity of human interferons was measured by inhibition of vesicular stomatitis virus (VSV) or encephalomyocarditis (EMC) virus yield, as previously described (Vandenbussche et al., 1981), except that the samples were titrated 18 hr after virus infection. Inhibition of DNA siynthesis in intact cells. The effect of interferon on cell growth was estimated by measuring the inhibition of DNA synthesis. Therefore, cells were cultivated in “Linbro” multiwell plates (24 wells) for 3 days in the presence or absence of interferon. Cell monolayers were washed twice with MEM and incubated in 0.2 ml of MEM containing 1 &i/ml of [63H]thymidine at 37” for 1 hr. The cells were then washed three times with cold
ACTIVITY
427
PBS and detached from plates by trypsinization. After sonication, TCA-precipitable radioactivity was countered in a toluene-based scintillant. RSa and Iii” cell extracts. For enzymatic assays, cells were cultivated in “Sterilin” multisquare wells (4 cm2 wells) and, when confluent, refed with MEM containing varying concentrations of interferons. Extracts were prepared essentially as previously described (Derynck et al., 1980). Cell monolayers were washed three times with cold 35 mM Tris-HCl buffer (pH 7.5) containing 140 mM NaCl. Cells were lysed in 50 ~1 of 20 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (Hepes) buffer (pH 7.4) containing 10 mlM KCl, 1.5 mM Mg(OAc)2, 0.5 m&f dithiothreitol (DTT), 1 mM phenylmethanesulfonyl fluoride, and 0.5% Nonidet (NP-40). After shaking vigorously for 20 min at 0”, the lysates were collected and centrifuged for 20 min at 10,000 g. The supernatants (SlO) were stored in liquid nitrogen for enzyme assays. Aliquots for 2’-phosphodiesterase determinations were prepared as previously described (Verhaegen et al., 1980) and, before freezing, they were adjusted to a final concentration of 7.5% (v/v) glycerol. Protein concentrations were determined by the method of Lowry et al., (1951). Assay of 2’,5’-oligoaden7dEate synthetase in RSa and IF r cells. Seven-microliter aliquots of cell extracts (about 2 mg protein/ ml) were incubated according to Minks et al. (1979) for 2 hr at 30” in a final volume of 12.5 ~1 containing 100 mMKOAc, 25 mM Mg(OAc)2, 10 mM Hepes-KOH (pH 7.4), 5 mM ATP, 4 mM fructose-l,&diphosphate, 1 mM DTT, 20 &ml of poly(1). poly(C) (dsRNA) (when present), and 2.5 &i of lyophilizied [c?P’JATP. The reaction was stopped by heating for 3 min at 95”. The samples were then treated with 150 U/ml of bacterial alkaline phosphatase for 1 hr at 37”. The whole samples were applied to 0.5 ml alumina columns, previously washed five times with 1 ml of 1 M glycine-HCl buffer (pH 2.0; elution buffer) as described by Revel et al. (1981). The 32P-labeled 2-5A was collected by applying 3 ml of elution buffer on the col-
428
VERHAEGEN-LEWALLE
umns and the radioactivity of the eluted samples was measured in the %-channel of a scintillation counter (Cerenkov effect). Assag of %‘-phmphacG&mme. The highly active (2’-5’)oligoisoadenylate synthetase (SlO) from interferon-treated HeLa cells was incubated in the presence of [cyq)ATP as previously described (Verhaegen et al., 1980). The =P-labeled 2-5A was isolated by DEAE-cellulose (Whatman DE-52) chromatography and concentrated by acetone precipitation. The pellet was resuspended in a minimum volume of water and this 2-5A preparation was used as substrate for the 2-phosphodiesterase assay. About 100,000 cpm of 2-5A were incubated in the presence of 4-~1 aliquots of SlO extract (5-10 mg protein/ml) or lysis buffer (control) for 1 or 2 hr at 30” in a final volume of 8 ~1 containing 100 mM KOAc, 25 mM Mg(OAc)a, 20 mM HepesKOH (pH 7.4), and 1 mM DTT. The reaction was stopped by heating for 3 min at 95”. Two-microliter aliquots were applied to thin-layer sheets of polyethyleneimine (Machery-Nagel) and chromatographed in 1 M acetic acid as previously described (Verhaegen et al., 1980). Thereafter, the sheets were submitted to autoradiography. RESULTS
Establishment
of an Antiviral
State by Hu-
man LY,p, and y Interferon Prepara-
tions HuIFN-a and HuIFN-/3 are equally effective in establishing the antiviral state in both RSa and IF’ cells (Kuwata et at., 1979). In the present study, we compared the ability of HuIFN-a and HuIFN-y to inhibit virus growth in RSa, IF’, and HEC1 cells (Fig. 1). The amounts of vesicular stomatitis virus (VSV) formed in the yieldreduction test on RSa cells were reduced by about 4.8 log and 2.8 log when the cells were treated with 100 U/ml of IFN-a or IFN-7, respectively. Likewise, HuIFN-7 inhibited the growth of encephalomyocarditis (EMC) virus in RSa cells (data not shown). Both HuIFN-CY and HuIFN-y also inhibited VSV growth in IF’ cells (Fig. I)
ET AL.
2
RSa
1 0
z-3-
FIG. 1. Antiviral activity of HuIFN-cy and HuIFNy in RSa, IF’, and HEC-1 cells. The inhibition of VSV growth by varying amounts of HuIFN-a! (0 0) or HuIFN-y (0 l ) was measured as described under Materials,and Methods. The VSV yield is expressed as percentage of virus yield (log) in control (without interferon treatment).
although the effect was less important than in the case of RSa cells. However, HuIFN-7 proved ineffective in inhibiting either VSV (Fig. 1) or EMC virus (not shown) in HEC-1 cells. The HEC-1 cell line has previously been found resistant to the antiviral action of HuIFN-8 (Verhaegen et al., 1980) and HuIFN-a (Chen et al., 1981) with either VSV, Sindbis, Vaccinia, or EMC virus as the challenge virus. It now appears that HEC-1 cells are also resistant to the antiviral action of HuIFN-7. Cell Growth Inhibition bg Human cx,P, and y Interferon Preparations Whereas RSa and IF’ cells are both sensitive to the antiviral action of interferons, they differ in their sensitivity to the anticellular actions of HuIFN-a and HuIFN/3 (Kuwata et al., 1979; Vandenbussche et al., 1981). On one hand, RSa cell growth is strongly inhibited by HuIFN-a, and even more so by HuIFN-P. On the other hand,
2-5A SYNTHETASE
429
ACTIVITY
sidered as sensitive to the anticellular action of HuIFN-7. The situation is quite different for HEC-1 cells which appeared to be resistant to the anticellular activity of HuIFN-7 (Fig. 2). Induction of 2-5A Synthetase by Human (Y,p, and y Interferon Preparations Both HuIFN-(U and HuIFN-P induce 25A synthetase activity in RSa and IF’ cells (Vandenbussche et al., 1981). 2-5A Synthetase is also inducible by HuIFN-y although the induction requires much higher doses of HuIFN--r than of HuIFN-/3 (Fig. 3). Moreover, the amplitude of the 2-5A synthetase activity induced by HuIFN-7 is much lower than that induced by HuIFN-@ (Fig. 3). In view of the possible
101~ "
1 100
1 10 lnlarfaron
1
1000
RSa
(U/ml)
FIG. 2. Anticellular activity of HuIFN-cy andHuIFNy in RSa, IF’, and HEC-1 cells. The inhibition of [aH]thymidine incorporation by varying amounts of HuIFN-(U (00) or HuIFN-y (0 0) was measured as described under Materials and Methods. The thymidine incorporation is expressed as a percentage of thymidine incorporation in control (without interferon treatment).
IF’ cells are relatively resistant to the anticellular activity of HuIFN-(I! and HuIFNp (Kuwata et al., 1979). We have compared the anticellular effects of HuIFN-(r and HuIFN-7 on RSa, IF’, and HEC-1 cells (which are resistant to the anticellular action of HuIFN-cx and HuIFN-/3 (Chen et al., 1981)]. HuIFN-r appeared to be more potent than HuIFN-(r in inhibiting the growth of RSa cells (Fig. 2). HuIFN-y proved also inhibitory to the growth of IF’ cells (Fig. 2), although much less so than to RSa cells. For HuIEN-a a dose of 1000 U/ml was required to inhibit IF’ cell growth by 50%, whereas 100 U/ml of HuIFN-y inhibited the growth of these cells to about 20% of the control. Although HuIFN--y was more inhibitory for RSa than for IF’ cells, the latter can be con-
IFr
; 8
lo-
5-
OC 0
0.1
1
Interferon
,/
100
10
100
0
(U/ml)
FIG. 3. Induction of 2-5A synthetase activity by HuIFN-j3 and HuIFN-y in RSa and IF’ cells. Cells were treated for 2.4 hr with varying amounts of HuIFN+(O __ 0) or HuIFN--y (0 0). The protein content in the assays was 14 pg, and 2-5A synthetase activity is expressed as radioactivity incorporated into [32p)2-5A oligomers (as described under Materials and Methods).
430
VERHAEGEN-LEWALLE
heterogeneity between human y interferons produced by leukocytes or splenocytes, we have determined the 2-5A synthetaseinducing ability with both “Leuven” and “Helsinki” HuIFN-y. For both y-interferons the level of 2-5A synthetase activity induced was 5- to lo-fold lower than that obtained following induction by HuIFNp at the same concentration (i.e., 200 U/ ml). From an inspection of the dose-response curves of HuIFN-8 and HuIFN-7 (Fig. 3), we could exclude the possibility that our HuIFN-y preparation contained a HuIFN-P contaminant at a concentration of 81% (De Ley et al., 1980). On the other hand, HuIFN-7 did not affect the level of 2-5A synthetase in HE&l cells (Table 1). However, it should be pointed out that HE&l cells contain high levels of 2-5A synthetase and 73K protein kinase and these levels are not enhanced by treatment with either HuIFN-a or HuIFN-8 (Verhaegen et al., 1980). Finally, we have observed that 2-5A synthetase of HeLa cells is equally inducible by HuIFN-7 and HuIFN-B (data not shown), indicating that our HuIFN-y preparations are fully capable of inducing 2-5A synthetase activity in susceptible cells. d’-Phosphodiesterase Levels in Cells Treated with Human /3 and y Intwfkrcm Prep arations In view of the comparison of the antiviral, anticellular activities and 2-5A synthetase-inducing ability of HuIFN-/3 and HuIFN--y, it was important to know whether the products of the 2-5A synthetase reaction were not degraded differently in cells treated with either HuIFNp or HuIFN-y. Therefore, we determined the degradation of 2-5A by 2’-phosphodiesterase in RSa cells that had not been treated or treated with HuIFN-P or HuIFN-y. The rate of degradation of 32Plabeled 2-5A, as measured either by the disappearance of [“2P]2-5A tri- or tetramer or by the apparition of r2P]AMP, was identical for all three extracts (data not shown) and the amount of released FPjAMP was a function of the protein concentration in the assay (Fig. 4). As
ET AL. TABLE
1
EFFECT OF INTERFERON-~ AND -7 ON 2-5A SYNTHETASE ACTIVITY IN HE&l CELLS= 3H cpm Interferon
No dsRNA
With dsRNA
Ab
0 j3 (200 U/ml) y (200 U/ml)
903 905 967
19,395 17,159 19,525
18,492 16,254 18,558
“Cells were treated for 24 hr with the indicated interferon. Methodology for cell extract preparation and enzyme assay was as previously described (Verhaegen et al., 1980). The incubations were carried out for 2 hr. The results are expressed as means from duplicate assays containing 225 pg of protein. b A = activity with dsRNA - activity without dsRNA.
shown in Fig. 4, neither HuIFN-/3 nor HuIFN-y increased or decreased 2-phosphodiesterase activity in RSa cells. Similar results were obtained with IF’ cells. Thus, the lower levels of 2-5A synthesized in response to HuIFN-7 than to HuIFNp do not result from an increased 2’-phosphodiesterase activity. Furthermore, in the conditions of the 2-5A synthetase assay (presence of 5 mM ATP and 4 mMfructose 1,6-diphosphate), we observed that the 2’-phosphodiesterase activity was completely blocked in both control and interferon-treated RSa cells (data not shown). DISCUSSION
We have compared three different human cell lines for their sensitivity to the anticellular and antiviral effects of IFN (Table 2). The HEC-1 cell line was completely resistant to the anticellular and antiviral activities of HuIFN-(u, +3, and -7. The RSa cell line was sensitive to both the antiviral and anticellular activities of HuIFN-CZ, -8, and -7, although the anticellular effect of HuIFN-y was about lofold more pronounced than that of HuIFNa! or -& if compared at an equivalent antiviral titer. This confirms the recent observations from Blalock et al. (1980) and Rubin and Gupta (1980). The IF’ cells were resistant to the anticellular activities of
2-5A SYNTHETASE
no
0
treatment
treatment
0.5
1
2.5
5
0
0.5
1
431
ACTIVITY
2.5
with
5
0
05
1
2.5
5
mg pmt. / ml extract FIG. 4. Dependence of Z-phosphodiesterase activity on cell extract concentration for untreated RSa cells, and RSa cells treated HuIFN-/3 or HuIFN-7. Interferon treatment was at 200 units/ml for 24 hr. The incubations were carried out for 1 hr as described under Materials and Methods. The spots corresponding to released $PwAMP were separated by thin-layer chromatography on polyethyleneimine-cellulose, visualized by autoradiography, and their radioactivity was measured in a scintillation counter.
IFN-(r or -@, yet responsive to the anticellular action of HuIFN-7 (Table 2). Table 2 also presents the levels of 2-5A synthetase induced in the different cell lines after treatment with HuIFN-(w, -8, or -7. Two major conclusions become apparent if the anticellular effects of HuIFN-or, -8, and -y are compared to their potentials to induce 2-5A synthetase activity: (1) RSa cells are much more sensitive to the anticellular action of IFN-cu or -/3
than IF’ cells; however, they acquire similar levels of 2-5A synthetase upon treatment with HuIFN-a or -/3 (Vandenbussche et al., 1981). (2) Although the anticellular activity of HuIFN-y is more pronounced than that of HuIFN-CY or -6 in both RSa and IF’ cells, HuIFN-r is clearly less efficient than HuIFN-a or -/3 in inducing 2-5A synthetase activity in these cells. Baglioni and Maroney (1980) have shown
432
VERI-IAEGEN-LEWALLE TABLE
ET AL. 2
EFFECTS OF (Y,P, AND y INTERFERON% SUMMARY Cell type RSa
IF’
Anticellular action*
I (Y
++d,e.h
+ +d+h
1
B
++=
++esh
11 Y
+d
+++d
+d ++h
IFN type
++”
++d,f,h
I
a
++d,e,h
&”
1
B
++e
f ”
Y
td
+td
-da
-da
-f@
-0
*d,f,i
-d
-d
*d.i
11
HEC-1
(2-5’)oligo A synthetase activity”
Antiviral action”
I a 1 B 11
Y
“Measured by VSV yield reduction (log,,) at 100 U/ml: ++ > 3.5 > + > 1 > - = 0. *Estimated by inhibition of pH]thymidine incorporation (percentage of incorporation 100U/ml:+++>95>++>60>+>40>~>10~-. ‘Estimated by an increase (fold) in activity at 500 U/ml: ++ z 10 > + > 3. d This report. ’ Kuwata et al. (1979). f Verhaegen et al. (1980). o Chen et al. (1981). h Vandenbussche et aE. (1981). i Constitutive activity, not enhanced by interferon treatment.
that the induction of the 2-5A synthetase by HuIFN-y in HeLa cells proceeds more slowly than with HuIFN-P, although the amplitude of the induction is similar. This notion cannot explain the weak 2-5A synthetase-inducing effect of IFN-7 in our cell systems since our cells were invariably treated with IFN for 24 hr. We can exclude the possibility that the low 2-5A synthetase levels observed in RSa and IF’ cells resulted from an artefact related to a stronger induction of 2’-phosphodiesterase by IFN--y, since (i) all extracts from either untreated or IFN-treated cells contained 2’-phosphodiesterase activity; and (ii) this activity was not induced after treatment with either IFN-/3 or-y (Fig. 4), in contrast with the observations of Kimchi et al. (1979a). (iii) In agreement with Minks et al. (1979), we found that the 2’-phosphodiesterase activity was completely blocked in the conditions of the 2-5A synthetase reaction (M. Verhaegen-Lewalle, unpublished data). HEC-1 cells proved totally refractory to
++d,f,h 4
in control ceils) at
the antiviral and anticellular actions of HuIFN-7 (Table 2). These findings can be regarded as an extension of our earlier observations with IFN-a and -/I in HEC1 cells (Verhaegen et al., 1980). The situation here is different from that in murine L1210R leukemia cells. These cells may lack (part of) the receptor for MuIFN-a and -/3 (Aguet, 1980). Since L1210R cells still respond to MuIFN-y (Ankel et al., 1980; Hovanessian et al., 1980a) one may assume that the ganglioside component (Ankel et al., 1980) of the murine cell receptor for MuIFN-y differs from the ganglioside component of the MuIFN-a and -0 receptor. Preliminary studies indicate a decreased binding of IFN-a and -/3 to HEC-1 cells (T. Kuwata, unpublished data). If this is the case and if human cells, like murine cells, contain separate receptors for HuIFN-y and HuIFN-a,@, then HEC1 cells may have lost either (i) the two kinds of receptors; (ii) a common component of the two receptors; (iii) any other regulatory or nonregulatory pathway that
25A SYNTHETASE
may be important for the control and the expression of the anticellular and antiviral effects of IFN-a, -p, and -y (Verhaegen et al., 1980). To our knowledge there are no quantitative data available on the number of different IFN receptors in human cells. Indirect evidence for the existence of different receptors for HuIFN-7 and HuIFN-a,B stems from the recent observations of Fleischman et al. (1979) .and De Ley et al. (1980) indicating a possible synergy in the antiviral actions of IFN-/3 and -7. The occurrence of tumor cells, like HEC1, which are completely refractory to any type of interferon, bears on the eventual clinical utility of interferon in the treatment of cancer. Our results point to the total absence of correlation between the anticellular effects of HuIFN-a, -0, and y and their capacity to induce 2-5A synthetase activity in RSa and IF’ cells. Whether this discordance is a general rule or an exception to the rule remains to be elucidated. It has been recently observed that HuIFN-a! and HuIFN-/3 induce high 2-5A synthetase levels in Raji cells, a human lymphoblastoid cell line which is virtually refractory to the anticellular action of IFN (Y. Tomita et al., personal communication). Other examples of such a discrepancy between the actions of interferon in human cell lines have been described by Meurs et al. (1981). The results presented here emphasize the need for further research on the molecular basis of IFN action and particularly the anticellular action of IFN-7. It would seem worthwhile to investigate alternative pathways responsible for the expression of the anticellular effect of IFN in these cells where 2-5A either does not seem to play a role or does not suffice to account for the anticellular action of IFN. ACKNOWLEDGMENTS We thank Dr. R. Derynck, Dr. R. Devos, and Prof. W. Fiers (Laboratory of Molecular Biology, University of Ghent) for the gift of crude HuIFN-y, Dr. J. Van Damme and Prof. A. Billiau (Rega Institute for Medical Research, University of Leuven) for the gift of HuIFN-fi, and Prof. J.-J. Cassiman (Division of Human Genetics, University of Leuven) for providing
433
ACTIVITY
the trisomic-21 cell strain LR. This work was supported by Grant 29606.79 from the Fonda de la Recherche Fondamentale Collective (Belgium) and, in part, by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture (Japan). REFERENCES AGUET, M. (1980). High-affinity binding of ‘%I-labelled mouse interferon to a specific cell surface receptor. Nature (London) 284.459-461. ANKU, H., KRISHNAMURTI, C., BESANCON, F., STEFANOS, S., and FALCOFF, E. (1980). Mouse fibroblast (type I) and immune (type II) interferons: Pronounced differences in affinity for gangliosides and in antiviral and antigrowth effects on mouse leukemia L-1210R cells. Proc. Nat Ad Sci USA 77, 2528-2532. BAGLIONI, C., and MARONEY, P. A. (1980). Mechanism of action of human interferons. Induction of 2’5’oligo(A) polymerase. J. Bid Chem 256,&390&X93. BLALOCK, J. E., GEORGIADES, J. A., LANGFORD, M. P., and JOHNSON, H. M. (1989). Purified human immune interferon has more potent anticellular activity than fibroblast or leukocyte interferon. CeU. Immud
49,390-394.
CHEN, H. Y., SAG, T., FUSE, A., KUWATA, T., and CONTENT, J. (1981). Resistance to interferon of a human adenocarinoma cell line, HEC-1, and its sensitivity to natural killer cell action. J. Gen virol. 52,177-181. CONTENT, J., and VERHAEGEN-LEWALLE, M. (1982). Molecular basis of interferon action. In “Intracellular Signals, Control of Growth and Differentiation,” (J. E. Dumont, ed.), pp. 275-298. Plenum, London. DE LEY, M., VAN DAMME, J., CLAEYS, H., WEENING, H., HEINE, J. W., BILLIAU, A., VERMYLEN, C., and DE SOMER, P. (1980). Interferon induced in human leukocytes by mitogens: Production, partial purification and characterization. Eur. J. Immunol 10, 877-883. DERYNCK, R., REMAUT, E., SANIAN, E., STANSSENS,P., DE CLERCQ,E., CONTENT,J., and FIERS, W. (1980). Expression of human fibroblast interferon gene in Escherichia
coli
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287,193-197.
EPSTEIN, L. B. (1979). The comparative biology of immune and classical interferons. In “Biology of the Lymphokines” (S. Cohen, E. Pick, and J. J. Oppenheim, eds.), pp. 443-514. Academic Press, New York. EVINGER, M., RUBINSTEIN, M., and PESTKA, S. (1986). Growth-inhibitory and antiviral activity of purified leukocyte interferon. Ann. N. I: Acad. Sci 350, 399-404.
FLEISCHMANN, W. R., JR., GEORGIADES, J. A., OsBORNE, L. C., and JOHNSON, H. M. (1979). Poten-
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tiation of interferon activity by mixed preparations of fibroblast and immune interferon. I$ect. Immun. 26,248~253. GLASGOW, L. A., CRANE, J. L., KERN, E. R., and YOUNGNER, J. S. (1978). Antitumor activity of interferon against murine osteogenic sarcoma in vitro and in vivo. Cancer !&eat. Rep 62, 1881-1888. GRESSER, I. (1977). On the varied biologic effects of interferon. CeU. Immurwl 34,406-416. GRESSER, I., DE MAEYER-GUIGNARD, J., TOVEY, M. G., and DE MAEYER, E. (1979). Electrophoretitally pure mouse interferon exerts multiple biologic effects. Proc Nat. Acud Sti USA 76, 53085312. HOVANESSIAN, A. G., LA BONNARDIERE, C., and FALCOFF, E. (1980a). Action of murine y (immune) interferon on fi (fibroblast) interferon resistant L1210 and embryonal carcinoma cells. J. Interferon Res. 1,X&135. HOVANESSIAN, A. G., MEURS, E., AUJEAN, O., VAQUERO,C., STEFANOS, S., and FALCOFF, E. (198Ob). Antiviral response and induction of specific proteins in cells treated with immune T (type II) interferon analogous to that from viral interferon (type I)-treated cells. virology 104, 195-204. H~VANESSIAN, A. G., WOOD, J., MEURS, E., and MONTAGNIER, L. (1979). Increased nuclease activity in cells treated with pppAZp5’ABp5’A. Proc Nat. Acad Sci USA 76,3261-3265. KIMCHI, A., SHULMAN, L., SCHMIDT, A., CHERNAJOVSKY, Y., FRADIN, A., and REVEL, M. (1979a). Kinetics of the induction of three translation-regulatory enzymes by interferon. Proc Nat. Acad Sti USA 76, 3208-3212. KIMCHI, A., SHURE, H., and REVEL, M. (1979b). Regulation of lymphocyte mitogenesis by (2-5’)oligoisoadenylate. Nature (London) 282,849-851. KIMCHI, A., SHURE, H., and REVEL, M. (1981). Antimitogenic function of interferon-induced (Z5’)oligo(adenylate) and growth-related variations in enzymes that synthesize and degrade this oligonucleotide. Eur. J. Biohem 114,5-10. KNIGHT, M., CAYLEY, P. J., SILVERMAN, R. H., WRESCHNER, D. H., GILBERT, C. S., BROWN, R. E., and KERR, I. M. (1980). Radioimmune, radiobinding and HPLC analysis of 2-5A and related oligonucleotides from intact cells. Nature (Lund& 288, 189192. KURAMOTO, H., TAMURA, S., and NOTAKE, Y. (1972). Establishment of a cell line of human endometrial adenocarinoma in vitro. Aw. J. Ob.stet. Gyn.ecoL 114,1012-1019. KUWATA, T., FUSE, A., and MORINAGA, N. (1976). Effects of interferon on cell and virus growth in transformed human cell lines. J. Gen ViroL 33,715. KUWATA, T., FUSE, A., SUZUKI, N., and MORINAGA, N. (1979). Comparison of the suppression of cell and virus growth in transformed human cells by
ET AL;
leukocyte and fibroblast interferon. J. Gen ViroL 43,435-439. LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. BioL Chem 193,265275. MEURS, E., HOVANESSIAN, A. G., and MONTAGNIER, L. (1981). Interferon-mediated antiviral state in human MRC5 cells in the absence of detectable levels of 2-5A synthetase and protein kinase. J. Inte&vm Res. 1,219-232. MINKS, M. A., BENVIN, S., MARONEY, P. A., and BAGLIONI, C. (1979). Synthesis of 2,5’-oligo(A) in extracts of interferon-treated HeLa cells. J. Bid Ch.em 254, 5058-5064. PAUCKER, K., CANTELL, K., and HENLE, W. (1962). Quantitative studies on viral interference in suspended L cells. III. Effect of interfering viruses and interferon on the growth rate of cells. Virology 17, 324-334. RESINGER, D. M., and MARTIN, E. M. (1980). The effect of interferon and AZp5’ AZp5’A on the growth of Daudi cells. Proceedings of the International Workshop on Interferons, held at the Memorial Sloan-Kettering Center (New York) on April 2224, 1979. REVEL, M., WALLACH, D., MERLIN, G., SCHATTNER, A., SCHMIDT, A., WOLF, D., SHULMAN, L., and KIMCHI, A. (1981). Interferon-induced enzymes: Microassays and their applications. In “Methods in Enzymology.” Academic Press, New York, in press. RUBIN, B. Y., and GUPTA, S. L. (1980). Differential efficacies of human type I and type II interferons as antiviral and antiproliferative agents. Proc Nat. Acad Sci. USA 77,5928-5932. STEWART, W. E., II (1979). Varied biologic effects of interferon. In “Interferon 1” (I. Gresser, ed.), pp. 29-51. Academic Press, New York. VANDENBUSSCHE, P., DIVIZIA, M., VERHAEGEN-LEWALLE, M., FUSE, A., KUWATA, T., DE CLERCQ, E., and CONTENT, J. (1981). Enzymatic activities induced by interferon in human fibroblast cell lines differing in their sensitivity to the anticellular activity of interferon. virology 111. 11-22. VERHAEGEN, I+., DIVIZIA, M., VANDENBUSSCHE, P., KUWATA, T., and CONTENT, J. (1980). Abnormal behavior of interferon-induced enzymatic activities in an interferon-resistant cell line. Proc Nat. Acad Sk USA 77,4479-4483. WILLIAMS, B. R. G., GILBERT, C. S., and KERR, I. M. (1979). The respective roles of the protein kinase and pppAZp5’A2’p5’A-activated endonuclease in the inhibition of protein synthesis by doublestranded RNA in rabbit reticulocyte lysates. Nucleic Acids Res. 6, 1335-1350. WILLIAMS, B. R. G., and KERR, I. M. (1978). Inhibition of protein synthesis by 2-5 linked adenine oligonucleotides in intact cells. Nature (London) 276,8890.
117,425-424
(1982)
2-5A Synthetase Activity Induced by Interferon a, 0, and y in Human Ceil Lines Differing in Their Sensitivity to the Anticellular and Antiviral Activities of These lnterferons M. VERHAEGEN-LEWALLE,* T. KUWATA,t Z.-X. ZHANG,* K. CANTELL,O AND J. CONTENT*,l
E. DECLERCQ,$
*D&nartement de Virobgie, Institut Pasteur du Bmbant, Rue du Remorqueur 28, B-1040 Brussels, Belgium, ~Lkpartment of Mtkdkbgp School of Medicine, Chiba University, Chiba 280, Japan, $.Rega Institute for Medical Research, Katholkke Universiteit Leuven, B-2000 L.euven, Belgium, and &%ntral Public Health Luboratwq, Helsinki, Finland Received July 8, 1981; accepted October 28, 1981 Two human cell lines (HEC-1, IF’) which are resistant to the anticellular and/or antiviral action of HuIFN-a or -,9 have been tested with respect to the anticellular and antiviral activities of HuIFN-y. HEC-1 cells were totally resistant to the antiviral activity of the IFN--y, with either EMC or VSV as challenge virus. Per antiviral unit HuIFN--y exerted a much stronger inhibitory effect on the growth of the interferon-sensitive cell line RSa than did IFN-a. IF’ cells were more resistant to the anticellular effect of IFNy than the wild-type cells (RSa), and HEC-1 cells were totally resistant to the anticellular effect of HuIFN-y. 2-5A synthetase was present at a high basic level in untreated HEC1 cells, as previously reported. This activity was not influenced by either HuIFN-fi or y. Similar levels of 2-5A synthetase were induced by HuIFN--y in IF’ and RSa cells; however, this induction was 10 times lower than that obtained for HuIFN-8. A 2’-phosphodiesterase activity was detected in control extracts from RSa and was not induced by either HuIFN-jl or -7. There was no correlation between the amplitude of 2-5A synthetase induction and extent of anticellular effect of either HuIFN-r in different cell lines or of different interferons in a given cell line. Finally, HEC-1 represents the first example of a human cell line which is resistant to all three types of interferon (HuIFNa, -8, and -7).
important role in the cytostatic effect of IFN (for review see Content and Verhaegen-Lewalle, 1981). The evidence in favor of this hypothesis is as follows: (1) The artificial introduction of 2-5A oligonucleotides into permeabilized Daudi cells (Williams and Kerr, 1978) or other cells (Hovanessian et al., 1979; Resinger and Martin, 1980) or the mere incubation of cells with high concentrations of 2-5A oligonucleotide cores (e.g., 2-5A oligonucleotides produced from ATP by the 2-5A synthetase and treated with alkaline phosphatase to remove the 5’-triphosphate) (Kimchi et al., 1979b, 1981) leads to an inhibition of DNA, RNA, and protein synthesis in these cells, concomitantly with the activation of the 2-SA-dependent en-
INTRODUCTION
Interferons (IFN) could be considered as a family of natural pleiotropic mediators of various cell functions (for review see Gresser, 19’77, and Stewart, 1979). Most of these effects, i. e., the cytostatic activity, are still poorly understood in molecular terms, although they have been known for a long time (Paucker et al., 1962) and obtained with both crude and pure interferon (Gresser et al., 1979; Evinger et al., 1989). Recently, several investigators proposed that one of the -interferon-induced, double-stranded (ds) RNA activated enzymes (the 2-5A synthetase) could play an ‘To whom requests for reprints dressed.
should be ad-
425
0042~6g22/82/040425-10$02.00/0 Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
426
VERHAEGEN-LEWALLE
donuclease (Williams et al., 1979; Hovanessian et al., 1979). (2) IFN-treated L cells contain at least 40-fold more 2-5A trimers, tetramers, and trimer cores than untreated cells (Knight et al., 1980), which implies that in some conditions the interferon-induced 2-5A dynthetase can function in the absence of a known source of double-stranded RNA. (3) For murine lymphocyte, 3T3 and BSC-1 cells, the balance between 2-5A synthesis and degradation, as monitored by the cytoplasmic levels of 2-5A synthetase and 2-phosphodiesterase, respectively, strongly correlates with the cell growth rate (Kimchi et al., 1981). However, as recently shown for a human fibroblast cell subline [IF’, originally isolated by Kuwata et al. (1976)], the induction of 2-5A synthetase by interferon is not always correlated with anticellular activity (Vandenbussche et al., 1981). IF’ cells differ from the parental (RSa) cells only by their resistance to the anticellular effect of interferon. In these two cell lines IFN induces similar levels of 2-5A synthetase, and 2-5A similarly activates the BdA-dependent endonuclease. Thus, the induction of the 2-5A pathway may not be a sufficient or universal requirement for the establishment of the anticellular effect of IFN. A similar conclusion could be reached from the results reported by other investigators for other human cell lines, as summarized elsewhere (Vandenbussche et al., 1981; Content and Verhaegen-Lewalle, 1981). Immune IFN (IFN-7) although antigenically and chemically distinct, shares some of the pleiotropic activities of IFN-a and $3 (Epstein, 1979). More interestingly, IFN-r may possess a higher antitumor activity in wivo (Glasgow et al., 1978) and a higher cytostatic activity in vitro (Blalock et al., 1980; Rubin and Gupta, 1980) when compared to IFN-a or -,9 on the same antiviral unit basis. Ankel et al. (1980) have reported that MuIFN-y binds to different gangliosides than MuIFN-ar or -B and that murine leukemia (L1210R) cells, while resistant to the antiviral and anticellular effects of MuIFN-a or -8, are sen-
ET AL.
sitive to the anticellular or antiviral effects of MuIFN-7. Hovanessian et al. (1980b) further established that MuIFNy, like MuIFN-(Y and -& induces a dsRNAdependent 67K protein kinase, a 2-5A synthetase, and several new proteins of unknown function in L929 mouse cells. Finally, MuIFN-y was found to induce significant levels of 67K protein kinase and the 2-5A synthetase activity in both L121OS and L1210R cells (Hovanessian et al., 1980a). We have now explored the antiviral and anticellular activity of HuIFN-7 in two different human cell lines known for their resistance to HuIFN-a and -p: (i) IF’, which is resistant to the anticellular activity of HuIFN-(Y and -@ (Kuwata et al., 1976; Vandenbussche et al., 1981); and (ii) HE&l, a human uterine cervix adenocarcinema-derived cell line, which is resistant to the anticellular and antiviral actions of IFN-a and -/3 and constitutive for both the protein kinase and 2-5A synthetase activities (Verhaegen et al., 1980). We have also measured the amplitude of the 2-5A synthetase activity induced by HuIFN-7, in an attempt to correlate this enzymatic response to the cytostatic activity of interferon. MATERIALS
AND
METHODS
Materials. Human leukocyte interferon (HuIFN-a), human fibroblast interferon (HuIFN-B), and human immune interferon [“Helsinki” HuIFN-7 (produced by human leukocytes stimulated with staphylococcal enterotoxin A) and “Leuven” HuIFN-7 (produced by human splenocytes stimulated with staphylococcal enterotoxin A)] were partially purified preparations. Specific activities of the interferon preparations (HuIFN-(u, HuIFN-8, “Helsinki” HuIFN-7, and “Leuven” HuIFN-7) were 106.‘, 105.‘, 104.‘, and 103.6 U/mg protein, respectively. These values are only indicative since for HuIFNy no international standard exists and the cell-virus system used for our titrations (VSV cytopathogenicity in LR, a strain of trisomic-21 cells) may not have been optimal. Three HuIFN samples, HuIFN-a
2-5A
SYNTHETASE
(BY5 reference units/ml), HuIFN-/3 (lo’.’ reference units/ml), and HuIFN-y (102.’ laboratory units/ml) gave 104.‘, 103.2, and 102.5 U/ml when titered on trisomic-21 cells, as compared to l@.‘, 102.‘, and lo’.’ U/ml, when titered on RSa cells. Thus, although the IFN titers were higher on trisomic-21 than on RSa cells, the relative order of activity of the three HuIFN types remained the same. The “Leuven” HuIFNy had been partially purified on controlled-pore glass bead columns and was cross-checked with the “Helsinki” HuIFNy for anticellular activity and ability to induce 2-5A synthetase. Both HuIFN-r preparations proved equally effective in this regard. Fetal calf serum was obtained from Gibco and bacterial alkaline phosphatase was from Boehringer. [6-‘H]Thymidine (20 Ci/mmol) was purchased from New England Nuclear Corporation (Boston, Massachusetts) and [&“!P]ATP (200 Ci/mmol) from the Radiochemical Centre (Amersham, England). CeU cdtures. HEC-1 cells (Kuramoto et al., 1972), and RSa and IF’ clonal transformed human cells (Kuwata et al., 1976) were grown as monolayers in Eagle’s minimal essential medium (MEM) containing 10% (v/v) fetal calf serum at 37” in a humidified 5% CO2 atmosphere. Confluent cultures were treated with interferon for 20-24 hr, as indicated in the text. Inhibition of vimLs growth. The antiviral activity of human interferons was measured by inhibition of vesicular stomatitis virus (VSV) or encephalomyocarditis (EMC) virus yield, as previously described (Vandenbussche et al., 1981), except that the samples were titrated 18 hr after virus infection. Inhibition of DNA siynthesis in intact cells. The effect of interferon on cell growth was estimated by measuring the inhibition of DNA synthesis. Therefore, cells were cultivated in “Linbro” multiwell plates (24 wells) for 3 days in the presence or absence of interferon. Cell monolayers were washed twice with MEM and incubated in 0.2 ml of MEM containing 1 &i/ml of [63H]thymidine at 37” for 1 hr. The cells were then washed three times with cold
ACTIVITY
427
PBS and detached from plates by trypsinization. After sonication, TCA-precipitable radioactivity was countered in a toluene-based scintillant. RSa and Iii” cell extracts. For enzymatic assays, cells were cultivated in “Sterilin” multisquare wells (4 cm2 wells) and, when confluent, refed with MEM containing varying concentrations of interferons. Extracts were prepared essentially as previously described (Derynck et al., 1980). Cell monolayers were washed three times with cold 35 mM Tris-HCl buffer (pH 7.5) containing 140 mM NaCl. Cells were lysed in 50 ~1 of 20 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (Hepes) buffer (pH 7.4) containing 10 mlM KCl, 1.5 mM Mg(OAc)2, 0.5 m&f dithiothreitol (DTT), 1 mM phenylmethanesulfonyl fluoride, and 0.5% Nonidet (NP-40). After shaking vigorously for 20 min at 0”, the lysates were collected and centrifuged for 20 min at 10,000 g. The supernatants (SlO) were stored in liquid nitrogen for enzyme assays. Aliquots for 2’-phosphodiesterase determinations were prepared as previously described (Verhaegen et al., 1980) and, before freezing, they were adjusted to a final concentration of 7.5% (v/v) glycerol. Protein concentrations were determined by the method of Lowry et al., (1951). Assay of 2’,5’-oligoaden7dEate synthetase in RSa and IF r cells. Seven-microliter aliquots of cell extracts (about 2 mg protein/ ml) were incubated according to Minks et al. (1979) for 2 hr at 30” in a final volume of 12.5 ~1 containing 100 mMKOAc, 25 mM Mg(OAc)2, 10 mM Hepes-KOH (pH 7.4), 5 mM ATP, 4 mM fructose-l,&diphosphate, 1 mM DTT, 20 &ml of poly(1). poly(C) (dsRNA) (when present), and 2.5 &i of lyophilizied [c?P’JATP. The reaction was stopped by heating for 3 min at 95”. The samples were then treated with 150 U/ml of bacterial alkaline phosphatase for 1 hr at 37”. The whole samples were applied to 0.5 ml alumina columns, previously washed five times with 1 ml of 1 M glycine-HCl buffer (pH 2.0; elution buffer) as described by Revel et al. (1981). The 32P-labeled 2-5A was collected by applying 3 ml of elution buffer on the col-
428
VERHAEGEN-LEWALLE
umns and the radioactivity of the eluted samples was measured in the %-channel of a scintillation counter (Cerenkov effect). Assag of %‘-phmphacG&mme. The highly active (2’-5’)oligoisoadenylate synthetase (SlO) from interferon-treated HeLa cells was incubated in the presence of [cyq)ATP as previously described (Verhaegen et al., 1980). The =P-labeled 2-5A was isolated by DEAE-cellulose (Whatman DE-52) chromatography and concentrated by acetone precipitation. The pellet was resuspended in a minimum volume of water and this 2-5A preparation was used as substrate for the 2-phosphodiesterase assay. About 100,000 cpm of 2-5A were incubated in the presence of 4-~1 aliquots of SlO extract (5-10 mg protein/ml) or lysis buffer (control) for 1 or 2 hr at 30” in a final volume of 8 ~1 containing 100 mM KOAc, 25 mM Mg(OAc)a, 20 mM HepesKOH (pH 7.4), and 1 mM DTT. The reaction was stopped by heating for 3 min at 95”. Two-microliter aliquots were applied to thin-layer sheets of polyethyleneimine (Machery-Nagel) and chromatographed in 1 M acetic acid as previously described (Verhaegen et al., 1980). Thereafter, the sheets were submitted to autoradiography. RESULTS
Establishment
of an Antiviral
State by Hu-
man LY,p, and y Interferon Prepara-
tions HuIFN-a and HuIFN-/3 are equally effective in establishing the antiviral state in both RSa and IF’ cells (Kuwata et at., 1979). In the present study, we compared the ability of HuIFN-a and HuIFN-y to inhibit virus growth in RSa, IF’, and HEC1 cells (Fig. 1). The amounts of vesicular stomatitis virus (VSV) formed in the yieldreduction test on RSa cells were reduced by about 4.8 log and 2.8 log when the cells were treated with 100 U/ml of IFN-a or IFN-7, respectively. Likewise, HuIFN-7 inhibited the growth of encephalomyocarditis (EMC) virus in RSa cells (data not shown). Both HuIFN-CY and HuIFN-y also inhibited VSV growth in IF’ cells (Fig. I)
ET AL.
2
RSa
1 0
z-3-
FIG. 1. Antiviral activity of HuIFN-cy and HuIFNy in RSa, IF’, and HEC-1 cells. The inhibition of VSV growth by varying amounts of HuIFN-a! (0 0) or HuIFN-y (0 l ) was measured as described under Materials,and Methods. The VSV yield is expressed as percentage of virus yield (log) in control (without interferon treatment).
although the effect was less important than in the case of RSa cells. However, HuIFN-7 proved ineffective in inhibiting either VSV (Fig. 1) or EMC virus (not shown) in HEC-1 cells. The HEC-1 cell line has previously been found resistant to the antiviral action of HuIFN-8 (Verhaegen et al., 1980) and HuIFN-a (Chen et al., 1981) with either VSV, Sindbis, Vaccinia, or EMC virus as the challenge virus. It now appears that HEC-1 cells are also resistant to the antiviral action of HuIFN-7. Cell Growth Inhibition bg Human cx,P, and y Interferon Preparations Whereas RSa and IF’ cells are both sensitive to the antiviral action of interferons, they differ in their sensitivity to the anticellular actions of HuIFN-a and HuIFN/3 (Kuwata et al., 1979; Vandenbussche et al., 1981). On one hand, RSa cell growth is strongly inhibited by HuIFN-a, and even more so by HuIFN-P. On the other hand,
2-5A SYNTHETASE
429
ACTIVITY
sidered as sensitive to the anticellular action of HuIFN-7. The situation is quite different for HEC-1 cells which appeared to be resistant to the anticellular activity of HuIFN-7 (Fig. 2). Induction of 2-5A Synthetase by Human (Y,p, and y Interferon Preparations Both HuIFN-(U and HuIFN-P induce 25A synthetase activity in RSa and IF’ cells (Vandenbussche et al., 1981). 2-5A Synthetase is also inducible by HuIFN-y although the induction requires much higher doses of HuIFN--r than of HuIFN-/3 (Fig. 3). Moreover, the amplitude of the 2-5A synthetase activity induced by HuIFN-7 is much lower than that induced by HuIFN-@ (Fig. 3). In view of the possible
101~ "
1 100
1 10 lnlarfaron
1
1000
RSa
(U/ml)
FIG. 2. Anticellular activity of HuIFN-cy andHuIFNy in RSa, IF’, and HEC-1 cells. The inhibition of [aH]thymidine incorporation by varying amounts of HuIFN-(U (00) or HuIFN-y (0 0) was measured as described under Materials and Methods. The thymidine incorporation is expressed as a percentage of thymidine incorporation in control (without interferon treatment).
IF’ cells are relatively resistant to the anticellular activity of HuIFN-(I! and HuIFNp (Kuwata et al., 1979). We have compared the anticellular effects of HuIFN-(r and HuIFN-7 on RSa, IF’, and HEC-1 cells (which are resistant to the anticellular action of HuIFN-cx and HuIFN-/3 (Chen et al., 1981)]. HuIFN-r appeared to be more potent than HuIFN-(r in inhibiting the growth of RSa cells (Fig. 2). HuIFN-y proved also inhibitory to the growth of IF’ cells (Fig. 2), although much less so than to RSa cells. For HuIEN-a a dose of 1000 U/ml was required to inhibit IF’ cell growth by 50%, whereas 100 U/ml of HuIFN-y inhibited the growth of these cells to about 20% of the control. Although HuIFN--y was more inhibitory for RSa than for IF’ cells, the latter can be con-
IFr
; 8
lo-
5-
OC 0
0.1
1
Interferon
,/
100
10
100
0
(U/ml)
FIG. 3. Induction of 2-5A synthetase activity by HuIFN-j3 and HuIFN-y in RSa and IF’ cells. Cells were treated for 2.4 hr with varying amounts of HuIFN+(O __ 0) or HuIFN--y (0 0). The protein content in the assays was 14 pg, and 2-5A synthetase activity is expressed as radioactivity incorporated into [32p)2-5A oligomers (as described under Materials and Methods).
430
VERHAEGEN-LEWALLE
heterogeneity between human y interferons produced by leukocytes or splenocytes, we have determined the 2-5A synthetaseinducing ability with both “Leuven” and “Helsinki” HuIFN-y. For both y-interferons the level of 2-5A synthetase activity induced was 5- to lo-fold lower than that obtained following induction by HuIFNp at the same concentration (i.e., 200 U/ ml). From an inspection of the dose-response curves of HuIFN-8 and HuIFN-7 (Fig. 3), we could exclude the possibility that our HuIFN-y preparation contained a HuIFN-P contaminant at a concentration of 81% (De Ley et al., 1980). On the other hand, HuIFN-7 did not affect the level of 2-5A synthetase in HE&l cells (Table 1). However, it should be pointed out that HE&l cells contain high levels of 2-5A synthetase and 73K protein kinase and these levels are not enhanced by treatment with either HuIFN-a or HuIFN-8 (Verhaegen et al., 1980). Finally, we have observed that 2-5A synthetase of HeLa cells is equally inducible by HuIFN-7 and HuIFN-B (data not shown), indicating that our HuIFN-y preparations are fully capable of inducing 2-5A synthetase activity in susceptible cells. d’-Phosphodiesterase Levels in Cells Treated with Human /3 and y Intwfkrcm Prep arations In view of the comparison of the antiviral, anticellular activities and 2-5A synthetase-inducing ability of HuIFN-/3 and HuIFN--y, it was important to know whether the products of the 2-5A synthetase reaction were not degraded differently in cells treated with either HuIFNp or HuIFN-y. Therefore, we determined the degradation of 2-5A by 2’-phosphodiesterase in RSa cells that had not been treated or treated with HuIFN-P or HuIFN-y. The rate of degradation of 32Plabeled 2-5A, as measured either by the disappearance of [“2P]2-5A tri- or tetramer or by the apparition of r2P]AMP, was identical for all three extracts (data not shown) and the amount of released FPjAMP was a function of the protein concentration in the assay (Fig. 4). As
ET AL. TABLE
1
EFFECT OF INTERFERON-~ AND -7 ON 2-5A SYNTHETASE ACTIVITY IN HE&l CELLS= 3H cpm Interferon
No dsRNA
With dsRNA
Ab
0 j3 (200 U/ml) y (200 U/ml)
903 905 967
19,395 17,159 19,525
18,492 16,254 18,558
“Cells were treated for 24 hr with the indicated interferon. Methodology for cell extract preparation and enzyme assay was as previously described (Verhaegen et al., 1980). The incubations were carried out for 2 hr. The results are expressed as means from duplicate assays containing 225 pg of protein. b A = activity with dsRNA - activity without dsRNA.
shown in Fig. 4, neither HuIFN-/3 nor HuIFN-y increased or decreased 2-phosphodiesterase activity in RSa cells. Similar results were obtained with IF’ cells. Thus, the lower levels of 2-5A synthesized in response to HuIFN-7 than to HuIFNp do not result from an increased 2’-phosphodiesterase activity. Furthermore, in the conditions of the 2-5A synthetase assay (presence of 5 mM ATP and 4 mMfructose 1,6-diphosphate), we observed that the 2’-phosphodiesterase activity was completely blocked in both control and interferon-treated RSa cells (data not shown). DISCUSSION
We have compared three different human cell lines for their sensitivity to the anticellular and antiviral effects of IFN (Table 2). The HEC-1 cell line was completely resistant to the anticellular and antiviral activities of HuIFN-(u, +3, and -7. The RSa cell line was sensitive to both the antiviral and anticellular activities of HuIFN-CZ, -8, and -7, although the anticellular effect of HuIFN-y was about lofold more pronounced than that of HuIFNa! or -& if compared at an equivalent antiviral titer. This confirms the recent observations from Blalock et al. (1980) and Rubin and Gupta (1980). The IF’ cells were resistant to the anticellular activities of
2-5A SYNTHETASE
no
0
treatment
treatment
0.5
1
2.5
5
0
0.5
1
431
ACTIVITY
2.5
with
5
0
05
1
2.5
5
mg pmt. / ml extract FIG. 4. Dependence of Z-phosphodiesterase activity on cell extract concentration for untreated RSa cells, and RSa cells treated HuIFN-/3 or HuIFN-7. Interferon treatment was at 200 units/ml for 24 hr. The incubations were carried out for 1 hr as described under Materials and Methods. The spots corresponding to released $PwAMP were separated by thin-layer chromatography on polyethyleneimine-cellulose, visualized by autoradiography, and their radioactivity was measured in a scintillation counter.
IFN-(r or -@, yet responsive to the anticellular action of HuIFN-7 (Table 2). Table 2 also presents the levels of 2-5A synthetase induced in the different cell lines after treatment with HuIFN-(w, -8, or -7. Two major conclusions become apparent if the anticellular effects of HuIFN-or, -8, and -y are compared to their potentials to induce 2-5A synthetase activity: (1) RSa cells are much more sensitive to the anticellular action of IFN-cu or -/3
than IF’ cells; however, they acquire similar levels of 2-5A synthetase upon treatment with HuIFN-a or -/3 (Vandenbussche et al., 1981). (2) Although the anticellular activity of HuIFN-y is more pronounced than that of HuIFN-CY or -6 in both RSa and IF’ cells, HuIFN-r is clearly less efficient than HuIFN-a or -/3 in inducing 2-5A synthetase activity in these cells. Baglioni and Maroney (1980) have shown
432
VERI-IAEGEN-LEWALLE TABLE
ET AL. 2
EFFECTS OF (Y,P, AND y INTERFERON% SUMMARY Cell type RSa
IF’
Anticellular action*
I (Y
++d,e.h
+ +d+h
1
B
++=
++esh
11 Y
+d
+++d
+d ++h
IFN type
++”
++d,f,h
I
a
++d,e,h
&”
1
B
++e
f ”
Y
td
+td
-da
-da
-f@
-0
*d,f,i
-d
-d
*d.i
11
HEC-1
(2-5’)oligo A synthetase activity”
Antiviral action”
I a 1 B 11
Y
“Measured by VSV yield reduction (log,,) at 100 U/ml: ++ > 3.5 > + > 1 > - = 0. *Estimated by inhibition of pH]thymidine incorporation (percentage of incorporation 100U/ml:+++>95>++>60>+>40>~>10~-. ‘Estimated by an increase (fold) in activity at 500 U/ml: ++ z 10 > + > 3. d This report. ’ Kuwata et al. (1979). f Verhaegen et al. (1980). o Chen et al. (1981). h Vandenbussche et aE. (1981). i Constitutive activity, not enhanced by interferon treatment.
that the induction of the 2-5A synthetase by HuIFN-y in HeLa cells proceeds more slowly than with HuIFN-P, although the amplitude of the induction is similar. This notion cannot explain the weak 2-5A synthetase-inducing effect of IFN-7 in our cell systems since our cells were invariably treated with IFN for 24 hr. We can exclude the possibility that the low 2-5A synthetase levels observed in RSa and IF’ cells resulted from an artefact related to a stronger induction of 2’-phosphodiesterase by IFN--y, since (i) all extracts from either untreated or IFN-treated cells contained 2’-phosphodiesterase activity; and (ii) this activity was not induced after treatment with either IFN-/3 or-y (Fig. 4), in contrast with the observations of Kimchi et al. (1979a). (iii) In agreement with Minks et al. (1979), we found that the 2’-phosphodiesterase activity was completely blocked in the conditions of the 2-5A synthetase reaction (M. Verhaegen-Lewalle, unpublished data). HEC-1 cells proved totally refractory to
++d,f,h 4
in control ceils) at
the antiviral and anticellular actions of HuIFN-7 (Table 2). These findings can be regarded as an extension of our earlier observations with IFN-a and -/I in HEC1 cells (Verhaegen et al., 1980). The situation here is different from that in murine L1210R leukemia cells. These cells may lack (part of) the receptor for MuIFN-a and -/3 (Aguet, 1980). Since L1210R cells still respond to MuIFN-y (Ankel et al., 1980; Hovanessian et al., 1980a) one may assume that the ganglioside component (Ankel et al., 1980) of the murine cell receptor for MuIFN-y differs from the ganglioside component of the MuIFN-a and -0 receptor. Preliminary studies indicate a decreased binding of IFN-a and -/3 to HEC-1 cells (T. Kuwata, unpublished data). If this is the case and if human cells, like murine cells, contain separate receptors for HuIFN-y and HuIFN-a,@, then HEC1 cells may have lost either (i) the two kinds of receptors; (ii) a common component of the two receptors; (iii) any other regulatory or nonregulatory pathway that
25A SYNTHETASE
may be important for the control and the expression of the anticellular and antiviral effects of IFN-a, -p, and -y (Verhaegen et al., 1980). To our knowledge there are no quantitative data available on the number of different IFN receptors in human cells. Indirect evidence for the existence of different receptors for HuIFN-7 and HuIFN-a,B stems from the recent observations of Fleischman et al. (1979) .and De Ley et al. (1980) indicating a possible synergy in the antiviral actions of IFN-/3 and -7. The occurrence of tumor cells, like HEC1, which are completely refractory to any type of interferon, bears on the eventual clinical utility of interferon in the treatment of cancer. Our results point to the total absence of correlation between the anticellular effects of HuIFN-a, -0, and y and their capacity to induce 2-5A synthetase activity in RSa and IF’ cells. Whether this discordance is a general rule or an exception to the rule remains to be elucidated. It has been recently observed that HuIFN-a! and HuIFN-/3 induce high 2-5A synthetase levels in Raji cells, a human lymphoblastoid cell line which is virtually refractory to the anticellular action of IFN (Y. Tomita et al., personal communication). Other examples of such a discrepancy between the actions of interferon in human cell lines have been described by Meurs et al. (1981). The results presented here emphasize the need for further research on the molecular basis of IFN action and particularly the anticellular action of IFN-7. It would seem worthwhile to investigate alternative pathways responsible for the expression of the anticellular effect of IFN in these cells where 2-5A either does not seem to play a role or does not suffice to account for the anticellular action of IFN. ACKNOWLEDGMENTS We thank Dr. R. Derynck, Dr. R. Devos, and Prof. W. Fiers (Laboratory of Molecular Biology, University of Ghent) for the gift of crude HuIFN-y, Dr. J. Van Damme and Prof. A. Billiau (Rega Institute for Medical Research, University of Leuven) for the gift of HuIFN-fi, and Prof. J.-J. Cassiman (Division of Human Genetics, University of Leuven) for providing
433
ACTIVITY
the trisomic-21 cell strain LR. This work was supported by Grant 29606.79 from the Fonda de la Recherche Fondamentale Collective (Belgium) and, in part, by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture (Japan). REFERENCES AGUET, M. (1980). High-affinity binding of ‘%I-labelled mouse interferon to a specific cell surface receptor. Nature (London) 284.459-461. ANKU, H., KRISHNAMURTI, C., BESANCON, F., STEFANOS, S., and FALCOFF, E. (1980). Mouse fibroblast (type I) and immune (type II) interferons: Pronounced differences in affinity for gangliosides and in antiviral and antigrowth effects on mouse leukemia L-1210R cells. Proc. Nat Ad Sci USA 77, 2528-2532. BAGLIONI, C., and MARONEY, P. A. (1980). Mechanism of action of human interferons. Induction of 2’5’oligo(A) polymerase. J. Bid Chem 256,&390&X93. BLALOCK, J. E., GEORGIADES, J. A., LANGFORD, M. P., and JOHNSON, H. M. (1989). Purified human immune interferon has more potent anticellular activity than fibroblast or leukocyte interferon. CeU. Immud
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