J. gen. Virol. (1983), 64, 2649-2653.
Printed in
Great Britain
2649
Key words: IFN resistance/IFN reeeptors/cGMP/2'-5'A synthetase/Daudi cells
Isolation of Daudi Cells with Reduced Sensitivity to Interferon. II. On The
Mechanisms of Resistance
By M I C H A E L G . T O V E Y , * M I C H E L D R O N , K N U D E. M O G E N S E N ,
B E R N A R D L E B L E U , 1 N A D I R M E C H T I 1 AND J A C Q U E L I N E
BEGONLOURS-GUYMARHO
Laboratoire d'Oncologie Virale, Institut de Recherches Scientifiques sur le Cancer, 94802 Villejuif
Cedex and 1Laboratoire de Biochimie des Protbines, Universit~ de Montpellier, France
(Accepted 25 August 1983)
SUMMARY
The mechanism of interferon resistance was studied in two clones of Daudi cells,
D I F 2 and DIF3, which exhibit respectively moderate and pronounced resistance to
both the antiviral and antiproliferative actions of h u m a n interferons-a and -]L Clones
D I F 2 and D I F 3 were found to possess specific high affinity interferon receptors similar
to those of parental Daudi cells. However, DIF2 cells, which have a tetraploid
karyotype, had approximately twice as m a n y interferon-binding sites as either D I F 3 or
parental Daudi cells. One of the first detectable changes in Daudi cells following
interferon treatment is a rapid increase in the intracellular concentration of cyclic
GMP. No increase in cyclic G M P was observed in D I F 2 or D I F 3 cells treated with
interferon-a. However, neither DIF2 nor DIF3 cells respond to sodium azide, a nonphysiological inducer of cyclic GMP. Interferon treatment was found to induce the
production of 2'-5'-oligo-isoadenylate synthetase in D I F 2 and D I F 3 cells in a m a n n e r
similar to parental Daudi cells, indicating that these cells possess functional interferon
receptors. The levels of 2'-5'-oligo-isoadenylate synthetase and 2'-5' A phosphodiesterase activity were similar in all three cell lines, suggesting that the interferon
resistance of clones DIF2 and D I F 3 was not due to a deficiency of pp(A2' p)nA.
INTRODUCTION
In the previous article (Dron & Tovey, 1983) we have described the isolation and
characterization of two clones of Daudi cells with markedly reduced sensitivity to both the
antiviral and anticellular actions of interferon-a and -ft. Clone DIF3 exhibits a slight decrease in
growth rate in the presence of 105 international units/ml of interferon-a, whereas the
multiplication of parental Daudi cells is completely inhibited by only 10 units/ml of interferon-a.
Clone DIF2 is of intermediate interferon sensitivity. In this article we describe experiments
designed to elucidate the mechanisms of interferon resistance of these cells.
METHODS
Cells and cell culture. The isolation and characterization of clones DIF2 and DIE3 were described by Dron &
Tovey (1983). All cells were cultivated in RPMI 1640 medium with 15~ foetal calf serum (Flow Laboratories,
Irvine, Scotland).
Interferon preparations. The interferon preparations used were the same as those described in the previous
article. Purification and radiolabellingof human interferon-~ from Namalwa cells were carried out as described by
Mogensen et al. (1981). 3zsI-IFN-c~had a titre of 16000 international units/ml and a radioactive content of 75
[tCi/~tg IFN protein at the time of the experiment. Exponentially multiplying cells were incubated for 2 h with
different concentrations of ~'-sI-IFN-c¢at 37 °C and then immediately placed on ice. Two ml of cell suspension per
point was centrifuged at 4 °C and washed twice with 4 ml of RPMI 1640medium containing 1~ foetal calf serum.
Cell pellets were transferred for counting (LKB Rackgamma 1270, 80~ efficiency). Bound radioactivity is
expressed per l06 cells.
0022-1317/83/0000-5716 $02.00
O 1983 SGM
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2650
M. G. TOVEY AND OTHERS
Assay of2"-5'-oligo-isoadenylate synthetase. Cell extracts were mixed with 50 i.tl of poly(rI)-(rC) agarose (P-L
Biochemicals) for 15 min at 30°C and the non-adsorbed material was removed by centrifugation. The
poly(rI)-(rC)-adsorbed material was incubated with 2.5 mxt-[ct-32p]ATP,400 Ci/mmol (Amersham International)
for 20 h at 30 °C, treated with bacterial alkaline phosphatase (Sigma) and then eluted from a column of acid
alumina (Sigma) (300 lad with 3-0 ml of 1 M-glycine-HCl buffer pH 2.0 as previously described (Merlin et al., 1981).
Results are expressed as pmol ATP incorporated per h per l0 s cells.
Cyclic nucleotide determination. One ml of cell suspension was treated with 1 ml of 12 ~ trichloroacetic acid for 15
min at 0 °C, centrifuged (2000g, 10 min) and the supernatant was extracted with ether, purified on a column of
Dowex AG1X8 (Murad et al., 1971) and assayed for cAMP content using a protein-binding assay as previously
described (Rochette-Egly & Castagna, 1977), and for cyclic GMP content using a radioimmune assay
(Amersham International). Recovery (70 to 80%) was monitored with 1 nCi of [3H]cyclic AMP or [3H]cyclic
GMP. Results are expressed as the mean of four replicates in pmol per 106 ceils.
RESULTS
Binding of tZSl-interferon-~ to Daudi cells and interferon-resistant clones
To determine whether the interferon resistance of clones DIF2 and DIF3 was due to the
absence of functional interferon receptors the direct binding at 37 °C of radiolabelled IFN-c< at
equilibrium to clones DIFz and DIF3 was compared with that to parental Daudi cells (Fig. 1).
Binding of ~2sI-IFN-ct to all three cell lines was saturable and was inhibited by an excess of
unlabelled interferon. Scatchard analysis of the binding of labelled interferon-or gave linear plots
and the binding constants for site saturation and affinity accorded well with the previously
published values for parental Daudi cells (Mogensen et al., 1981). The binding curves for clone
DIF3 and wild-type Daudi cells were indistinguishable. Clone DIF2 however, bound
I
x
I
I
I
1
I
fl
20
a
Z
-~
10
e~
(
100
I
I
I
I
I
200
300
400
500
600
Free IFN (IU/ml)
Fig. 1. Direct binding of ~2Sl-interferon-ct to Daudi, DIFz and DIF3 cells. The curves show the binding
of human IFN-ct (Namalwa, sp. act. ~>2 x l0 s reference units/rag protein) at 2 h equilibrium to
parental Daudi cells (O), clone DIF2 (A), and clone DIF3 (O). The symbols indicate the actual
experimental values and the curves were calculated from the linear regressions of the Scatchard plots
(Scatchard, 1949). The background of non-specific binding ( < 5 ~ of specific binding) has been
subtracted from the values presented.
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Mechanism of interferon resistance
2651
Table 1. The effect of human interferon-~ on the intracellular concentration of cyclic G M P in
cloned Daudi cells*
Cell type
Daudi
DIF2
DIF 3
Treatment
Untreated
Interferon
Sodium azide
Untreated
Interferon
Sodium azide
Untreated
Interferon
Sodium azide
Cyclic GMP (pmol/106 cells)
2.13 + 0.25"~
3.04 ± 0.09J r < 0.001"t-~
3.76 ± 0 . 2 3 . ,
P < 0.001
1.92 ± 0.14~
2.16 ± 0.36fNS'~
2.23 ± 0.44
fNS
2.00 + 0.20
1.08 ± 0.29
1-10 + 0.17
* Each cell type (2 x 106 cells/ml) was treated with either 104 units/ml of electrophoretically pure human
interferon-co(sp. act. 2 x 108 reference units/mg protein) in phosphate-buffered saline (PBS), or with an equal
volume of PBS alone, or with 10 mM-sodiumazide. Samples (1.0 ml) of cell suspension were then taken at various
times after treatment and assayed for cyclic GMP content as described in Methods. The cyclic GMP concentrations shown in the table are the values obtained at 5 min after treatment.
t Determined by Student's t-test.
:l:NS, Not significant.
approximately twofold more interferon than either clone DIF3 or parental Daudi cells. The
increased binding on DIF2 cells m a y be related to the tetraploid karyotype of these cells
(Mogensen et al., 1982; Epstein et al., 1982).
Determination of the influence of soluble factor(s)produced by DIF 3 cells on the interferon
sensitivity of Daudi cells
To determine whether the phenotype of interferon resistance was due to the production by
DIF3 cells of a soluble factor capable of either inactivating interferon at the cell surface or
inhibiting its action, Daudi cells were treated with interferon in the presence of increasing concentrations of nutrient medium in which D I F 3 cells had been cultivated. This treatment had no
effect on the sensitivity of Daudi cells to the antiproliferative action of interferon (data not
shown).
Induction of cyclic nucleotides
The binding of interferon to its specific cell surface receptor is followed by a rapid increase in
the intraceUular concentratioh of cyclic G M P (Tovey et al., 1979; Tovey & Rochette-Egly, 1981 ;
Tovey, 1982). Treatment of Daudi cells with electrophoretically pure I F N - ~ caused a rapid
increase in the intracellular concentration of cyclic G M P within 1 to 5 min (Table 1). A similar
increase in the intracellular concentration of cyclic G M P was observed when D a u d i cells were
treated with sodium azide, a non-physiological activator of guanylate cyclase (Table 1). N o
increase in cyclic G M P was observed when clones DIF2 and DIF3 were treated with either I F N
or sodium azide (Table 1). Indeed, some decrease in the intraceUular concentration o f cyclic
G M P was observed in DIF3 cells following treatment with either interferon or sodium azide
(Table 1).
2'-5'-Oligo-isoadenylate synthetase activity in Daudi cells and interferon-resistant clones
In agreement with previous reports (Krishnan & Baglioni, 1980; T o m i t a et al., 1982;
Silverman et al., 1982) Daudi cells were found to contain high levels of endogenous 2'-5'-oligoisoadenylate synthetase activity (Fig. 2), which was enhanced several-fold following treatment
with highly purified human interferon-or (Fig. 2). The level of endogenous 2'-5'-oligoisoadenylate synthetase activity in DIF2 and DIF3 cells was similar to that found in parental
- Daudi cells although in all three cell lines the basal level of the enzyme varied considerably from
one experiment to another. A n increase in 2'-5'-oligo-isoadenylate synthetase activity was also
observed in both DIF2 and DIF3 cells following interferon treatment (Table 2). The level o f 2'-5'
A phosphodiesterase activity was similar in all three cell lines and did not change significantly
following interferon treatment (data not shown).
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2652
M.G.
2000
(a)
TOVEY AND OTHERS
(b)
(c)
1800
_~ 1600
1400
1200
1000
og
o~
O<
800'
600
400 [-
0
B
1
10
X\x
100 100(
0
l0
X\"
100 1000
0
Inter~ron(IU/ml)
11
10
100 1000
Fig. 2. The effect of interferon-~ on the level of 2'-5'-oligo-isoadenylate synthetase in Daudi and DIF 3
cells. Daudi or DIF 3 cells were seeded at 2 x 105 cells/ml in RPMI 1640 medium with 10% foetal calf
serum containing the concentration of electrophoretically pure human interferon-or (sp. act. 2 × 108
reference units/rag protein) indicated. After 24 h treatment, 5 x 106 cells per sample were centrifuged
(9000 rev/min, 2 min) and resuspended in 500 ~tl of cold lysis buffer (20 mM-HEPES pH 7.5, 5 mMMgCl2, 120 mM-KC1, 7 mrcl-dithiothreitol, 10~ glycerol, 0-5 ~ Nonidet P40) and incubated for 2 min on
ice. The samples were then centrifuged (9000 rev/min, 8 min, 4 °C) and the supernatant was recovered
and assayed for 2'-5'-oligo-isoadenylate synthetase activity as described in Methods. (a) Parental Daudi
cells, (b) clone DIF2, (c) clone DIF3; Fq, untreated, [], interferon-treated.
DISCUSSION
Cells have been isolated in which the phenotype of interferon resistance has been shown to be
due to the lack of a functional receptor for interferons-a and -fl (Aguet, 1980; Aguet &
Blanchard, 1981). Receptor binding studies carried out at 37 °C, where binding is a function of
the activity of the interferon-receptor complex (Mogensen & Bandu, 1983), suggest that the
interferon-carrying capacity of the cellular receptors is unimpaired in the interferon-resistant
Daudi cell clones, DIF2 and DIF3.
The interferon-resistant clones, in contrast to parental Daudi cells, do not exhibit elevated
levels of cyclic GMP following interferon treatment. However, these cells do not respond either
to sodium azide a non-physiological inducer of cyclic GMP, and it remains to be established
whether these changes are related to the phenotype of interferon resistance.
Interferon induces a number of cellular proteins including 2'-5'-oligo-isoadenylate synthetase,
a double-stranded RNA-activated enzyme that synthesizes a series of oligonucleotides of
general structure ppp(A2'p)nA. The products of the synthetase activate at subnanomolar concentrations an endoribonuclease leading to inhibition of protein synthesis. This system has been
implicated in both the antiviral and antiproliferative actions of interferon (Kerr et al., 1981 ;
Lengyel, 1981 ; Lebleu & Content, 1982). The levels of both endogenous and interferon-induced
2'-5'-oligo-isoadenylate synthetase activity, and ppp(A2'p),A degradation are similar in Daudi
cells and the interferon-resistant clones, suggesting that the decreased interferon sensitivity of
DIF2 and DIF3 cells is not due to a lack of ppp(A2'p),A. However, this remains to be
established by direct measurement of intracellular ppp(A2'p)nA in virus-infected or growtharrested cells. Silverman et al. (1982) were unable to detect ppp(A2'p)nA in interferon-treated
wild-type or resistant Daudi cells isolated by a different procedure, again:arguing against an
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Mechanism o f interferon resistance
2653
i n v o l v e m e n t o f t h e p p p ( A 2 ' p ) . A s y s t e m in t h e i n t e r f e r o n r e s i s t a n c e o f D a u d i cells. W e a r e
currently investigating whether any correlation exists between the production of other
i n t e r f e r o n - i n d u c e d p r o t e i n s a n d t h e loss o f i n t e r f e r o n s e n s i t i v i t y o f D I F 3 cells.
We are indebted to Dr Ion Gresser for his continued advice and encouragement and we thank Dr Kari Cantell
for the gift of HulFN-~ from leukocytes and HulFN<t from Namalwa cells (the latter was originally prepared by
Drs K. H. Fantes and M. D. Johnston, Burroughs Wellcome, Beckenham Court, Kent, U.K.). This work was
supported in part by grants from DGRST (contract no. 82 L 0074) and D R E T (contract no. 80-34-522) and the
Richard Lounsbery Foundation. One of us, M.D., is grateful to the Ligue Nationale Franqaise contre le Cancer for
fellowship support.
REFERENCES
AGUET,M. (1980). High-affinity binding of ~zSI-labelled mouse interferon to a specific cell surface receptor. Nature,
London 284, 459-461.
AGUET,M. & BLAN¢HARD,B. (1981). High affinity binding of ~2St-labelled mouse interferon to a specific cell surface
receptor. II. Analysis of binding properties. Virology 115, 249 261.
DRON, M. & TOVEY,M. G. (1983). Isolation of Daudi cells with reduced sensitivity to interferon. I. Characterization.
Journal of General Virology 64, 2641-2647.
EPSTEIN, C. J., McMANUS, N. H., EPSTEIN, L. B., BRANCA, A. A., D'ALESSANDRO, S. B. & BAGLIONI, C. (1982). Direct
evidence that the gene product of the human chromosome 21 locus IFRC, is the interferon ~ receptor.
Biochemical and Biophysical Research Communications 107, 1060-1066.
KERR, I. M., WRESCHNER, D. FI., SILVERMAN,R. H., CAYLEY, P. J. & KNIGHT, M. (1981). The 2-5A (pppA2'p5'A2'p5'A)
and protein kinase systems in interferon-treated and control ceils. In Advances in Cyclic Nucleotide Research,
vol. 14, pp. 469-478. Edited by J. E. Dumont, P. Greengard & G. A. Robison. New York: Raven Press.
KRISHNAN, I. & BAGLIONI, C. (1980). Increased levels of (2'-5') oligo(A) polymerase activity in human lymphoblastoid cells treated with glycocorticoids. Proceedings of the National Academy of Sciences, U.S.A. 77, 65066510.
LEBLEU, B. & CONTENT, J. (1982). Mechanisms of interferon action: biochemical and genetic approaches. In
Interferon 4, 1982, pp. 47-94. Edited by I. Gresser. London: Academic Press.
LENGYEL, P. (1981). Mechanisms of interferon action : the (2'-5')(A), synthetase-RNase L pathway. In Interferon 3,
1981, pp. 78 99. Edited by I. Gresser. London: Academic Press.
MERLIN, G., REVEL, M. & WALLACH, D. (1981). The interferon-induced enzyme oligo-isoadenylate synthetase: rapid
determination of its in vitro products. Analytical Biochemistry ll0, 190-196.
MOGENSEN, g. E. & 8ANDU, M-T. (1983). Kinetic evidence for an activation step following binding of human
interferon ct2 to the membrane receptors o1" Daudi cells. European Journal of Biochemistry (in press).
MOGENSEN, K. E., BANDU, M-T., VIGNAUX, F., AGUET, M. & GRESSER, I. (1981). Binding of t251-1abelled human ct
interferon to human lymphoid cells. International Journal of Cancer 28, 575-582.
MOGENSEN, K. E., VIGNAUX, F. & GRESSER, I. (1982). Enhanced expression of cellular receptors for human interferonct on peripheral lymphocytes from patients with Down's syndrome. FEBS Letters 140, 285-287.
MURAD, F., MANGANELLO,V. & VAUGHN, M. (1971). A simple sensitive protein-binding assay for guanosine 3'-5'monophosphate. Proceedings of the National Academy of Sciences U.S.A. 68, 736-739.
ROC,ETTE-E~LY, C. a CASTA~NA, M. (1977). A simultaneous protein-binding assay for adenosine 3'-5'monophosphate and guanosine 3'-5'-monophosphate in biological materials. Biochemical and Biophysical
Research Communications 74, 1287 1296.
SCAT¢.ARD, G. (1949). The attraction of proteins for small molecules and ions. Annals of the New York Academy o/
Sciences 51, 660-672.
SILVERMAN,R. H., WATLING, D., BALKWILL,F. R, TROWSDALE,J. & KERR, I. M. (1982). The ppp(A2'p),A and protein
kinase systems in wild-type and interferon resistant Daudi cells. European Journal of Biochemistry 126, 333341.
TOMITA, Y., CANTELL,K. & KUWATA,T. (1982). Effects of human 7 interferon on cell growth, replication of virus and
induction of 2'-5' oligoadenylate synthetase in three human lymphoblastoid cell lines and K652 cells.
International Journal of Cancer 30, 161 165.
TOVEY, M. G. (1982). Interferon and cyclic nucleotides. In Interferon 4, 1982, pp. 23 46. Edited by I. Gresser.
London: Academic Press.
TOVEY, M. G. & ROCHETTE-EGLY,C. (1981). Rapid increase in guanosine 3'-5' cyclic-monophosphate in interferontreated mouse leukemia L I210 cells: relationship to the development of the antiviral state and inhibition of
cell multiplication. Virology 115, 272 281.
TOVEY, M. G., ROCHETTE-EGLY, C. & CASTAGNA, M. (1979). Effect of interferon on concentrations of cyclic
nucleotides in cultured cells. Proceedings of the National Academy of Sciences, U.S.A. 76, 3890-3893.
(Received 7 April 1983)
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