[CANCER RESEARCH 45, 2539-2544, June 1985]

Synergistic Effect of Human Immune Interferon and Double-Stranded RNA

against Human Colon Carcinoma Cells in Vitro

Mrunal S. Chapekar and Robert I. Glazer1

Applied Pharmacology Section, Laboratory of Medicinal Chemistry and Pharmacology Developmental Therapeutics Program, Division of Cancer Treatment, NationalCancer Institute, Bethesda, Maryland 20205

ABSTRACT

The cytocidal activity of human immune interferon (IFN-7) incombination with the synthetic double-stranded RNA, poly(l)-poly(C), was investigated in human colon carcinoma cell line HT-29. Three days of treatment with IFN-7 (10 to 25 units/ml)

resulted in 30 to 40% reduction in colony formation, whereaspoly(l)-poly(C) (25 to 100 tig/ml) reduced cell viability by 10 to20% of control. The lethal effect of the combination of IFN-7 andpolyO)-poly(C) was synergistic wherein 70 to 90% reduction incolony formation was observed. Measurements of DNA, RNA,and protein synthesis after IFN-7 and poly(l)-poly(C) treatmentshowed a dose-dependent reduction in all three parameters.Recombinant IFN-7 in combination with poly(l) •poly(C) exhibited

a similar effect. Studies evaluating the molecular mechanism ofIFN-7 and poly(l) •poly(C) toxicity indicate a lack of involvementof the double-stranded RNA-dependent (2',5')oligoadenylate~

RNase L and protein kinase pathways; however, the effectappears to be related to the inhibition of ribosomal RNA transcription in this cell line.

INTRODUCTION

IFNs2 are classified as types a, 7, and 7 on the basis of theirantigenic properties (28). Whereas IFN-«and -ft are produced in

several types of cells following treatment with viral or chemicalstimuli (19), IFN-7 is produced by lymphocytes in response to

antigens or mitogens (15). A comparison of the anticellular effectsof the 3 types of IFNs revealed that IFN-7 was a more potentantiproliferative agent than either IFN-a or IFN-0 in certain mouse

(9) and human tumor (3, 27) systems and was effective againstIFN-/7-resistant L1210 leukemia (1,18). Our recent investigationsof human IFNs have shown IFN-7 to have cytocidal activity inhuman colon carcinoma cell line HT-29 (6) in comparison to theweak antiproliferative effect of IFN-/8 or IFN-aA (5).

The synthetic dsRNA, poly(l)-poly(C), is a potent IFN inducer

and is active against the development of murine leukemia (20)and the growth of a number of transplanted tumors (2,34). Mostof these effects were mediated by the induction of IFN by poly(l) •poly(C) since Gresser ef al. (16) demonstrated that coadministra-tion of anti-IFN antibodies with poly(l) •poly(C) eliminated the

antitumor effect in mice.

1To whom requests for reprints should be addressed, at National Cancer

Institute, Building 37, Room 5D02, Bethesda, MD 20205.2The abbreviations used are: IFN, interferon; dsRNA, double-stranded RNA;

IFN-a, leukocyte interferon; IFN-/3, fibroblast interferon; IFN-f, immune interferon;IFN-aA, recombinant leukocyte interferon species A; poly(l) •poly(C), polyriboinosinicacid-polyribocytidylic acid; poly(l)-poly(Cm), polyriboinosinic-polyribo-2'-O-methyl-cytidylic acid; (2',5')-oligo(A), pppA(2'pA)n; HEPES, 4-<2-hydroxyethyl)-1-pipera-

zineethanesulfonic acid; TCA, trichloroacetic acid; PBS, phosphate-buffered saline(6.3 ITÃŒMNa2HP04-0.8 mu KH2P04-0.154 M NaCI, pH 7.4); LCW, concentration

producing a 50% reduction in cell viability.Received 9/6/84; revised 1/8/85; accepted 3/8/85.

When IFN-/3-treated mouse L-cells were exposed to dsRNA,a pronounced toxic effect was produced which resulted in cyto-

lysis (10,29,31). This phenomenon was also observed in mouseembryonic cells (29), human foreskin fibroblasts (33), humanfibrosarcoma HT-1080, and epidermal carcinoma RT-4 (21). In

our previous report (5), treatment of human colon carcinoma cellline HT-29 with either IFN-0 or IFN-aA and poly(l)-poly(C) re

sulted in a significant growth inhibition in comparison to themarginal cytostatic effect of IFN-0, IFN-aA, or poly(l)-poly(C)

treatment alone.In the present investigation, we report the synergistic cytocidal

activity of the combination of IFN-7 and poly(l)-poly(C) in HT-29cells and the relationship of this effect to the (2',5')oligo(A)-

RNase L and protein kinase pathways believed to be involved inthe growth-inhibitory effects of IFNs (7).

MATERIALS AND METHODS

[mef/7y/-14C]Thymidine (53 mCi/mmol), [2,8-3H]adenosine (32 Ci/mmol), [3H]teucine (140 Ci/mmol), and [14C]uridine (506 mCi/mmol) were

purchased from New England Nuclear (Boston, MA), andpppA(2'pA)3[32P]pCp (3000 Ci/mmol) was purchased from Amersham/Searle Corp. (Arlington Heights, IL). (2',5')Oligo(A) and poly(l)•poly(C)

were purchased from Pharmacia-PL Biochemicals (Milwaukee, Wl). In-

domethacin, dexamethasone, nordihydroguiaretic acid, quinacrine,spermine tetrahydrochloride, and spermidine trihydrochloride were purchased from Sigma Chemical Co. (St. Louis, MO). IFN-7 (10s units/mg)

was purchased from Cellular Products (Buffalo, NY). Human recombinantIFN-7 was kindly provided by Dr. Sidney Pestka, Roche Institute of

Molecular Biology (Nutley, NJ).Cells and Incubation. HT-29 cells originally derived from a human

colon carcinoma (12) were grown under an atmosphere of air and 5%COj in RPMI 1640 supplemented with 10% heat-inactivated fetal calf

serum, 40 mw HEPES buffer (pH 7.4), and gentamicin, 50 ¿ig/ml.Cellinocula were KfylO ml in 25-sq cm flasks and 10$/100 ml in 150-sq cm

flasks.Colony Formation and Cell Growth. Cell viability was determined by

the soft agar clonogenic assay as described previously (13). Briefly, cellswere washed with Hanks' balanced salt solution, without Ca*2 or Mg+2and containing 20 HIMEDTA, and trypsinized with 0.1 % trypsin in Hanks'balanced salt solution at 37°C for 10 min. Cells were then plated at

concentrations of 100 to 1000 cells/5 ml of RPM11640 containing 20%fetal calf serum and 0.1% agar. Colonies were counted after 14 days.Loss of cell viability is defined as the inability of a single cell to forni acolony after 14 days of incubation in drug-free soft agar medium. At thetime of plating, greater than 90% of control or drug-treated cells excluded

trypan blue. Cell growth was monitored by counting cells with a ModelZB Coulter Counter.

DNA, RNA, and Protein Synthesis. Logarithmically growing cells in25-sq cm flasks were incubated for 1 h with either 2 nC\ of [3H]adenosine(0.5 Ci/mmol) plus 10 6 M deoxycoformycin, an adenosine deaminaseinhibitor, and 1 ¡iC\of [mef/>y/-14C]thymidine (53 mCi/mmol) or 10 ¿iCiof[3H]leucine (2.6 mCi/mmol). Cold TCA-precipitable radioactivity was col

lected on glass fiber discs, and radioactivity was determined by liquid

CANCER RESEARCH VOL. 45 JUNE 1985

2539

on March 19, 2020. © 1985 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

SYNERGISM OF IFN-7 AND dsRNA IN COLON CARCINOMA CELLS

scintillation spectrometry. [3H]Leucine incorporation was measured ashot (90°Cfor 10 min) TCA-precipitable radioactivity collected on Millipore

filters {type HA; pore size, 0.45 Mm).(2',5')Oligo(A) Synthetase Assay. Logarithmically growing cells in

150-sq cm flasks were exposed to IFN-7 and/or poly(l)-poly(C) for 1 or

3 days, and cell extracts were prepared after lysing of the cells with 20HIM HEPES (pH 7.4)-5 mw MgCI2-120 mw KCI-1 mw dithiothreitol-10%(v/v) glycerol-0.5% Nonidet P-40. Cell extracts containing 21 ¿igofprotein/10 v\ were assayed for (2',5')oligo(A) synthetase activity asdescribed previously (5). One unit of (2',5')oligo(A) synthetase is thatamount which synthesizes 1 nmol of (2',5'X>ligo(A)n/h at 30°C.

(2',5')Oligo(A) Levels in Intact Cells. Intracellular (2'5')oligo(A) levels

were determined in logarithmically growing cells in 150-sq cm flasksfollowing 1 day of treatment with IFN-7 (25 units/ml) and/or poly(l)-

poly(C) (100 //g/ml). Cells were harvested by trypsinization, washed withcold PBS, and extracted with 100 ¡Aof TCA. Extracts were neutralizedby shaking with 2 volumes of 0.5 M trioctylamine in trifluorotrichloro-ethane. (2',5')Oligo(A) levels in neutralized extracts were measured by

the radio-binding assay as described previously (9). Using our assaysystem, the minimum amount of (2',5')oligo(A) detectable was 20 fmol/10" cells.

RNA Extraction and Electrophoresis. Logarithmically growing cellsin 150-sq cm flasks were prelabeled with 2.5 ¿iCiof ["CJuridine and then

treated with IFN-7 (25 units/ml) and/or poly(l)-poly(C) (100 /¿g/ml)for 1day. Cells were pulse-labeled for 2 h with 50 nCi of [3H]adenosine (0.5Ci/mmol) and 10"* M deoxycoformycin and harvested by scraping in cold

PBS. Cells were then washed once with cold PBS and lysed with 2 mlof 0.02 M sodium acetate (pH 5)-0.14 M NaCI-polyvinyl sulfate (10 /ig/ml)-0.2% sodium dodecyl sulfate. Cell lysate was extracted 3 times withwater-saturated phenol containing 0.1% 8-hydroxyquinoline. The emul

sion was clarified by centrifugation at 10,000 x g for 10 min, the upperphase was removed, and the RNA was precipitated overnight at -20°C

with 3 volumes of 2% potassium acetate in 95% ethanol. The precipitatewas washed once with 95% ethanol and dissolved in 200 n\ of 40 mwTris-HCI (pH 7.6)-20 mM potassium acetate-3 rriM EDTA-10% glycerol-

0.3% sodium dodecyl sulfate (running buffer).RNA was electrophoresed in composite gels containing 1.9% poly-

acrylamide-0.6% agarose-40 mu Tris-HCI (pH 7.6)-20 mM sodium acetate-3 mM EDTA-10% glycerol. Gels were run at 6 ma/tube for 3 h. Thegels were sliced into 2-mm sections, dissolved in 70% perchloric acid,

neutralized with NaOH, and mixed with 10 ml of scintillation fluid, andthe radioactivity was determined.

Protein Phosphorylation. Logarithmically growing cells in 150-sq cmflasks were exposed to IFN-7 (25 units/ml) and/or poly(l)-poly(C) for 1

day, and extracts were prepared after lysing the cells with 20 ITIMHEPES(pH 7.4)-5 mM MgClj-120 mM KCI-1 mM dithiothreitol-10% (v/v) glycerol-0.5% (v/v)-Nonidet P-40. Cell extracts containing 21 ^g of protein/10 /il

were assayed for the phosphorylation of endogenous proteins in theabsence and presence of polyamines (0.5 mM spermine tetrahydrochlo-ride plus 0.5 mM spermidine trihydrochloride) or poly(l)-poly(C), 1 //g/ml

as described previously (6).

RESULTS

Cell Growth and Viability. Exponentially growing HT-29 cellswere treated with IFN-7 (10 to 25 units/ml) and/or poly(l) •poly(C)

(25 to 100 Mg/ml) for 1 or 3 days, and cell growth and cell viabilityvia a soft agar clonogenic assay were determined (Chart 1). IFN-7 or poly(l)-poly(C) alone produced a marginal inhibition ofgrowth; however, the combination of IFN-7 and poly(l)-poly(C)resulted in a synergistic effect (Chart 1A). IFN-7 at 10 to 25units/ml or poly(l)-poly(C) (100 fig/m\) alone produced 20%growth inhibition but, when used in combination, 40% growth

60

A. CELL COUNT B. COLONY FORMATIONTREATMENTPoly

Ill-Poly (ClIFN 7. lOu/ml.»PolyIll-Poly 1C)IFN >, 25u/ml+Poly (Il-Poly (Cl1

DAYO

D

A3

DAYS•

•

A

IFN 7. 10-25u/ml1 (toy -C—- IFN>. 10u/ml.

Iday[FM -y. 25u/ml.

I «y

-IFN-y. 10u/ml. 3 dovi

~IFN y. 25u/ml. 3 days

25 50 75 100 25 50 75 100

Poly Ill-Polv (CI (tg/ml

Chart 1. Effect of IFN-7 and/or poly(l) •pdy(C) on cell growth and viability of HT-29 cells. Log-phase cells were treated for 1 or 3 days with IFN--»,and poly(l).

poly(C) and cell number (A) or colony formation (B) was determined. Each value isthe mean of 3 experiments, where the standard error did not exceed 5%. u, units.

inhibition after 1 day and 90% growth inhibition after 3 days oftreatment were observed (Chart ^A). Similarly, IFN-7 (10 to 25units/ml) and poly(l)- poly(C) (100 ¿¿g/ml)resulted in 40 and 20%

loss of cell viability, respectively; however, when added together,90% loss of cell viability occurred after 3 days of continuousexposure (Chart 10).

DNA, RNA, and Protein Synthesis. Cells were pulse-labeledwith [14C]thymidine, [3H]adenosine, and [3H]leucine as measuresof DNA, RNA, and protein synthesis, respectively. [3H]Adenosinewas selected over [3H]uridine as a measure of RNA synthesissince intracellular [3H]UTP levels were significantly lower inpoly(l) •poly(C)-treated cells pulse-labeled with [3H|undine in com

parison to untreated cells. These pool effects were due to thebreakdown of the C-strand of poly(l) •poly(C) by intracellularnucleases resulting in higher levels of cytidine and subsequentinhibition of uridine phosphorylation. Intracellular [3H]ATP levelsafter a [3H]adenosine pulse were unchanged in IFN-7 and/or

poly(l)-poly(C)-treated cells (results not shown). Reduction in

DNA, RNA, and protein synthesis after 3 days of treatment withthe combination of IFN-7 and poly(l)-poly(C) paralleled growthinhibition (Chart 2). Three days of treatment with IFN-7 (25 units/ml) and poly(l)-poly(C) (100 Mg/ml) reduced DNA, RNA, and

protein synthesis by 95,90, and 70%, respectively.Effect of Sequential Addition of IFN-7 and Poly(l) Poly(C)

on Cell Growth and Viability. To examine if the continuouspresence of both IFN-7 and poly(l)-poly(C) was required for the

expression of synergism, cells were first incubated with eachdrug, washed, and then incubated with the second compound.IFN-7 pretreatment sensitized the cells to the cytotoxic effect ofpoly (I)-poly (C) wherein 90% loss of cell viability was observed(Chart 3A). However, poly(l)-poly(C) pretreatment and subsequent IFN-7 treatment failed to generate a similar toxic effect(Chart 33). These results suggest the possibility that poly(l)-

poly(C) is degraded by intracellular nucleases and that the continuous presence of dsRNA in the medium is essential to producethe cytotoxic effect. On the other hand, once IFN-7 ¡sbound to

the cell membrane, it initiates events which are necessary forthe development of cytotoxicity by subsequent treatment withpoly(l)-polv(C).

CANCER RESEARCH VOL. 45 JUNE 1985

2540

on March 19, 2020. © 1985 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

SYNERGISM OF IFN-v AND dsRNA IN COLON CARCINOMA CELLS

A. I14C] THYMIDINE, 1 HR3 DAY TREATMENT

100

80

8 60

40

-IFN y, lOulml

-IFN y. 25u/ml

20

Poly (l)-Poly 1C)

IFN y, 25u/mlPoly (Il-Poly ICI

IFN y, lOu/ml + Poly Ill-Poly (CI

B. 13H]ADENOSINE, 1 HR

IFN y. 1O-25 u/ml

Poly Ill-Poly ICI

IFN y. 10u/mH-

Ill-Poly (CI

C. [3H] LEUCINE, 1 HR

Poly Ill-Poly ICIIFN 7, 10-25u/ml

IFN y. 10u/ml + Poly Ill-Poly ICI

k

IFN y. 25u/ml + Poly Ill-Poly ICI

25 50 75 100 25 50 75 100 25 50 75 100

Poly ID-Poly (C) /¿g/mlChart 2. Effect of IFN-7 and/or poly(l)•poly(C)on 114C|thymidine,|3H|adenosine,and |3H|leucineincorporation.Log-phasecells were pulse-labeledwith the appropriate

radioiabeledprecursor, and TCA-insolubteradioactivity was measured. Eachvalue is the mean of 3 experiments, where the standard error did not exceed 5%. u, units.

Chart 3. Effect of sequential treatment withIFN-7 and poly(l) -poly(C) on cell growth and cellviability of HT-29 cells. Log-phase cells wereexposed to IFN-7 or poly(l)-poly(C) for 1 day,washed, and subsequently treated with poly(l) •poiy(C) or IFN-7, respectively, and cell growthand colony formation were determined. Eachvalue is the mean of 3 experiments where thestandard error did not exceed 5%.

100

_j 80Ooc

8 60

40

20

A. IFN 7, Days 0-1Poly (l)-Poly (C) Days 1-4

Cell Count Q

Colony Formation ^

BPoly (l)-Poly (C) Days 0-1

IFN 7 Days 1-4

Polydl-Poly (C) +IFN 7, 25 units/ml - + -»•

Effect of Recombinant IFN-7 and Poly(l) Poly(C) on CellViability. HT-29 cells were exposed to IFN-7 and/or poly(l)-poly(C) for 3 days, and Å“il viability was determined (Table 1).Recombinant IFN-7 reduced cell viability in a dose-dependentmanner at a LCso of 5 units/ml. IFN-7 (5 to 10 units/ml) incombination with poly(l)-poly(C) (100 ng/m\) resulted in a syner-

gistic cell kill after 3 days of treatment, wherein cell viability wasreduced to 5 to 10% of control.

Effect of the Combination of IFN-7 and Either Poly(l)

Poly(Cm) or CMP plus IMP on Cell Viability. To examine if thelethality produced by IFN-7 and poly(l)-poly(C) is specific fordsRNA, cells were incubated continuously for 3 days with IFN-7

and either poly(l)-poly(Cm) or CMP plus IMP, and colony formation was determined. Poly(l)-poly(Cm) failed to generate a syn-ergistic cytotoxic effect in combination with IFN-7, and CMP plusIMP did not augment the lethality produced by IFN-7 in these

cells (results not shown).Effect of Antibodies to IFN-«and IFN-0 on the Cytotoxicity

of IFN-7 and Poly(l) Poly(C). Since dsRNA induced a low levelof IFN production (40 units/ml) in HT-29 cells (5), it was possiblethat the IFN induced by poly(l)-poly(C) in these cells togetherwith IFN-7 generated the synergistic cytotoxic response. Toexamine this possibility, cells were incubated with anti-IFN-«andanti-IFN-/3 antibodies during 3 days of treatment with IFN-7 and

CANCER RESEARCH VOL. 45 JUNE 1985

2541

on March 19, 2020. © 1985 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

SYNERGISM OF IFN-7 AND dsRNA IN COLON CARCINOMA CELLS

Table1Effect of recombinant human IFN-y and poly(l) -poly(C) on cell viability

Logarithmicallygrowing cells were treated continuously for 3 days with recombinant IFN-y and/or poly(l)-poly(C), and colony formation was determined afterremovalof iFN->and poly(l)•poly(C)from the mediumas describedunder 'Materialsand Methods."

TreatmentPoly(l).poly(C)

25 /ig/ml100/ig/mlIFN-7

5 units/ml10 units/ml25 units/ml50 units/ml

100units/mlIFN-7

(5 units/ml) + poly(l)-poly(C)(25 »ig/ml)IFN-y (5 units/ml) + poly(l).poly(C) (100 vg/rri)IFN-7 (10 units/ml) + poly(l)-poly(C)(25 jig/ml)IFN-y (10 units/ml) + poly(l)-poly(C)(100 ^g/ml)Cotony

formation(% ofcontrol)94±8a

87±751

±338 ±326 ±314 ±410±215±2

8±217±4

8±1''Mean ±SDof duplicate samples from 3 experiments.

poly(l)-poly(C). Antibody concentrations up to 400 neutralizingunits/ml did not inhibit the cytotoxic effect of IFN-7 and poly(l)-

poly(C) (results not shown). These results suggest that augmentation of the cytotoxicity of IFN-7 by poly(l)-poly(C) is not due toendogenous IFN secretion in dsRNA-treated cells.

Effect of Agents Inhibiting Prostaglandin Biosynthesis onthe Cell Lethality Produced by IFN-7 and Poly(l) Poly(C).

Wallach and Revel (33) reported that cell lysis mediated bydsRNA in human fibroblasts pretreated with IFN-/3 was abro

gated by agents which interfere with prostaglandin biosynthesis.To test this possibility, cells were treated with agents whichinhibit prostaglandin biosynthesis concurrently with IFN-7 andpoly(l) •poly(C) for 3 days, and colony formation was determined.Our results indicate that dexamethasone, as well as the cycloox-

ygenase inhibitor indomethacin or the lipooxygenase inhibitorsnordihydroguiaretic acid and quinacrine, did not protect thesecells from the lethal effect of IFN-7 and poly(l)-poly(C) (results

not shown).(2',5')Oligo(A) Synthetase Activity and (2',5')Oligo(A) Lev

els in IFN-7 and Poly(l) Poly(C)-treated Cells. To determine ifthe dsRNA-activated (2',5')ol¡go(A)-RNase L pathway is involvedin the cytotoxic effect of IFN-7 and poly(l)-poly(C), (2',5')oligo(A)synthetase activity and (2'5')oligo(A) levels were measured in

cells treated with IFN7 and/or poly(l)-poly(C). One to 3 days ofexposure to IFN-7 (25 units/ml) resulted in a 4- to 6-fold inductionof (2',5')oHgo(A) synthetase activity, and poly(l)•poly(C) (100 ngfml) under similar conditions induced (2',5')oligo(A) synthetase

by 2-fold. The combination of IFN-7 (25 units/ml) and poly(l)-

poly(C) (100 /ug/ml) resulted in a synergistic induction of(2',5')oligo(A) synthetase activity which was 10- and 18-fold

greater than control activity after 1 and 3 days of continuoustreatment, respectively (Table 2). However, no measurableamount of (2',5')oligo(A) was detected by radioligand assay in

HT-29 cells after 1 or 3 days of treatment with IFN-7 (25 units/ml) and/or poly(l)-poly(C), despite the ability of this method todetect concentrations as low as 20 fmol of (2',5')oligo(A)/106

cells (results not shown).rRNA Synthesis in IFN-7 and Poly(l) Poly(C)-treated Cells.

Since the incorporation of [3H]adenosine into nucleic acids wassignificantly inhibited after 3 days of treatment with IFN-7 and

Table 2(2',5')Oligo(A) synthetaseactivity following treatmentof HT-29 cells with IFN-y

andpoly(l).poly(C)

1 day oftreatmentTreatmentControl

Poly(l)-poly(C)(100Mg/ml)IFN-y (25 units/ml)IFN-7 (25 units/ml) + poly(l)-

poly(C)(100Mg/ml)(2',5')Oligo(A)

synthetase(units/mg)6.4

±0.2a

12.4 ±0.524.2 ±1.066.9 ±5-Fold1.0

1.93.810.43

days oftreatment(2',5')Oligo(A)

synthetase(units/mg)5.3

±0.310.1 ±1.330.7 ±3.293.4 ±4.6-Fold1.0

1.95.8

17.6

Mean ±SD of duplicate assays from 2 experiments.

10

Q.Û

10

32S

45S |28S 18SI M I

3H/'4C = 0.9

IFN 7, 25 u/ml

I II I

(93!

=0.93 (99)

Sa_

Poly (l)-Poty 1C), 100 ¿lg/ml

IFN 7. 25 u/ml + Poly Ill-Poly ICI,

100 (ig/ml

3H/'*C»0.13 (231

EL

20 30 40 50 10 20 30 40 50

FRACTION NUMBER

Chart 4. rRNAsynthesis in HT-29cells treated with IFN-y and/or poly(l)•poly(C).Cells were prelabeledwith [14C]uridinefor 2 days and subsequently treated withIFN-y and/or poly(l)-poly(C)for 1 day. Cells were pulse-labeledfor 2 h with [3H]-adenosine before the end of treatment, and RNA was extracted and separatedetectrophoreticallyas described under •Materialsand Methods." Numbersin parentheses indicate percent of control 3H/14Cratios.

poly(l) •poly(C) at an interval where cell viability was reduced, thesynthesis of rRNA was measured 1 day after treatment, whereina minimal effect on cell viability was observed. RNA extracted

CANCER RESEARCH VOL. 45 JUNE 1985

2542

on March 19, 2020. © 1985 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

SYNERGISM OF IFN-7 AND dsRNA IN COLON CARCINOMA CELLS

from cells prelabeled with [14C]uridine and pulsed for 2 h with[3H]adenosine was resolved in polyacrylamide-agarose gels. Oneday of exposure to IFN-7 (25 units/ml) produced no effect onthe incorporation of [3H]adenosine into 28S and 18S rRNA, and

poly(l) •poly(C) (100 MQ/ml) inhibited rRNA formation by only 20%(Chart 4). In contrast, the synthesis of 28S and 18S rRNA wasinhibited by 75 to 80% after HT-29 cells were treated concurrently with IFN-7 and poly(l)-poly(C) (Chart 4). The effect of IFN-

7 and/or poly(l)-poly(C) on rRNA maturation was also investigated by pulse-labeling cells for 24 h with [3H]adenosine. No

buildup of 45S rRNA precursor was evident after treatment withIFN-7 and poly(l)-poly(C), indicating that inhibition of [3H]adeno-

sine incorporation into 28S and 18S rRNA is due to inhibition oftranscription rather than of the processing of rRNA.

Protein Phosphorylation. We recently reported that treatmentof HT-29 cells with IFN-7 stimulated the polyamine-dependent

Phosphorylation of a M, 70,000 polypeptide (6). No additionalchanges in polyamine-dependent pnosphorylation were observed in cells treated with IFN-7 and poly(l)-poly(C) (results not

shown).

DISCUSSION

The present study has examined the effect of IFN-7 andpoly(l)-poly(C) used alone and in combination in human coloncarcinoma cell line HT-29. Previously, IFN-7 was shown to be

highly toxic to the cell line at a LC50of 15 units/ml (6). We nowreport that virtually nontoxic concentrations of IFN-7 ¡ncombination with poly(l)-poly(C) result in a synergistic cytocidal effect.This effect is specific for IFN-7 since the combination of IFN-«or IFN-/Õwith poly(l) •poly(C) resulted in only an additive cytostaticeffect and a marginal effect on cell viability in HT-29 cells (5).Since poly(l)-poly(C) induces low amounts of Interferon production in HT-29 cells (5), it was possible that the observed effectwas due to the synergism between IFN-7 and the IFN producedin dsRNA-treated cells. This possibility was ruled out sinceaddition of excess anti-IFN-0 and anti-IFN-a to IFN-7- and poly(l) •poly(C)-treated cells did not block the synergistic cytotoxic effect.

The observed effect is also specific for dsRNA with an unmodifiedC-strand, since poly(l)-poly(Cm) in combination with IFN-7 did

not generate a synergistic cytotoxic effect in these cells. Inaddition, the breakdown products of poly(l)-poly(C), CMP andIMP, did not augment the cell lethality produced by IFN-7.

The synergism produced by the combination of IFN-7 andpoly(l) •poly(C) appears to be related to the sensitivity of the cellsto the lethal effects of IFN-7 since another human colon carci

noma cell line, BE, which was marginally sensitive to the cytocidaleffect of IFN-7 (LCuo > 600 units/ml) exhibited a moderate

enhancement in sensitivity by the combination regimen, whereasthe human promyelocytic leukemia cell line HL-60 which is totallyresistant to IFN-7 failed to show any response after treatmentwith IFN-7 and poly(l)-poly(C).3 Although the activity of the

combination of IFN-7 and poly(l)-poly(C) was not studied previously, a synergistic effect was observed by poly(l)-poly(C) incombination with IFN-0 in murine L929 cells and embryonic

fibroblasts (10. 29, 30), as well as in human embryonic andforeskin fibroblasts (33). A similar cytotoxic effect was noted inIFN-jS-resistant and -sensitive human HT-1080 fibrosarcoma and

3Unpublished results.

RT-4 epidermal carcinoma cell lines (21).

Several investigators have examined the mechanisms underlying poly(l)-poly(C) toxicity in IFN-treated cells. Heremans ef a/.(17) observed that, in L929 cells pretreated with IFN-/3 andsubsequently exposed to poly(l)-poly(C), several ultrastructuralchanges were apparent such as disorganization of the nucleusand endoplasmic reticulum. Cooper ef al. (8) suggested that celldivision is unnecessary for the development of toxicity in L929cells treated with IFN-/3 and poly(l)-poly(C) but that the nucleus

is essential for the initiation of cytotoxicity of IFN, but not for thedevelopment of toxicity after poly(l)-poly(C) treatment. Stewartef a/. (30) observed poly(l) •poly(C) toxicity in cells treated withIFN-/8despite the presence of actinomycin D or cycloheximide in

the culture medium, which suggests that transcriptional andposttranscriptional processes were not involved.

Wallach and Revel (33) reported that the cytolytic effect ofpoly(l) •poly(C) in human foreskin fibroblasts treated with IFN-/Ì

was significantly inhibited by dexamethasone, suggesting thatthe mechanism of cytolysis may be related to the inhibition ofprostaglandin biosynthesis. However, our results do not supportthis concept since the toxic effect of the combination of IFN-7and poly(l) •poly(C) was unaffected by various agents that inhibit

prostaglandin biosynthesis.Induction of the dsRNA-dependent (2',5')oligo(A)-RNase L

pathway in IFN-treated cells has been con-elated with the growth-

inhibitory effects of IFN (7). Nilsen ef al. (25) reported previouslythat (2',5')oligo(A) levels were elevated in HeLa cells treated

with IFN-ßfollowing reovirus infection or in uninfected HeLa cellsafter treatment with poly(l)-poly(C) and IFN-/1 Goswami andSharma (14) also observed elevated (2' ,5')oligo(A) levels in cells

treated with IFN-/3 and poly(l) •poly(C) but not in cells treatedwith IFN-0 alone. In the present study, no detectable amountsof (2',5')ol¡go(A) and no rRNA degradation were observed in HT-

29 cells treated with IFN-7 and/or poly(l)-poly(C), despite thesynergistic induction of (2',5')oligo(A) synthetase activity by the

combination of IFN-7 and poly(l)-poly(C). A lack of involvementof dsRNA-dependent pathways in the action of IFN was also

reported by Faure ef al. (11), who observed identical(2',5')oligo(A) synthetase and protein kinase induction in mouse

fibroblasts sensitive and resistant to treatment with IFN-0 andpoly(l) •poly(C). Recent reports have also questioned the relation

ship between the antiproliferative and antiviral effects of IFN and(2' ,5')oligo(A) synthetase induction (31,32). In the latter studies,IFN-0 induced (2',5')oligo(A) synthetase activity to an equal

magnitude in 2 human fibroblast cell lines, one of which wasresistant to IFN-0. Rice ef al. (26) observed high levels of(2',5')oligo(A) in vaccinia virus-infected HeLa, L929, and CVI

cells with little or no inhibition of viral replication.The role of transcription in the antiproliferative activity of IFN

has not been addressed previously. However, inhibition of transcription of vesicular stomatitis virus in the presence of IFN hasbeen reported in human (22), chick (23), and monkey cells (24).Recently, Brennan and Stark (4) observed an inhibition of theonset of early transcription in IFN-pretreated cells infected with

SV40. However, there are no studies of the inhibition of transcription in IFN- and poly(l)-poly(C)-treated cells. Therefore, it isof significance that, in HT-29 cells treated with IFN-7 and poly(l)-

poly(C), the only event which correlated with the onset of cytotoxicity was inhibition of rRNA transcription in the absence of aneffect on the processing of nucleolar precursors to rRNA. Al-

CANCER RESEARCH VOL. 45 JUNE 1985

2543

on March 19, 2020. © 1985 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

SYNERGISM OF IFN-y AND dsRNA IN COLON CARCINOMA CELLS

though, we have not determined the IFN-7- or poly(l)-poly(C)

sensitive component to this effect, we plan to pursue this infuture studies.

REFERENCES

1. Ankel. H., Krishnamurti, C., Besancon, F., Stefancs, S., and Falco«.E. Mousetibroblast (type I) and immune (type II) interferons: pronounced differences inaffinity for gangliosides and antiviral and antigrowth effects on mouse leukemiaL-1210R cells. Proc. Nati. Acad. Sci. USA, 77: 2528-2532,1980.

2. Bart, R. S., and Kopf, A. W. Inhibition of the growth of murine malignantmelanoma with synthetic double-stranded ribonucleic acid. Nature (Lond.),224:3-12,1969.

3. Blalock, J., Georgiades, J. A., Langford, M. P., and Johnson, H. M. Purifiedhuman Interferon has more potent anticellular activity than fibroblast or leukocyte interferon. Cell. Immunol., 49: 390-394,1980.

4. Brennan, M. B., and Stark, G. R. Interferon pretreatment inhibits simian virus40 infection by blocking the onset of early transcription. Cell, 33: 811-816,

1983.5. Chapekar, M. S., and Glazer, R. I. Effect of fibroblast and recombinant

leukocyte interferons and double-stranded RNA on ppp(2' ,5')A«synthesis and

cell proliferation in human colon carcinoma cells in vitro. Cancer Res., 43:2683-2687,1983.

6. Chapekar, M. S., and Glazer, R. I. Effects of human immune interferon on cellviability, (2',5')oligoadenylate synthesis and polyamine-dependent protein

phosphorylation in human colon carcinoma cells in vitro. Cancer Res., 44:2144-2149,1984.

7. Chapekar, M. S., and Glazer, R. I. Growth inhibitory effects of Interferons and(2',5')oligoadenylate and its analogs. In: R. I. Glazer, (ed.), Developments in

Cancer Chemotherapy, pp. 207-224. Boca Raton, FL: CRC Press, 1984.8. Cooper, J. A., Morser J., Colby, M. S., and Burke, D. C. The toxic effect of

double-stranded RNA for ¡nterferon-treated cells: evidence for a heterogeneouscellular response and the rote of the cell nucleus. J. Gen. Virol., 43: 553-561,

1979.9. Crane, J. L., Jr., Glasgow, L. A., Kern, E. R., and Younger, J. S. Inhibition of

murine osteogenic sarcomas by treatment with type I or type II interferon. J.Nati. Cancer Inst., 6Õ:871-874,1978.

10. De Ctercq, E., and De Somer, P. Are cytotoxicity and Interferon Inducingactivity of poly(l)-poly(C) invariably linked in interferon-treated L cells? J. Gen.Virol., 27:35-44,1975.

11. Paure, S., Milhaud, P. G., De Laclos, H. F., Mechti, N„Silhol, M., Huez, G.,and Lebleu, B. Isolation and biological characterization of mouse LM fibroblastvariants resistant to the cytotoxicity of poly riboinosinic-poly ribocy tidy lie acidafter interferon treatment. J. Gen. Virol., 65: 617-627,1984.

12. Fogh, J., and Trempe, G. New human tumor cell lines. In: J. Fogh (ed.), HumanTumor Cells m Wfro, pp. 115-159. New York: Plenum Publishing Corp.. 1975.

13. Glazer, R.I., Hartman, K. D., and Richardson, C. L. Cytokinetic and biochemicaleffects of 5-iminodaunorubicin in human colon carcinoma in culture. CancerRes., 42: 117-121,1982.

14. Goswami, B. B., and Sharma, O. K., Degradation of rRNA in interferon-treatedvaccinia virus infected cells. J. Biol. Chem., 259:1371-1374, 1984.

15. Green, J. A., Cooperband, S. R., and Kibrick, S. Immune specific induction ofinterferon production in cultures of human blood lymphocytes. Science (Wash.DC) 164: 1415-1417, 1969.

16. Gresser, I., Maury, C., Bandu, M. T., Tovey, M., and Maunoury, M. T. Rote of

endogenous interferon in the antitumor effect of poly(l)-poly(C) and statoloneas demonstrated by the use of antimouse interferon serum. Int. J. Cancer, 21:72-77,1978.

17. Heremans, H., Stewart, W. E., II, Billiau, A., and DeSomer, P. Toxicity ofpoly(rl)-poly(rC) in interferon treated cells—an ultrastructural evaluation evaluation. J. Gen. Virol., 30:131-135,1976.

18. Hovanessian, A. G., La Bonnardiere, C., and Falcoff, E. Action of murine y(immune)interferon on ß(fibroblast)-interferon resistant L1210 and embryonalcarcinoma cells. J. Interferon Res., Õ:125-135,1980.

19. Krim, M. Towards therapy with interferons. Part l. Interferons: production andproperties. Blood, 55. 875-884,1980.

20. Larson, V.M., Clark, W. R., Dagte, G. E., Hilleman, M. R. Influence of syntheticdouble stranded ribonucleic acid (poly I-C) on Friend leukemia in mice. Proc.Soc. Exp. Biol. Med., Õ32:602-607,1969.

21. Un, S. L., Greene, J. J., Ts'o, P. O. P., and Carter, W. A. Sensitivity and

resistance of human tumor cells to interferon and In Cn. Nature (Lond.), 297:417-419,1982.

22. Manders, E. K., Tilles, J. G., and Huang, A. S. Interferon-mediated inhibition ofvirion-directed transcription. Virology, 49: 573-581,1972.

23. Marcus, P. I., Englehardt, D. L., Hunt, J. M., and Sekellick, M. J. Interferonaction: inhibition of vesicular stomatis virus RNA synthesis induced by virion-bound polymerase. Science (Wash. DC) 174: 593-598,1966.

24. Marcus, P. I., and Sekellick, M. J. Cell killing by viruses III. The interferonsystem and inhibition of cell killing by vesicular stomatitis virus. Virology, 69:378-393,1976.

25. Nilsen, T. W., Maroney, P. A., and Baglioâ„¢,C. Double-stranded RNA causessynthesis of (2'.5')oligo-(A) and degradation of messenger RNA in interferon-

treated cells. J. Bid. Chem., 256:7806-7811,1981.26. Rice, A. P., Roberts, W. K., and Kerr, I. M. 2',5'A accumulates to high levels

in interferon-treated vaccinia virus-infected cells in the absence of any inhibitionof virus replication. J. Virol., 50: 220-228,1984.

27. Rubin, B. Y., and Gupta, S. L. Differential efficacies of human type I and typeII interféronsas antiviral and antiproliferative agents. Proc. Nati. Acad. Sci.USA, 77: 5928-5962,1980.

28. Stewart, W. E., Blalock, J. E., Burke, D. C., Chany, C., Dunnick, J. F., Falcoff,E., Friedman, R. M., Galasso, G. J., Joklick, W. K., Vilcek, J. T., Younger, J.S., and Zoon, K. C. Interferon nomenclature. Nature (Lond.), 286:110,1980.

29. Stewart, W. E., Il, De Ctercq, E., Billiau, A., Desmyter, J., and De Somer, P.Increased suceptibility of cells treated with interferon to the toxicity of polyri-boinosinic-polyribocytidylic acid. Proc. Nati. Acad. Sci. USA, 69:1851-1854,

1972.30. Stewart, W. E., De Ctercq, E., and De Somer, P. Specificity of interferon-

induced enhancement of toxicity for double stranded ribonucleic acids. J. Gen.Virol., 18: 237-246,1973.

31. Vandenbussche, P., Divizia, M., Verhaegen-Lewalle. M., Fuse, A., Kuwata, T.,De Ctercq, E., and Content, J. Enzymatic activities induced by interferon inhuman fibroblast cell lines differing in their sensitivity to the anticellular activityof ¡nterferon.Virology, 111:11 -22,1981.

32. Verhaegan-Lewalle, M., Kuwata, T., Zhang, Z.-X., De Clercq, E., Cantell, R.,and Content, J. (2-5) Synthetase activity induced by interferon »,0, and r inhuman cell lines differing in their sensitivity to the anticellular and antiviralactivities of these interferons. Virology, 117: 425-434,1982.

33. Wallach, D., and Revel, M. Hormonal protection of interferon treated cellsagainst double-stranded RNA induced cytolysis. FEBS Lett., 707: 364-368,

1979.34. Zeleznick, L. D., and Bhuyan, B. K. Treatment of leukemia (L1210) mice with

double-stranded polyribonucleotides. Proc. Soc. Exp. Biol. Med., 730: 126-

128,1969.

CANCER RESEARCH VOL. 45 JUNE 1985

2544

on March 19, 2020. © 1985 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

1985;45:2539-2544. Cancer Res   Mrunal S. Chapekar and Robert I. Glazer  Vitro

inDouble-Stranded RNA against Human Colon Carcinoma Cells Synergistic Effect of Human Immune Interferon and

  Updated version

  http://cancerres.aacrjournals.org/content/45/6/2539

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/45/6/2539To request permission to re-use all or part of this article, use this link

on March 19, 2020. © 1985 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from