20007 Received 19 August 1982/Accepted 16September1982 DNA synthesis, as measured by incorporation of[3H]TTP, was inhibited in Swiss mouse 3T3 cells treated with interferon and subsequen
Trang 1Vol 45, No 2 JOURNAL OF VIROLOGY, Feb 1983, p 895-897
0022-538X/83/020895-03$02.00/0
Copyright C 1983, American Society for Microbiology
Persistence of Interferon Action in Mouse Cells Permeabilized
with Lysolecithin
ELSBETH J LEEt AND THAZEPADATH SREEVALSAN*
DepartmentofMicrobiology, Georgetown University, Schools ofMedicine andDentistry, Washington,D.C.
20007 Received 19 August 1982/Accepted 16September1982
DNA synthesis, as measured by incorporation of[3H]TTP, was inhibited in
Swiss mouse 3T3 cells treated with interferon and subsequently permeabilized
with lysolecithin The degree of inhibition observed was similar in intact or
permeabilized cells The interferon-induced antiviral state was retained in
per-meabilized cells
Interferon (IFN) induces pleiotropic effectson
mammalian cells, including the inhibition of
DNAsynthesis and cell growth (7) The
mecha-nisms mediating these effects, however, remain
poorly definedatpresent Incorporationof
exo-geneouslyadded [3H]thymidineinto
acid-insolu-ble material is the usualtechnique employedfor
thedetermination of DNA synthesis in
mamma-lian cells Using thistechnique, several authors
haveshown that there is significantinhibition of
theincorporation of labeledthymidine into
cel-lularDNA in IFN-treated cells (1, 5, 6, 8) In
experiments with L1210cells,however,
Brouty-Boye and Tovey (2) have reported that IFN
inhibits the uptake of thymidine into cells, a
finding which has been confirmed with Swiss
mouse 3T3 cells (T Sreevalsan, unpublished
data) Therefore, a reduction in uptake could
accountfor the observed inhibition in the
incor-poration of [3H]thymidine into acid-insoluble
material in Swiss 3T3 cells treated with IFN(1,
6) Inthisreport, weexamined the effect of IFN
on DNAreplicationin permeabilized cells, into
which[3H]TTPcanbeintroduced directly If the
mechanism of inhibition of DNA synthesis is
dependentontheeffect ofIFN ontheability of
cellstotakeupthymidine,onewouldexpectthe
incorporation of[3H]TTP into cellular DNA in
permeabilized cells to be unaffected by IFN
Our results indicated that the inhibitory effects
ofIFN on DNA synthesis persisted even after
the cells werepermeabilized
The procedure used to permeabilize Swiss
3T3 cells was a modification of a method
de-scribedrecently (3, 4).Cells tobepermeabilized
were seeded in 33:mm dishes at 105 cells per
dish When cells wereconfluentand quiescent,
t Present address: Laboratory of Tumor Virus Genetics,
National Cancer Institute, National Institutes of Health,
Be-thesda, MD 20205
cultureswere stimulatedfor18 hwith Dulbecco modified Eagle medium containing 10% calf
serum asdescribedpreviously(6) Unstimulated culturesreceived medium containingno serum Thecells were permeabilized by using lysoleci-thin, and DNAsynthesis was measured by the
incorporation of[3H]TTPintotheacid-insoluble fraction as described by Castellot et al (3) Preliminary experiments indicated that,
com-pared with unstimulated cells, there is an
in-crease(50-to100-fold) in the ability of
stimulat-ed cells to synthesize DNA Additionally, [3H]TTPincorporation was linearup to 20 min
after permeabilization ofthe cells Thereafter, the ability of permeabilized cells to synthesize
DNA was gradually lost These findings were
similarto those reportedpreviously (3, 4)
Theresults in Table1 show thatincorporation
of[3H]thymidine by intact cells or [3H]TTP by permeabilized cellswasimpaired inmouse IFN-treated cultures The degree of inhibition of
DNA synthesis was similar in both intact and
permeabilized cells There was some (ca 20%) reduction of DNA synthesis when cells were treated with human lymphoblastoid IFN rather than mouse IFN (results not shown) This was notsurprising since it isnowknownthat human
IFN can act in cells of heterologous species,
includingmice (9) The results presented in Fig
1 show that the observed inhibitory effect of mouse IFN on DNA synthesis was dose depen-dent Again, comparable levels on inhibition
were observed inintact or permeabilized cells at
agiven doseofIFN
Experiments were performed to determine whether the antiviral state induced by IFN in cells could persist after permeabilization The results (Table 2) showed that the IFN-induced
antiviral state, as judged by resistance to
en-cephalomyocarditis virus replication, was
re-895
Trang 2896 NOTES
TABLE 1 Inhibitory effect of IFN on DNA
synthesis in intact or permeabilized 3T3 cellsa
Radioactivity in:
Treatment ~Intactcells Permeabilized
Treatment (CpMXcells cells(cpm x
106 cells) 104/MgOf
protein) Unstimulated 0.04 ± 0.01 0.05 ± 0.007
Serum + mouse 0.35 ± 0.02 0.58 ± 0.16
IFN
a Quiescent Swiss mouse 3T3 cells in 33-mm Nunc
dishes were prepared and then stimulated with fresh
medium containing serum as described previously (6).
Mouse IFN (specific activity, 2 x 105 U/mg of
pro-tein), prepared by described methods (6), was used at
1,000 U/ml The cultures were incubated for 18 h, at
which time DNA synthesis was ascertained Cultures
to be permeabilized were chilled by placing the dishes
on a metal tray in ice and then washed two times with
cold phosphate-buffered saline (pH 7.4) containing 2
mM CaC12 Dishes were then washed with cold buffer
containing 35 mMMgCI2and 1 mM CaC12
Monolay-ers were permeabilized in the same buffer (1 ml) with
60 to 80 ,ug oflysolecithin (Sigma Chemical Co., St.
Louis, Mo.) per ml for 2 min at 0°C (The
concentra-tion oflysolecithinrequired for optimal
permeabiliza-tion [>95%] varied depending on the batch of cells
used Therefore, for each set of experiments the
optimum concentration of lysolecithin required was
determined.) The degree of permeabilization was
as-sessed bydeterminingthe uptake of trypan bluebythe
cells After removal of theoverlyingsolution,
permea-bilized monolayers were incubated with the buffer
described above (1ml),withoutCaC12, containing0.25
mM dCTP, 0.25 mM dGTP, 0.25 mM dATP, 0.12 mM
GTP,0.12 mM UTP, 0.12 mMCTP,1.25 mMATP,10
mM phosphoenolpyruvate, and 5 ,uCi/ml of 60 ,uM
[methyl-3H]TTP tetrasodium salt (specific activity,
77.4 Ci/mmol; New England NuclearCorp., Boston,
Mass.) Intact cultures were incubated for 30 min with
0.25 ,uCi of 1 t±M[3H]thymidine (specificactivity, 52
Ci/mmol, New England Nuclear Corp.)
Permeabi-lized cells were incubated for 20 min at 37°C in
humidified 10% CO2 Monolayers were washed with
cold phosphate-buffered saline containing 2 mM
CaC12, followedbya 15-min incubation with cold 5%
trichloroacetic acid containing 0.2 M sodium PPi
Cultures were washed five times with cold 5%
tri-chloroacetic acid and then washed three times with
absolute ethanol The air-dried monolayers were
dis-solved in 0.1 N NaOH at 37°C for 30 min, and the
amount of radioactivity was determined by liquid
scintillation counting in acidified Ready Solv
(Beck-man Instruments, Inc., Palo Alto, Calif.) Values
represent the mean ± the standard error of the mean of
three experiments.
tained even after the cells were permeabilized
with lysolecithin Although the synthesis of
en-cephalomyocarditis virus-specific RNA in
con-trolpermeabilized cellswas lowcomparedwith
thatin intactcells,therewas asignificant
reduc-
100r J
0
c-z
0
LL
0
0 -J
w
60k
401-20k
10
F~~~~~~~~V
UNITS OF IFN
FIG 1 Inhibitory effect of IFN on DNA synthesis
in permeabilized cells Quiescent cultures (33 mm)
were prepared and stimulated as described in Table 1 Some of the cultures received mouse IFN (specific activity, 2 x 105 U/mg of protein) at the indicated concentration After 18 h of incubation, sets of
cul-tures were permeabilized, and DNA synthesis in cells
was measured by methods identical to those described
in Table 1 Values are expressed as a percentage of the yield of the control stimulated cells and represent the mean the standard error of the mean of three experiments Symbols: O, intact cells; 0, permeabi-lized cells.
TABLE 2 Persistence of IFN-induced antiviral activity in permeabilized 3T3 cells'
Radioactivity (cpm x103
of [3H]uridine per culture Treatment per 45min)in:
Intact Permeabilized cells cells
Serum + actinomycinD 10.70 1.90
+ virus Serum + actinomycin D 0.24 0.07 + virus + IFN
a Quiescent cultures (33 mm) were prepared and stimulated as described in Table 1 Wherever
indicat-ed, cultures received mouse IFN (1,000 U/ml)at the time of stimulation Afterapproximately15 h of
incu-bation,cultures werechallengedwith
encephalomyo-carditis virus and subsequently incubated for 6 h in media containing 2% dialyzed calf serum Cultures receiving 1p.gof actinomycin D per ml were incubated with the drug at this time After incubation, cultures were permeabilized as described in Table 1 This was followed by a 45-min pulse with [3H]uridine (1 ,uCi;
specific activity, 77 Ci/mmol). Cell-specific(no actino-mycin D) orvirus-specific(plusactinomycinD) acid-insoluble counts were expressed as counts per minute incorporated per culture.
J VIROL.
Trang 3NOTES 897
tion in the amount of viral RNA synthesis in
IFN-treated cultures Theseresultssuggestthat
theantiviralactivity induced by IFN in cellscan
persist evenafterthe cellsarepermeabilized
Themajormetabolic pathway for thedenovo
synthesis ofthymidylic acid in cells involves the
transfer of the methylene group from N5,N10
methylene-tetrahydrofolic acid to position 5 of
deoxyuridylic acid and is catalyzed by the
en-zymethymidylate synthetase Thesalvage
path-wayinvolves the transfer of the -y-phosphate of
ATPtothe5'position of thymidylic acid and is
catalyzed by theenzymethymidine kinase The
presentresultsclearly show thatintroduction of
TTP directly into cells did not abrogate the
inhibitory effects of IFN on DNA synthesis
This suggests that the anti-mitogenic effect of
IFN should reside, at least partly, on
mecha-nisms other than the reduced uptake of
thymi-dine Our results also show that the antiviral
activity induced by IFN in cells persisted even
afterthecellswerepermeabilized Thissuggests
that the factors mediating the inhibitory effects
of IFN are molecules which may be tightly
bound intracellularly
This work was supported by grant CD#56 from the
Ameri-can Cancer Society.
LITERATURE CITED
1 Balkwill, F R., and J Taylor-Papadimitriou 1978 Inter-feron affects both G0 and S+G2 in cells stimulated from quiescence to growth Nature (London) 274:798-800.
2 Brouty-Boye, D., and M G Tovey 1978 Inhibition by interferon of thymidine uptake in chemostat cultures of L1210 cells Intervirology 9:243-252.
3 Castellot, J J., Jr., M R Miller, D M Lehtomaki, and
A B Pardee 1979 Comparison of DNA replication and repair enzymology using permeabilized baby hamster kid-ney cells J Biol Chem 234:6904-6908.
4 Miller, M R., J J Castellot, Jr., and A B Pardee 1978 A permeable animal cell preparation for studying macromo-lecular synthesis DNA synthesis and the role of deoxyri-bonucleotides in S phase initiation Biochemistry 17:1073-1080.
5 Sokawa, Y., Y Watanabe, and Y Kawada 1977 Suppres-sive effects of interferon on the transition from a quiescent
to a growing state in 3T3 cells Nature (London) 268:236-238.
6 Sreevalsan, T., E Rozengurt, J Taylor-Papadimitriou, and
J Burchell 1980 Differential effect of interferon on DNA synthesis, 2-deoxyglucose uptake and ornithine decarbox-ylase activity in 3T3 cells stimulated by polypeptide growth factors and tumor promoters J Cell Physiol 104:1-9.
7 Taylor-Papadimitriou, J 1980 Effect of interferon on cell growth and function, p 13-46 In I Gresser (ed.),
Interfer-on 1980 Academic Press, Inc., New York.
8 Tovey, M G., D Brouty-Boye, and I Gresser 1975 Early effect of interferon on mouse leukemia cells cultivated in a chemostat Proc Natl Acad Sci U.S.A 72:2265-2269.
9 Weck, P K., E Rinderknecht, D A Estell, and N Steb-bing 1982 Antiviral activity of bacteria-derived human alpha interferon against encephalomyocarditis virus infec-tion in mice Infect Immun 35:660-665.
VOL.45, 1983