[CANCER RESEARCH 34, 3497-3500,
December 19741
Brief Communication
Enhancement of Simian Virus 40 Transformation and
Integration by 4Nitroquinoline 1.Oxide'
K. Hirai,2 V. Defendi,2and L. Diamond
The Wistar Institute ofAnatomy
and Biology, Philadelphia, Pennsylvania 19104
the surviving fraction of 4NQO-treated cells was one-fourth to
one-half that of untreated cells, as determined by the
SUMMARY
Treatment of Chinese hamster embryo cells with 4-nitro
quinoline 1-oxide prior to infection with SV4O increases the
frequency of cell transformation,
the level of nuclear
penetration
of SV4O, and the amount of SV4O DNA
integrated into cell DNA.
Pretreatment
of animal cells with X-ray (4, 14, 18),
thymidine analogs (19), and chemical carcinogens (2, 3, 9)
increases the sensitivity of survivingcells to transformation by
DNA-oncogenic viruses. The property that many of these
agents share is the abifity to induce strand breaks in cellular
DNA. If viral transformation is dependent upon integration of
viral DNA into cellular DNA, then the enhancement of
transformation may be mediated through facilitation of the
integration of viral DNA at the breakpoints of cell DNA. We
have tested this possibility in the nonpermissive SV4O ChH3
cell transformation
system (1 1) in combination with the
chemical carcinogen, 4NQO.
4NQO induces single- or double-strand scissions of mamma
lian cell DNA, and the scissions produced by low concentra
tions of 4NQO are completely rejoined after incubation of
cells in medium without 4NQO (1). Enhancement of SV4O
and simian adenovirus 7 transformation by 4NQO has been
described
in different
cell systems
including
ChH (5 , 9 , 16).
We have shown previously that SV4O DNA becomes integrated
into the DNA of CMI cells at 15 to 20 hr p.i. (8).
The conditions of SV4O infection, 4NQO treatment, and
the assay of transformation have been described (5), as has
been the procedure for hybridization of SV4O complementary
RNA with cell DNA to measure the extent of viral DNA
integration (8). Several experiments demonstrate that treat
ment of CMI cells with 4NQO (0.4 @g/ml)2 hr before SV4O
infection induces a 2- to 8-fold increase in the transformation
frequency
published
I Supported
(Table
methods
in
part
1), as calculated
according to previously
(3, 5, 10). At this concentration
by
American
Cancer
Society
Grants
of 4NQO,
VC-73
and
PRP-45 and by USPHS Research Grants CA-10815 and CA@08936from
the National Cancer Institute.
2Present address: New York University Medical Center, 550 First
Avenue, New York, N. Y. 10016.
3The abbreviations used are: ChH, Chinese hamster embryo; 4NQO
4-nitroquinoline 1-oxide; p.i., postinfection.
Received August 20, 1974; accepted September 17, 1974.
colony-forming
efficiency
in liquid
medium.
There
is no
preferential killing by 4NQO of noninfected cells since the
percentage of I-antigen-positive
cells in 4NQO-treated or
nontreated culture is the same up to 48 hr p.i. (Chart 1). It has
also been shown that enhancement
is not due to selection
of
cells in the original population that are resistant to 4NQO
since cells transformed by 4NQO + SV4O are as susceptible to
4NQO cytotoxicity as is the parental untransformed popula
tion (5). Cells from the colonies that grew in agar were positive
for the SV4O—Tantigen and no colonies appeared in agar after
treatment with 4NQO alone, indicating that only SV4Otransformed colonies are detected in this system.
The same chronological sequence of 4NQO treatment and
SV4O infection as that shown in Table 1 was used in the
experiments described below. Cells were treated with 4NQO,
(0.4 j.tg/ml) for 2 hr just prior to SV4O infection and
subcultured 24 hr later to bring both treated and untreated
cultures to the same density. After another 24 hr of
incubation, nuclei were isolated from the cells of the infected
culture and layered on alkaline glycerol gradients to separate
cell DNA from SV4O DNA. The cell DNA was hybridized with
SV4O complementary RNA-3H. As shown in Table 2, the
number of “integrated―
SV4O genome equivalents per diploid
cell at 48 hr p.i. was greater in 4NQO-treated cells than in
untreated infected cells. This was also verified in experiments
in which the genome equivalent was determined by DNA-DNA
reassociation kinetics (7). Although there were variations in
the degree of enhancement of integration, they were similar to
the variations observed in the enhancement of transformation
frequency.
The fInding that there were more genome equivalents
integrated in the 4NQO-treated cells is compatible with the
hypothesis that the strand scission of cell DNA produced by
4NQO facilitates integration. However, 4NQO treatment could
have other effects on the cells that may be related to the
enhanced integration or transformation. For example, 4NQO
treatment could render the nonpermissive CHH cells permis
sive for viral DNA replication, thereby increasing the number
of SV4O DNA molecules available for integration ; however,
when thymidine-3 H was added to the 4NQO-treated cultures,
we were not able to detect any newly synthesized SV4O DNA
by ethidium bromide-CsCl density gradient equilibrium centri
fugation. Another possibility is that 4NQO modifies the
DECEMBER 1974
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3497
K. HiraietaL
Table 1
Enhancement ofSV4O-induced transformation by 4NQO
The preparation of the ChH cells and the transformation assay have been described (5). Cell
monolayers at a density of 1.5 to 2 X 106 cells/iS sq cm (Falcon plastic flask) were treated
with complete medium containing 4NQO (0.4 pg/ml). After 2 hr, the 4NQO-containing
medium was removed, and the monolayers were washed twice with phosphate-buffered saline
and infected with SV4O at a multiplicity of 25 to 50 plaque-forming units/cell. After a 2-hr
adsorption period, the monolayers were washed twice with phosphate-buffered saline and refed
with complete medium. Twenty-four hr later, the cells were trypsinized and plated into liquid
medium to determine the colony-forming efficiency and into soft agar medium to determine
the transformation frequency.
colonies
%
transforma
Experi
in agar
ment4NQO
factor10 (jig/mI)Surviving
fractionaNo.of medium (%)bCalculatedtioncEnhancement
0.1324.820 0.41.0
0.2556(0.028)67 (0.033)0.028
0.0622.330 0.41.0
0.3155(0.027)40(0.020)0.027
0.0062.040 0.41.0
0.466(0.003)
0.0648.050 0.41.0
0.3015
(0.008)
38(0.019)0.008
.0
0.468
(0.004)
14 (0.007)0.004
0.41
a The
surviving
fraction
was
calculated
6 (0.003)0.003
from
the
0.0153.8
colony-forming
efficiency
in
liquid
medium.
The colony-forming efficiency of untreated cells was 5 to 10%.
b Two X 10' cells plated in soft agar medium.
C%of coloniesin agarmedium/surviving
fraction.
—
@
100
/_@
4NQO
CONTROL
50
10
20
30
40
50
TIME AFTER INFECTION (HR)
Chart 1. Effect of 4NQO pretreatment on T-antigen induction. Cell
monolayers were treated with complete medium containing 4NQO (0.4
@tg/ml).After 2 hr the monolayers were washed with phosphate-buf
fered saline and infected with SV4O at a multiplicity of 50
plaque-forming units/cell. At various times thereafter, immunofluores
cence assay for SV4O T-antigen was performed as previously described
(11). ., no 4NQO;£,4NQO(0.4 pg/mi).
uptake of SV4O. To test this possibility, the amount of SV4O
DNA in the cell nuclei at 5 hr pi. was measured by
RNA-DNA hybridization. The amount of SV4O DNA per
nucleus of 4NQO-pretreated cells was about twice as high as
the amount in the nuclei of untreated cells (Table 3), showing
known. It has been reported (17) that the DNA repair
synthesis induced in Syrian hamster cells by 4NQO is
completed by 8 to 10 hr. SV4O DNA becomes integrated in an
alkaline stable form into the DNA of ChH cells at 15 to 20 hr
p.'- (8) and, therefore, most of the carcinogen-induced damage
that 4NQOpretreatment
enhances
thepenetration
of SV4O to DNA may have been repairedprior to virus integration.
into cell nuclei. Although no overt toxic effect of 4NQO is
apparent at 5 hr p.i. or 7 hr post-4NQO, it is possible that the
increased virus uptake reflects an early alteration of membrane
permeability.
At present, the mechanisms for enhancement of viral
penetration into cell nuclei and of viral integration are not
3498
However, it may be that, in ChH cells, the parental strands
still contain unrepaired 4NQO-induced breaks at the time
SV4O DNA becomes integrated.
Since SV4O infection induces polynucleotide ligase activity
in monkey kidney cells at 24 hr p.i. (15), it is possible that
integration of SV4O DNA may be facilitated at sites of
CANCER RESEARCH VOL. 34
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4NQO Enhancement
ofSV4O Transfonnation
and Integration
Table 2
Enhancement by 4NQO ofSV4O integration into 0tH cell DNA at 48 hr p.L
At 48 hr p.i., nuclei (S X 106) were isolated by the Penman (1 3) method using the non-ionic,
detergent, NP-40, suspended in 1 ml of 0.01 M Tris (pH 7.4)-0.0l M NaCI-0.OOlS M MgCI.@
buffer containing 0.5% NP-40 and 0.5% sodium deoxycholate and layered on an alkaline
glycerol gradient (10 to 30%) in 0.3 N NaOH, 0.01 M methylenediaminetetraacetate, and 0.5 M
NaCI. After storage at 4°overnight to allow complete liberation of the DNA, the solution was
centrifuged for 4.5 hr at 63,000 x g in a Beckman Spinco SW 25.1 rotor. The fractions were
collected from the top by an ISCO density gradient collector. The cell DNA fractions were
pooled and the pH of the solution was neutralized. The cell DNA was immobilized on filters
and hybridized with SV4O complementary RNA-3 H that had been made in vitro by Escherichia
coli DNA-dependent RNA polymerase as previously described (6). The radioactivity of SV4O
complementary RNA-3 H hybridized with cell DNA was normalized to cpm/100 pg DNA after
subtraction of the amount bound to normal ChH DNA. The numbe@ of SV4O genome
equivalents per diploid cell was calculated from the radioactivity (Experiment 1 348 cpm;
Experiment 2 = 328 cpm; Experiment 3 = 363 cpm; Experiment 4 = 274 cpm/lOO pg of DNA)
of the SV4O complementary RNA-3 H hybridized with the DNA of SV3T3 T2 -cells that contain
2.2 copies of SV4O genome per diploid cell (12) and with the DNA of ChH-Cl 5 and CI 21,
which contain, respectively, 1.2 and 19.2 equivalents of SV4O genome per diploid cell (7).
SV4O
Experi
complementary RNA-3H
factor10 (pg/mI)Hybridized
(cpm,I100 pg DNA)SV4O
ment4NQO
82.05.420
0.42,395
13,03015.1
15.01.830
0.41,220
2,2218.2
30.57.340
0.4693
5,0204.2
0.41,030
5,6898.6
Table 3
genome
equivalents!
diploid cellEnhancement
47.55.5
ACKNOWLEDGMENTS
Enhancement
by4NQO ofSV4Openetration
into
nuclei
ofChH cells
The expert technical assistance of R. Knorr is gratefully acknowl
At 5 hr p.i., the nuclei (106) were isolated from SV4O-infected CHH
cells and the DNA was extracted by the sodium dodecyl sulfate-phenol edged.
method and hybridized with SV4Ocomplementary
(6).Hybridized
RNA-' H
REFERENCES
SV4O complementary
nuclei)ExperiRNA-'H (cpm/lO'
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ment 1
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1059
2653
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6123
1028
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The positive correlation between the increase in the amount
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to determinewhether, after 4NQO treatment, there aremore
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CANCER RESEARCH VOL. 34
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Enhancement of Simian Virus 40 Transformation and Integration
by 4-Nitroquinoline 1-Oxide
K. Hirai, V. Defendi and L. Diamond
Cancer Res 1974;34:3497-3500.
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