J. gen. ViroL ( 1 9 6 9 ) , 4, 29-36
With I plate
Printed in Great Britain
29
Transformation of Rat Embryo Cells by Adenovirus Type 1
By R. M. M c A L L I S T E R , * M. O. N I C O L S O N , * A. M. L E W I S , J u s . , t
I. M A C P H E R S O N : ~ AND R. J. H U E B N E R t
* Children's Hospital of Los Angeles, Los Angeles, Calif. 90o27. ~Laboratory
of Viral Diseases, National Institute of Allergy and Infectious Diseases,
Bethesda, Md. 20014, ~ Institute of Virology, University of Glasgow, Scotland
(Accepted 24 June, 1968)
SUMMARY
Primary rat embryo cells were transformed by adenovirus I. The transformed cells formed foci of multilayered growth in monolayer cultures; cell
lines were derived from such foci. A virus stock containing I.I x lO9 virus
particles and 1.5 × lO8 p.f.u./ml, contained 2-3 x lO2 focus-forming units/ml.
when assayed in rat embryo cells. Approximately 4"8 × lO6total virus particles
or 6"5 x lO5 p.f.u, were required to induce one focus of transformed cells. The
transformation rate was o'o0o3 ~o for cells exposed to about 2 p.f.u./cell. The
transformed cells contained adenovirus I specific tumour antigens when
tested by immunofluorescent and complement-fixation reactions. They also
synthesized adenovirus I specific RNA which forms RNase-resistant hybrids
with adenovirus I DNA.
INTRODUCTION
Previous studies by Freeman et al. (I 967a) demonstrated the capacity of adenovirus
(Ad) types 2 and 5, hitherto not suspected to be oncogenic types, to transform primary
cultures of rat embryo cells in vitro. These investigators found that pulse-labelled RNA
isolated from Ad-2 transformed cells hybridized to a significant extent with the DNA
of adenovirus types I, 2 and 5. They therefore suggested that in addition to subgroup
A viruses (types I2, 18, 30, which are highly oncogenic in hamsters, and the
weakly oncogenic subgroup B (types 3, 7, 14, 16, 2 I ) , a third group, subgroup C, composed of types I, 2 and 5, had demonstrated (types 2, 5) or suspected (type 1) transforming potential in vitro.
This paper describes the transformation in vitro of rat embryo cells by adenovirus
type I.
METHODS
Virus. Adenovirus type I (MOSTstrain) was recovered from a human lymphoma and
propagated once in HeLa cells (McAllister, Landing & Goodheart, 1964). The virus
was typed using antiserum from the Research Reference Reagents Branch (National
Institute of Allergy and Infectious Diseases); the typing was confirmed by Dr Wallace
P. Rowe (personal communication). The stock contained I.I x lO9 virus particles/ml.
when counted by electron microscopy (Watson, Russell & Wildy, 1963) and 1.5 × IO8
p.f.u./ml, when assayed on human embryo kidney cells. No adenovirus-associated
virus was detected in the stock by electron microscopy.
:~ Present address: The Imperial Cancer Research Fund, Lincoln's Inn Fields, London.
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30
R.M. MCALLISTER AND OTHERS
Cell cultures. Near-term embryos from inbred Hooded Lister rats were trypsinized
and cultures seeded with 3 x lO5 cells/ml, in Eagle's basal medium containing antibiotics, twice the normal concentration of amino acids and vitamins, and supplemented
with IO % foetal calf serum. Plastic Petri dishes (5 cm. diameter) were seeded with
5 ml. of the cell suspension, incubated at 37 ° in 5 % CO2 in air, re-fed on the third day,
and used when confluent 6 days after seeding.
Liquid and agar media. Transformation assays were made in Eagle's medium without
calcium and with twice the normal concentration of amino acids and vitamins,
supplemented with 5 % calf serum and 2 % foetal calf serum (F medium). Media for
agar suspension cultures (Macpherson & Montagnier, I964) consisted of Eagle's
medium supplemented with 2o % foetal calf serum and either o'33 % or o'5 % agar.
Transformation assays. The method used was described in detail by McAllister &
Macpherson (1968) and was similar to that of Freeman et aL (I967a) and Freeman
et al. (I967b), except that Petri dish cultures were used instead of tube cultures.
Confluent monolayers of rat embryo cells (approx. 1.4 x Io 6 cells]culture) in 5 cm.
plastic Petri dishes were rinsed with tris + saline solution (pH 7"4), exposed to o. I ml.
of virus suspension for 3 hr at 37° and fed with 4 ml. of F medium. The cultures were
incubated at 37 ° in a humidified atmosphere constantly gassed with 5 % CO2 in air.
The medium was replaced every 2 to 3 days for 50 days. Foci of transformed cells were
counted after the medium had been poured off and the cultures fixed with methanol
and stained with Giemsa stain.
Tests for infectious virus. The transformed cell lines were tested for infectious virus
by passing lO5 viable cells on each of IO confluent human embryo kidney cultures. The
cultures were observed for 14 days when both o-z ml. of supernatant fluid and lO5 cells
were subcultured into fresh human embryo kidney cell cultures which were observed
for a further 21 days. There was no evidence of cytopathic effect (CPE).
Cultivation of transformed cells in agar medium. Trypsin-dispersed transformed cells
(5 x lO5) were mixed in 1.5 ml. of o'33 % agar medium and the mixture added to 5
cm. Petri dishes containing a pre-set base of 0"5 % agar medium (Macpherson &
Montagnier, 1964; McAllister & Macpherson, 1968). After I day, 2 ml. of Eagle's
medium containing 20 % foetal calf serum was pipetted on top of the agar and replaced weekly. Cell colonies were counted after 21 days.
Histological studies. Colonies of transformed cells were removed from the agar
medium 19 days after plating, fixed in fresh Bouin's solution, stained with haematoxylin and eosin (McAllister, Reed & Huebuer, I967) and examined by Drs George
Reed, Hart Isaacs, Benjamin Landing, and Howard Igel.
Serological tests. The Ad-I transformed rat embryo cell line used for preparing
antigens for immunofluorescent tests was in the I8th to the 21 st tissue culture passage.
Cells were grown on cover-glasses, fixed in cold acetone, and stained by the indirect
method of Pope & Rowe (1964) using the appropriate goat anti-hamster, sheep antirabbit, or horse anti-human conjugate diluted in tissamine rhodamine-conjugated
bovine serum albumin as a counter-stain.
For the complement-fixation tests (Huebner et al. I963) frozen and thawed extracts
of 20 % cell suspensions (11th tissue culture passage) were used for antigens.
Individual sera were obtained from animals bearing transplanted tumours induced
initially by Ad-I-SV 40 and Ad-2-SV 40 hybrid virus populations (Lewis et al. 1966;
Black et al. I967). Additional sera were obtained from hamsters immunized as
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Transformation o f rat embryo cells by adenovirus type 1
3I
weanfings with extracts of transplanted tumours induced by Ad-I-SV 40 hybrid
(Gilden, Beddow & Huebner, 1967) and a child with an Ad-i infection who subsequently developed antibody to both Ad-I T and viral antigen (Lewis, Wiese &
Rowe, I967). Serum pools were prepared from animals bearing transplanted tumours
induced by Ad-7 (PINCKNEY strain), Ad-12 (HUIEstrain), SV 40 (strain 776) or the
AHLSTROMstrain of Rous sarcoma virus. The antigens used in determining the fluorescent antibody titres of the serum specimens were homologous adenovirus-induced
T or V antigen-containing cells fixed on cover-glasses (Lewis et al. 1966; Black
et al. 1967). The 8426 line of rat embryo cells transformed by Ad-3 which was
used as a positive control was kindly provided by Dr A. E. Freeman (Freeman et al.
I967c).
Studies of nucleic acid homology. Transformed cells in their 12th tissue culture
passage were incubated for 30 min. at 37 ° in the presence of IOO/zc/ml. of [3~p]_
orthophosphate. The cells were harvested and whole-cell RNA extracted by amodification of the method of Scherrer, Latham & Darnell (I963), employing sodium dodecyl
sulphate (SDS) and hot phenol. No carrier RNA was added. The purified RNA was
treated with DNase (electrophoretically purified, Worthington Biochemicals) and
re-extracted with hot phenol+ SDS. The RNA was precipitated several times with
ethanol, dissolved in o'o15 M-NaC1, and o.oo16 M-Naa citrate, and dialysed overnight.
Specific activity of the whole-cell RNA was 12oo counts/min.//zg.
Purified adenovirus-I DNA was kindly supplied by Dr Maurice Green. Escherichia
coli [SH]DNA was prepared according to Marmur (1961) from stocks grown in
[ZH]thymidine. DNA and RNA were hybridized on membrane filters by the methods
of Gillespie & Spiegelman (I 965) and Fuj inaga & Green (I 966). Virus-specific [3~P]RNA
was identified by the formation of RNase-resistant hybrids with Ad-t DNA.
Transplantation studies. Sixty-four newborn inbred Hooded Lister rats, 24 to
48 hr old, were injected subcutaneously with I to 5 x lO6 transformed cells.
RESULTS
Transformation of rat embryo cells
When confluent monolayers of rat embryo cells were exposed to virus, loci of
multilayered growth were visible 25 days after infection (P1. 12 a). The loci were
easily recognized against a monolayer background of untransformed cells and were
identical with those induced in rat cells by adenovirus types 2, 3 and 12 (Freeman
et al. I967a, b, c; McAllister & Macpherson, I968). No areas on morphologically
altered cells were seen in uninoculated control cultures (Table 1). The smaller number
of loci appearing in cultures exposed to lO p.f.u./cell compared with those exposed to
2 p.f.u./cell may have been due to the slight cytopathic effect noted in cultures
exposed to the larger dose of virus.
The number of Ad-1 virus particles or infectious units required to induce one focus
was similar to that required by type I2 (McAllister & Macpherson, 1968) (Table 2).
Freeman et al. (1967 a) showed that IO7.3 ID 50 of Ad-2 induced loci in 17 of 27 tubes.
Our studies with Ad-2 also showed that it was less effective than our Ad-I strain in
transforming rat cells (Table 2).
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Journal of General Virology, Vol. 4, No. ~
Plate I
a. Focus of transformed rat embryo cells 2 5 days after infection. Unstained.
b. Colony of Ad-l transformed rat ceils from agar suspension culture. The cytological characteristics
resemble those of cells transformed by other adenoviruses; these include the uniform cell type,
nuclear characteristics and occasional giant cells. Haematoxylin and eosin.
c. Higher magnification of 2.
d. Malignant lymphoma from which the Ad-i was isolated. Haematoxylin and eosin.
R. M. McALLISTER AND OTHERS
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(Facll'lg
p. 3I)
32
R . M. M C A L L I S T E R
AND
OTHERS
Cell line derivation
Two foci of Ad-I transformed cells appearing in one culture 25 days after infection
were picked with finely drawn Pasteur pipettes and each was subcultured in a plastic
Petri dish in Eagle's medium supplemented with Io% foetal calf serum. Both loci
yielded cell lines with an epithelioid morphology typical of adenovirus transformed
cells. In another experiment a culture with foci of transformed cells was passaged.
After the 5th passage, the culture consisted mainly of epithelioid cells.
Table I. Transformation of rat embryo cells by adenovirus I
N u m b e r o f foci p e r m o n o l a y e r c u l t u r e (I-4 x i o s cells/
culture exposed to virus)
~
- - ~
,
Expt I
Expt 2
Virus input
(p.f.u./cell)
Io'7
2.I
i.i
o'5
4, 4
9, 7, 3, i
ND*
4, I, i , o, o
4, 4, 3, o
8, 8, 7, 5, 5, 3, 2, I, I
6, 4, 4, 2, 2, 2, i , i , i , o
ND*
Control
o, o
o. o
* ND = Not done.
Table z. Transformation of rat embryo cells by adenoviruses*
Ad-I
Focus forming units
(f.f.u.)/ml. v i r u s s t o c k )
Virus particles/f.f.u.
p.f.u./f.f.u.
2"3
× IO z
4"8 × IOs
6"5 x I o 5
Ad-2
50
2-4 × t o a
4 × 107
Ad-12
I ×
I0 3
5X
lO s
I ' I X IO~
* A d - I = I"~ x IO 9 v i r u s p a r t i c l e s / r n l , a n d I ' 5 x i o 8 p . f . u . / m l .
A d - z = I-2 x i o 1° v i r u s p a r t i c l e s / m l , a n d 2 x i o 9 p . f . u . / m l .
A d - ] z = 5 x i o 9 v i r u s p a r t i c l e s / m l , a n d I . I x xo 9 p.f.u./ml.
Cultivation in agar medium
The transformed cells in 8th passage formed colonies in agar medium with a plating
efficiency of 5 %. When the colonies were processed by standard pathological techniques, they were found to have the cytological characters of adenovirus-induced
hamster tumours (Berman, x967) or of agar suspension colonies derived from tumour
cells (McAllister et al. I967) (PI. i b, c). The cytological characters of the colonies
also resembled those of the human lymphoma from which the Ad-I was recovered
(PI. I d); however, the significance, if any, of this observation is obscure and may
well be a coincidence, especially since adenovirus-induced hamster tumours do not
appear to be derived from the reticulo-endothelial system (Dr H. Igel, personal
communication).
Antigenic analysis of Ad-I transformed cells
Immunofluorescence tests. The cells contained at least one antigen reacting specifically with sera from hamsters with high titre Ad-I T antibody. Appropriate controls
were not stained by the reacting sera and the Ad-I transformed rat embryo cells
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Type of serum
4
5
3
5
F A pool 3
CF pool 23
I> 16or
NT~
/> Ioo
/> Iooo
/> t o o
I6o*
16o*
Io
x60
640
160
40
16o
64o
o
o
I/5
1/5
ffIO
~/5
o
o
o
I/Io
o
i/IO
0
o
o
I/IO
0
I/IO
i/io
i/4o
o
o
o
t/5
I/5
I/5
o
I/5
Normal
rat embryo
Ad-~-SV 4o
Ad-2-SV 4o
Ad-5-SV 40
^
SV 40
Ot
0
O
0
32-64
o
t 0 - < 4 . N D = not done.
Adq2
Hamster tumour antisera
o
o
o
o
o
8o-9o
20-30
< I
5-I o
o
80-90
4o-50
8o-9o
8o-9o
A d q trans.
rat embryo
Specificity of adenovirus tumour antigen as determined by complement fixation
A d q transformed rat cells
32-64*
I6-32
16-32
Ad-2 transformed rat cells
4-8
4-8
ND
Ad-12 transformed rat cells
o
o
ND
* Reciprocal of complement fixing antigen titre (2o ~ cell extracts).
Antigen
T a b l e 4.
438 x-H
438I-H
Io88-H
Io88-H
R 726-H 2
R 726-H 3
Acute
Convalescent
LRAS- i i
F A pool G
F A pool H
F A pool D
H
H
R
R
Serum
Screening
dilution
0
0
0
SCHMIDT-RUPPIN
NT
NT
7o-80
o
20-30
o
o
NT
NT
NT
o
o
I-5
o
Ad-3 trans.
rat embryo
A
homologous
antigen
Antigens (positive nuclei, ~ )
V,
r
F A titre
* Titrated against Ad-i T antigen containing cells.
t Titrated against SV 4o T antigen induced by E 46 + .
+ CF antigen of I/8o against 4-8 units of AHLSTROMhamster tumour antigen.
Ad-I hyperimmune-rabbit antiserum
F r o m hamster with Ad-i2 induced
tumour
F r o m hamster with Ad-7 induced
tumour
F r o m hamster with SV 4o induced turnout
F r o m hamster with AHLSTROMRSV
induced tumour
F r o m 2rid weanling hamster passage of
A d q - S V 40 hybrid-induced tumour
Serum from hyperimmune hamsters
immunized with extracts of A d q - S V 40
hybrid-induced turnout
F r o m 4th weanling hamster passage of
Ad-2-SV 40 hybrid induced tumour
Serum from patient with Ad-I infection
h. . . . . .
Antisera
Immunofluorescent studies of Ad-I transformed rat embryo cells
......
T a b l e 3.
.~
t~
I:1
34
R.M. MCALLISTER
AND OTHERS
failed to react with sera containing other specific, high titre viral and T antibodies
(Table 3). Morphologically, the nuclear staining pattern was indistinguishable from the
staining of Ad-I and -2 T antigens in human embryo kidney cells (Black et al. 1967) and
that described by Freeman et al. (I967a) for the Ad-2 transformed rat embryo cells.
Most nuclei contained small irregular particles or dots which gave a dust-like appearance quite distinct from the thin needle-like nuclear flecks seen in Ad-7 or Ad-I2
transformed hamster or rat cells. In addition to the dust-like pattern some nuclei
contained small dots or ovoid bodies and IO % to 20 % of the cells contained I or 2
highly staining ovoid bodies in the cytoplasm. The nuclear staining with human
convalescent serum, while morphologically similar, was less frequent and less intense
than that with sera from hamsters bearing tumours induced by hybrid virus. The
cytoplasmic bodies were not stained by either of the two convalescent human sera
tested. These findings suggest a different range of antigens in the Ad-I transformed
rat embryo cells from those in Ad-I infected human and monkey ceils.
Complement fixation tests. Antigens prepared from the Ad-I transformed cells
reacted with sera from hamsters bearing tumours induced by Ad-I-SV 40, Ad-2-SV 40
or Ad-5-SV 4o hybrid viruses but not with sera from hamsters bearing tumours induced by Ad-I2, SV 40 or SCHMIDT-RUPPINviruses (Table 4).
Table 5. Nucleic acid homology
DNA
Adenovirus-i, 3 #g.
Escherichia coli K 996, 3 #g.
Ad-i transformed
whole-cell
RNA input
RNA bound
(counts/rain.)
(counts/min.)*
26,9o0
2o0
269,00o
746
26,9OO
24
269,000
3
Input
bound (%)
0"74
0.28
O-O8
O'0OI
* Blank value: filter incubated in [82P]RNAin absence of DNA was subtracted from each sample.
Nucleic acid homology
The results are shown in Table 5. It is readily apparent that Ad-I transformed cells
synthesized Ad-I specific R N A which formed RNase-resistant complexes with Ad-I
DNA. By comparison, hybrids were formed with Escherichia coliDNA only to alimited
extent. In the range of R N A concentration examined, a tenfold increase in R N A
resulted in about a fourfold increase in hybrid formed. Although a saturation plateau
cannot be determined from two points, the data suggest thatthe viralRNA represented
a small fraction of the total transformed cell RNA.
Transplantation studies
Forty-eight of the 64 Hooded Lister rats which were injected with Ad-I transformed
cells survived infancy and failed to develop tumours during I2 months' observation.
DISCUSSION
An unexpected result of our studies as well as those of Freeman et al. (I967a) has
been the failure of Ad-I or Ad-2 transformed cells to induce tumours in rats. Why
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T r a n s f o r m a t i o n o f r a t e m b r y o cells b y a d e n o v i r u s t y p e I
35
A d - i t r a n s f o r m e d cells d o n o t induce t u m o u r s is u n k n o w n a n d o f interest since r a t
cells t r a n s f o r m e d b y A d - 3 a n d A d - I 2 d o i n d u c e t u m o u r s ( F r e e m a n et al. i 9 6 7 b , c).
A d - I a n d A d - 2 m a y p r o d u c e strong t r a n s p l a n t a t i o n antigens in the ceils they transf o r m , thus m i n i m i z i n g their chances o f initiating a focus o f t u m o u r cells. Tests t o
m e a s u r e adenovirus-specific surface antigens o n these cells are in progress.
T h e a u t h o r s wish to t h a n k D r E. A. C. F o l l e t t for the electron m i c r o s c o p y a n d M i s s
J o a n Beveridge, M r s L i n d a G o l d e n , M r s M a r y Peer, Miss M a r y A n n Gaffey a n d M r s
L i a n n e D i x o n for technical assistance.
This investigation was s u p p o r t e d b y Public H e a l t h Service R e s e a r c h G r a n t no.
CA-o4865 f r o m the N a t i o n a l C a n c e r Institute a n d P o s t d o c t o r a l Scholarship G r a n t
no. PS-32 f r o m the A m e r i c a n C a n c e r Society.
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R.M. MCALLISTER AND OTHERS
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