TRANSFORMA TION OF PRIMARY RA T EM1BRYO CEI LS BY ADENOVIRUS TYPE 2* By AARON E. FREEMANI PAUL H. BLACK,t EUSTACE A. VANDERPOOL,t PATRICK H ...
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TRANSFORMA TION OF PRIMARY RA T EM1BRYO CEI LS BY
ADENOVIRUS TYPE 2*
By AARON E. FREEMANI PAUL H. BLACK,t EUSTACE A. VANDERPOOL,t
PATRICK H. HENRY,§ JOAN B. AUSTIN,t AND ROBERT J. HUEBNERt
MICROBIOLOGICAL ASSOCIATES, INC., AND NATIONAL INSTITUTES OF HEALTH,
BETHESDA, MARYLAND
Communicated July 25, 1967
Based upon their oncogenic properties in newborn hamsters, the adenoviruses
have been divided into three subgroups:" 2 subgroup A, highly oncogenic adeno-
virus types 12,3 18,4 and 31;5 subgroup B, weakly oncogenic adenovirus types
3,6 7,7 14, 16, and 21 ;8 and an unclassified subgroup including the presumably non-
oncogenic adenoviruses.9-" The transplantable tumors produced by the oncogenic
serotypes were found to be free of infectious virus, but to contain virus subgroup
specific complement-fixing (CF)'2 and fluorescent antibody (FA) T (tumor) anti-
gens13 which reacted with sera from animals having tumors. Cell cultures de-
rived from the adenovirus-induced tumors'4 were shown to be sensitive to calcium
at concentrations generally used in tissue culture media."' 16 When the calcium
concentration was reduced to 0.1 mM, cell growth was greatly facilitated.16
In vitro transformation of hamster and rat cells by adenovirus type 12 (AD-12)
had been reported previously;"3 17, 18 however, use of low calcium medium per-
mitted more reproducible quantitation of the transformation event in rat cells
and facilitated the derivation of transformed cell lines.'9 Subsequently, using
low calcium medium, we succeeded in producing transformation of rat cells with
adenovirus type 3 (AD-3)20 and adenovirus type 7 (AD-7).21 It seemed reason-
able, therefore, to use the low calcium medium in attempts to transform rat cells
with several of the nononcogenic adenoviruses. The purpose of this note is to
present evidence of transformation of rat embryo cells by adenovirus type 2 (AD-2).
Materials and Methods.-Virus: Adenovirus type 2, prototype strain Adenoid 6, was obtained
from Dr. Wallace P. Rowe and passed once in human embryonic kidney (HEK) cultures. The
HEK-grown pool (titer 109.2 I5Do/ml) was subdivided into ampoules and stored in the vapor phase
above a liquid nitrogen reservoir. Purity of the virus was confirmed by "breakthrough" neutrali-
zation tests22' 23 against Ad-2 specific antisera in HEK cultures. By means of CF tests, the virus
stock was also found to be free of adenovirus-associated virus (AAV) types 14.24
Cell cultures: Embryos, delivered by caesarian section from near-term inbred Fisher rats, were
decapitated, minced, washed, trypsinized, and planted at 2 X 105 cells per milliliter in Eagle's
basal mediums with 10% fetal bovine serum, 2 mM glutamine, and penicillin and streptomycin
in concentrations of 100 units and 100 iMg/ml, respectively. These cultures were incubated at
370C under 5% CO2 in air, were fed on the third day, and used when confluent, usually 5 days
after seeding.
Media: Transformation studies were carried out in Eagle's minimum essential medium21
formulated without calcium, and supplemented with 5% dialyzed calf serum, 2% fetal bovine
serum, 2 mM glutamine, 0.2 mM nonessential amino acids, and antibiotics.16 From a 0.5 M stock
of calcium chloride, calcium was added to the medium at a final concentration of 0.1 mM.
Transformation studies: Confluent stationary tube cultures were drained and inoculated with
0.1 ml of virus serially diluted in the low calcium growth medium. Twenty-seven cultures per
dilution were inoculated, the inoculum per tube representing 108.2 or 107.2 infectious units of virus.
Virus was adsorbed at 370C for 4 hr, with manipulation of the culture every 15 min to assure
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optimal contact with the cells. After the adsorption period, the cultures were fed by adding 1
1205120}626.TliROBIOJOGY: YI'EEAI .1.\N 7'A TALN.
Pit.,c. N. A. S.9
11 of medium which was replaced every other day throughout the course of the experiment,
approximately 60 days. The cultures were maintained in a stationary position at 370C.
Tests for infectious virus: Transformed cell lines were tested for infectious virus by passing
viable cells and/or supernatant fluid on confluent HEK cultures. In addition, an equivalent of
2 X 106 cells was added in extract form (20% uniclarified, 3 X frozen and thawed) to each of nine
tube cultures of HEK. Cultures were observed up to 21 days, at which time 0.2 ml of super-
natant fluid was passed from each negative culture into a new HEK culture which was also ob-
served for a 21-day period. There was no evidence of cytopathic effect (CPE).
Calcium sensitivity tests: Replicate cultures of transformed cells were fed and maintained in
media containing 0.1 and 5.0 mM calcium. Cultures were considered calcium-sensitive if retrac-
tion, clumping, or sloughing occurred within 6 days in the medium containing 5.0 mMd calcium.
Antigenic analysis: For FA tests, cells grown on glass cover slips were fixed in cold acetone and
stained by the indirect procedure described by Pope and Rowe.'3 The following sera were used at
dilutions of 1:5 to 1: 10: sera from hamsters bearing transplanted tumors induced with the
Ad-1-SNV4 hybrid virus,27 the Ad-2-SV4o hybrid virus,'7 and a line of hamster kidney cells (Ad-2++
HK-1)28 transformed with the Ad-2-SV4o hybrid virus. Also used were an Ad-2 hyperimmune
rabbit serum, and acute and convalescent human sera from a patient with a proved Ad-1 virus
infection and a subsequent rise in Ad-1 T antibody.29' 30 Control sera were obtained from normal
hamsters and from hamsters bearing transplanted tumors induced with Ad-7, Ad-12, SV40, and
the Schmidt-Ruppin strain of Rous sarcoma virus. Goat antihamster gamma globulin conju-
gated with fluorescein-isothiocyanate was used with the hamster sera, while sheep antirabbit and
horse antihuman conjugates were utilized in FA tests carried out with the rabbit and human
sera, respectively.
For the CF tests,12' 31 antigen was obtained from thrice-frozen and thawed extracts of 20%O cell
pack preparations. Approximately 4-8 units of both antigen and antibody were utilized.
Nucleic acid homology studies: The cells were incubated as a spinner culture (2 X 106 cells/ml)
for 3 hr at 370C in the presence of 10 Muc/ml each of uridine-5-H3, 25.4 c/mM (Nuclear Chicago),
and adenine-H3, 3.6 c/mM (New England Nuclear). The cells were harvested and RNA was
extracted by a sodium dodecyl sulfate (SDS)-phenol procedure as described by Scherrer and
Darnell.3' The RNA was washed with 3 M sodium acetate, pH 6.0, and treated with DNase
prior to using it in the hybridization studies. Nucleic acid homology studies were performed
rising a modification of the membrane filter method of Nygaard and Hall.33 The rat liver DNA
used in these studies was extracted by a previously described modification of the Marmur method.34
The virus DNA was extracted by papain digestion of the viruses followed by SDS: phenol ex-
traction.35 Aliquots corresponding to 6 jig of the virus and 10 ,ug of the host cell DNA's were
reacted with approximately 2.5 X 106 cpm of radioactive whole cell RNA. Triplicate deter-
minations were made with each RNA-DNA combination. The blanks contained 4,ug of E. coli
I)NA per vial.
Results.--Morphological alteration: At the time of inoculation with Ad-2, the
cultures consisted of a smooth monolayer of fibroblastic cells with occasional multi-
layered areas caused by local retraction of the cell sheet. The condition of the
cultures remained essentially the same for the first few days after inoculation, but
on the fourth to sixth days, there were signs of adenovirus infection marked by cell
degeneration and aggregation of rounded cells. The CPE progressed until by the
seventh to tenth days, very few cells remained. These remaining cells multiplied,
yielding a secondary culture consisting of fibroblasts similar to those in the uninoc-
ulated cultures. There were one to three distinct episodes of CPE followed by
regrowth of the cultures. A few isolated foci of small epithelioid cells were found
in cultures as early as 30 days after inoculation with either 108-2 or 107-2 infectious
doses of virus. These foci increased rapidly in size with piling up of the cells to
form colonies with domed centers (Fig. 1). Transformed colonies were clearly dif-
ferentiated from multilayered areas caused by retraction because the cells on the
margins of foci were oriented circumferentially whereas cells on the margins of
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0\
V
a.4
*.d
:i
46
8.%0 .4
* 94 , .1,
¾ 641
¾
is C
0
0
0
.f. ..I
FIG. 1.-Focus of transformed rat embryo cells in a tube inoculated
40 days before with adenovirus type 2. X90.
explants tended to orient radially to the colony. Transformed foci were found in
18 of 27 cultures inoculated with 108.2 IDw and in 17 of 27 cultures inoculated with
107 2 ID50. When compared with Ad-3 and Ad-12 transformed rat embryo cells
(Table 1), the cultures were found to be very similar regarding both the cell and
colonial morphology. No areas of morphologically altered cells were seen in 27
uninoculated control cultures.
Cell line derivation: From six attempts, six cell lines were obtained from trans-
formed tube cultures. For the first ten passages, these lines consisted of varying
percentages of the transformed epithelioid cells which seemed to grow on top of a
fibroblastic background. Although the number of fibroblasts gradually decreased
with passage, they were remarkably persistent, surviving in passage longer than
the uninoculated controls which grew very slowly after the sixth passage. After
the tenth passage, however, each of the transformed cell lines consisted mainly of
epithelioid cells.
Growth characteristics compared with Ad-3 and Ad-12 transformed cells: When
the established Ad-2 transformed cell lines were compared with Ad-3 and Ad-12
transformed rat lines (Table 1), they were found to be similar in that the cells in
all three types of culture had lost contact inhibition with the development of
high cell population densities. Further, all three types of transformed cultures
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appeared to grow indefinitely in vitro. To date, the Ad-12 transformed cultures120)8 0MICROBIOLOGY: FREEMAN ET AL. P)Roc. N. A. S.
TABLE 1
EVIDENCE OF TRANSFORMATION BY ADENOVIRUS TYPE 2
AS COMPARED WITH ADENOVIRUS TYPES 3 AND 12
Criteria for AAd-2 transformed Ad-3 transformed Ad-12 transformed
transformation rat cells rat cells2° rat cells's
Nolispecific criteria:
Morphological Same as Ad-12 Same as Ad-12 Same round undiffer-
alteration-cell tr ansformed cells transformed cells entiated epitheloid
cells
Morphological Same as Ad-12 Same as Ad-12 Compact, fried-egg
alteration-colony transformed cells transformed cells appearance
Loss of contact Same as Ad-12 Same as Ad-12 Yes, monolayer popu-
inhibition triansformed cells transformed cells lation density-5 X
10' cells/cm2
Indefinite life span To adate, over 50 To date, over 100 To date, over 200
in culture dc)ublings doublings doublings
Growth in suspen- Yes, 30 hr generation Yes,* generation time Yes, 24 hr generation
sion culture tiime not studied time
Virus-free Yes Yes Yes
Transplantability On ttest Yes, 30% newborn rats Yes, 90% of newborn
50-60 days after in- rats, 30-35 days
oculation with 106 after inoculation
cells with 106 cells
Adenovirus group
specific criteria:
Calcium sensitivity Yes Yes Yes
Adenovirus subgroup
specific criteria:
Tumor antigen-CF Yes, t titers to 1:8 Yes, titers to 1:16 Yes, titers to 1:64
Tumor antigen-FA Yes, t 60-100%o of cells Yes, 90-100% of cells Yes, 90-100%', of cells
Adenovirus Yes, , Ad-1,2,5 specific Yes, Ad-3,7,21 specific Yes, Ad-12,18,31 spe-
messenger RNA cific
* These studies were performed in the laboratory of Dr. Klaus Schell.43
t Tested against sera from hamsters bearing tumors induced by Ad-1-SV40 or Ad-2-SV4O hybrid viruses.
have doubled over 200 times, the Ad-3 transformed cultures have doubled over 100
times, and the Ad-2 transformed cultures have doubled over 50 times. Each type
of transformed cell has been established in suspension culture. The Ad-2 trans-
formed cells divide with an average generation time of 30 hours as compared with
24 hours for Ad-12 transformed cells. Experiments are in progress to determine
whether the Ad-2 transformed rat cells can be transplanted into newborn rats.
Calcium sensitivity: When grown in 5 mM calcium, five of the six Ad-2 trans-
formed lines retracted, clumped, or sloughed off the glass within six days. Usually
retraction occurred within 48 hours and the cultures appeared to consist of inter-
connecting fibrous bands. As retraction continued, the cultures appeared to con-
sist of isolated islands of cells tending to form free floating aggregates. In the final
stages, almost all of the cells, although still viable, were floating in the medium.
Antigenic analysis of Ad-2 transformed cells: FA tests: The results of fluorescent
antibody studies carried out with one cell line of Ad-2 transformed rat embryo cells
are detailed in Table 2. The transformed cells were stained with sera from hamsters
bearing transplanted tumors induced with the Ad-1-SV40 and Ad-2-SV40 hybrid
viruses, with Ad-2++ HK-1 transformed cells, and with a convalescent serum from
a patient with a proved Ad-i virus infection. For the most part, the staining was
confined to the nucleus; however, some cytoplasmic staining was also observed.
The nuclear staining, which occurred in 10-100 per cent of the cells, was in the form
of small dots, short flecks, ovoids, and small balls. Frequently, nuclei were filled
with tiny dots, giving a speckled or dustlike appearance. in general, the nuclear
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fluorescence was similar to the staining of Ad-i and Ad-2 T antigens, in HEK cells,VOL. 58, 1967 MICROBIOLOGY: FREEMAN ET AL. 1209
TABLE 2
SPECIFICITY OF ADENOVIRUS TYPE 2 TUMOR ANTIGEN AS DETERMINED BY FA ANALYSIS
Cells Stained (%)
Antisera Ad-2 Transformed Normal Rat Cells
Virus Type of serum Nucleus Cytoplasm Nucleus Cytoplasm
Ad-l-SV4 hybrid Hamsters with transplants of
virus-induced tumors* 60-100 10-20 0
Ad-2-SV4o hybrid A hamster with a transplanted
virus-induced tumor 60-100 10-20 0
Ad-2-SV4 hybrid Hamsters carrying transplants of
in vitro transformed cells (Ad-
2++HK-1 Line)t 10-20 5-10 0
Ad-1 Human infection-acute 0 0 0
Ad-i Human infection-convalescent 10-20 5-10 0
Ad-2 Antiviral rabbit 0 0 0
None Normal hamster 0 0 0
Ad-7 Hamsters with transplants of
virus-induced tumor 0 0 0
Ad-12 Hamsters with transplants of
virus-induced tumor 0 0 0
SV40 Hamsters with transplants of
virus-induced tumor 0 0 0
Schmidt-Ruppin Hamsters with transplants of
strain of Rous virus-induced tumor 0 0 0
sarcoma
* Three separate hamster tumor sera wvere tested.
t Serum pool from three hamsters was tested.
with sera from hamsters bearing tumors induced by Ad-l-SV40 or Ad-2-SV40 hybrid
viruses.36 The cytoplasmic staining, which occurred in 5-20 per cent of the cells,
was in the form of one or two highly fluorescent ovoid or fleck-shaped bodies. Not
infrequently, cells with cytoplasmic staining were devoid of nuclear fluorescence.
The cytoplasmic staining was reminiscent of a characteristically large fleck-shaped
antigen seen in Ad-12 transformed cells stained with Ad-12 hamster tumor sera."3
The Ad-2 transformed cells did not stain with Ad-2 hyperimmune serum, normal
hamster serum, or sera from hamsters bearing tumors induced with Ad-7, Ad-12,
SV4o, or with the Schmidt-Ruppin strain of Rous sarcoma virus. Normal rat
cells were not stained by any of the sera tested. In addition, Ad-3 and Ad-12
transformed rat cells were hot stained with sera from hamsters bearing tumors in-
duced with Ad-1-SV40 or Ad-2-SV40 hybrid viruses.
CF tests: Antigens prepared from the Ad-2 transformed cultures were found to
react with sera from hamsters bearing tumors induced by the Ad-1-SV40 or Ad-2-
SV40 hybrid viruses but not with sera from hamsters bearing Ad-3, Ad-12, SV40, or
Schmidt-Ruppin virus-induced tumors (Table 3). Since adenovirus-infected cells
also produce T antigens, extracts of the Ad-2 transformed cells were passed in
HEK cultures and there was no evidence of the presence of infectious virus.
Nucleic acid homology studies: Recently, Fujinaga and Green have shown that
adenovirus-transformed cells contain virus-specific RNA which hybridizes with the
DNA's of members within the same subgroups. Thus Ad-12 transformed cells
produced an RNA which hybridized with the DNA's of Ad-12, -18, or -31;37 Ad-3
transformed cells produced RNA which hybridized with the DNA's of Ad-3, -7,
or -21.38 There was no hybridization of Ad-12 or Ad-3 RNA with the DNA's of
the nononlcogenic adenoviruses. Studies in our laboratory demonstrated that RNA
derived from Ad-2 transformed cells hybridized with Ad-2 DNA but not with Ad-3,
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Ad-7, or Ad-12 DNA. Ill addition, no hybridization occurred with Ad-2 trans-1ICROBIOLOGY:
1210 FREEMAN ET AL. PROC. N. A. S.
TABLE 3
SPECIFICITY OF ADENOVIRUS TUMOR ANTIGEN AS DETERMINED BY CF TEST
Hamster Tumor Antisera
Schmidt-
Antigen Ad-3 Ad-12 Ad-1-SV4o Ad-2-SV40 SV40 Ruppin
Ad-2 transformed rat cells 0* 0 4-8t 4-8 0 0
Ad-3 transformed rat cells 8-16 0 0 0 0 0
Ad-12 transformed rat cells 0 32-64 0 0 0 0
*0 =VOL. 58, 1967 MICROBIOLOGY: FREEMAN ET AL. 1211
supported by the homology experiments which showed that the Ad-2 transformed
cells produced RNA which hybridized with the DNA's of adenovirus types 1, 2, and
5, but not adenovirus types 3, 4, or 12, or with SVm. The subgroup specificity of
this reaction provided evidence of a third group of oncogenic adenoviruses pre-
viously included among those which were regarded as lacking oncogenic activity.
It would seem logical to characterize this third group as subgroup C.
It is possible that other members of this apparent subgroup may be oncogenic.
In support of this view, we have derived cell lines morphologically transformed by
Ad-5.40 It is also quite possible that under the proper conditions, additional ad-
enoviruses will prove to have oncogenic activity.
By age five, as many as 90 per cent of urban children have been reported as having
been infected with adenovirus types 1, 2, or 5.41 These viruses, moreover, were
also recovered from the adenoids and tonsils of the majority of children studied.42
Thus infections occur early in life and persist in the nasopharyngeal and lymphoid
tissue for an undetermined number of years.
It has been suggested that sera from human cancer patients and matched con-
trols should be tested to determine if antibodies to T antigens of the A and B group
can be correlated with certain neoplastic diseases.' The transformation of rat
embryo cells with Ad-2 provides complement-fixing T antigens representative of a
third (C) subgroup of oncogenic adenoviruses for possible use in such seroepidemio-
logical surveys. Similarly, the ready demonstration in rat cells transformed by
Ad-2 of an RNA complementary to the DNA's of three members of the C subgroup
of adenoviruses makes it worthwhile to test human tumors for the presence of the
RNA representative of 11 adenoviruses having demonstrated or suspected onco-
genic activity.
We thank Mr. Horace C. Turner and Mr. Ronald G. Wolford for their valuable assistance.
* This work was supported in part by U.S. Public Health Service contract PH143-63-81.
t Department of Virus Research, Microbiological Associates, Inc., Bethesda, Maryland.
t National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Be-
thesda, Maryland.
§ National Cancer Institute, National Institutes of Health, Bethesda, Maryland.
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12 Huebner, R. J., W. P. Rowe, H. C. Turner, and W. T. Lane, these PROCEERDINGS, 50 379
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13 Pope, J. H., and W. P. Rowe, J. Exptl. Med., 120, 577 (1964).1212 MICROBIOLOGY: FREEMAN ET AL. PROC. N. A. S.
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publication.
21 Freeman, A. E., and I. Archie, unpublished data.
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30 We thank Dr. A. M. Lewis, Jr., for providing the Ad-1-SV40 and Ad-2-SV40 tumor sera, the
hyperimmune Ad-2 antiviral serum, and the acute and convalescent Ad-1 human sera.
31 Black, P. H., W. P. Rowe, H. C. Turner, and R. J. Huebner, these PROCEEDINGS, 50, 1148
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32 Scherrer, K., and J. E. Darnell, Biochem. Biophys. Res. Commun., 7, 486 (1962).
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37 Fujinaga, K., and M. Green, J. Virology, 1, 576 (1967).
38 Fujinaga, K., and M. Green, these PROCEEDINGS, 57, 806 (1967).
39We thank Dr. M. Green and Dr. K. Fujinaga for allowing us to use this unpublished data.
40 Freeman, A. E., E. Vanderpool, and R. J. Huebner, unpublished data.
41 Huebner, R. J., J. Bell, and W. P. Rowe, in Cellular Biology, Nucleic Acids and Viruses, ed.
T. M. Rivers, Special Publications, vol. 5 (N.Y.: The Academy, 1957), p. 393.
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43 Schell, K., and W. Case, unpublished data.
44 Gillespie, D., and, S. Spiegelman, J. Mol. Biol., 12, 827 (1965).
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