[CANCER RESEARCH 45, 2670-2680,
June 1985]
Properties of Rat Cells Transformed by DMA Plasmids Containing Adenovirus
Type 12 E1 DNA or Specific Fragments of the E1 Region: Comparison of
Transforming Frequencies1
Phillip H. Gallimore,2 Philip J. Byrd, Joanne L. Whittaker,3 and Roger J. A. Grand
Cancer Research Campaign Laboratories, Department of Cancer Studies, University of Birmingham, The Medical School, Birmingham, B152TJ, England
In this paper, we describe for the first time the transformation
of normal rat cells by DMA equivalent to adenovirus type 12 Early
Region 1 (E1A). This DMA was 30-fold less efficient at transfor
for a morphologically transformed phenotype.
Our present study was undertaken: (a) to compare the trans
forming ability of Ad-12 recombinant DNA plasmids (17) that
represent the entire Ad-12 E1 region (pAsc 2, 0 to 16.5 m.u.)
and subgenomic fragments of Ad-12 E1 (pAsc 10.3, 0 to 10.3
mation than DMA encoding the entire E1 region. Those estab
lished lines expressing a full complement of adenovirus type 12
E1 proteins were phenotypically indistinguishable from adenovi
rus type 12 virus-transformed cell lines. However, cell lines
m.u.; pAsc 6.8, 0 to 6.8 m.u.; and pAsc 4.7, 0 to 4.7 m.u.); (b)
to examine the In vitro and in vivo properties of cell lines derived
from a number of unique transformation events induced by each
plasmid; and (c) to determine which Ad-12 E1 proteins were
produced by plasmids carrying subgenomic fragments of E1
DMA and therefore not expressing E1B M, 52,000 protein took
longer to establish and produced tumors only after a protracted
latent period. A Giemsa-banding study showed that adenovirus
transformation can occur without disruption of the normal rat
karyotype.
expressed in each cell line in an attempt to define specific roles
for individual polypeptides.
ABSTRACT
MATERIALS
AND
METHODS
Tissue Culture and Transformation
All of the experiments carried out in this study utilized cells prepared
from HL rats. HL REB cells were prepared as previously described (18).
HL BRK cells were initiated from pooled 7-day-old rat kidneys dispersed
INTRODUCTION
Several rodent species have been shown to be susceptible to
human Ad-124 oncogenesis (1-3), and more recently, Mukai ef
al. (4) reported that baboons, inoculated intraocularly as newboms, may develop retinoblastoma-like neoplasms. From a num
ber of studies, evidence has accumulated that the retention and
expression of genes encoded by Ad-12 E1, 0 to approximately
11.2 m.u., are sufficient to induce malignant transformation of
both rat (5-7) and human (8, 9) cells in tissue culture.
For the nononcogenic serotypes Ad-5 and Ad-2, it has been
reported (10, 11) that normal rat cells can be atypically trans
formed by DMA representing the E1A transcription unit (0 to 4.5
m.u.). Similar studies carried out with Ad-12 E1A DMA failed to
observe any transformation events (12). However, it had previ
ously been shown that Ad-12 E1A DNA could atypically trans
form an already immortalized rat cell line, 3Y1 (13). In the absence
of the E1A transcription unit, E1B DNA (4.6 to 11.2 m.u.) has no
transforming activity (14). Normal rat cells have also been suc
cessfully transformed by the Ad-12 Hind\\\G DNA fragment (0 to
6.8 m.u.), but the immortalized cell lines derived from these
experiments were not tumourigenic even when inoculated into
athymic nude mice (15). These authors concluded that expres
sion of the large E1B protein (M, 54,000 from the DNA sequence)
(16) was essential for a malignant phenotype but nonessential
1The Cancer Research Campaign, England, funded this research.
2 Cancer Research Campaign Life Fellow.
3 Present address: ICI/University Joint Laboratory, Adrian Building, University
Road, Leicester, LE1 7RH, England.
•Theabbreviations used are: Ad-12, adenovirus type 12 (other adenoviruses
are defined similarly); PCS, fetal calf serum; DME, Dulbecco's modified Eagle's
medium; BSA, bovine serum albumin; E1, Early Region 1; m.u., map units; HL,
hooded Lister; BRK, baby rat kidney; REB, rat embryo brain; SDS, sodium dodecyl
sulfate; hr, host range; bp, base pair; kbp. kilobase pair.
Received 9/18/84; revised 2/28/85; accepted 3/5/85.
CANCER RESEARCH
using dispase (1.25 units/ml) after removal of the kidney capsule. Both
REB and BRK cultures were grown using DME supplemented with 10%
PCS, and exponentially growing cultures (2 to 8 x 105 cells/5-cm dish)
were used for transformation studies. DNA transfection was carried out
in the absence of carrier DNA, using the glycerol boost modification (19)
of Graham and van der Eb's (20) transfection technique. Transformed
foci (one per dish) were picked using a finely drawn Pasteur pipet 4 to 6
weeks after transfection. Cell lines were passaged using versene (0.01 %,
w/v) in 0.85% saline. Cell lines were maintained and passaged using
DME plus 10% PCS as growth medium. In the text, each BRK line is
referred to in an abbreviated form, e.g., ACC.H1 for the first pAsc 4.7
BRK cell line isolated. In Table 5, each line is referred to in a complete
form, i.e., Ad-12 AcclH/HL BRK1.
Plating Efficiencies on Plastic or in Semisolid Medium
Cells were removed from the dishes using versene in 0.85% saline,
washed, counted, and plated onto 5-cm dishes at 200 cells per dish in
either 10 or 1% FCS-supplemented DME. Plating experiments in semisolid medium were carried out as previously described (21), except that
DME/methylcellulose medium supplemented with 10% PCS was used.
All plating experiments were carried out using 4 dishes per experimental
group, colonies were counted after 3 weeks of incubation at 37°C, and
the results were calculated from duplicate experiments.
Isolation, Growth, and Purification of Ad-12 Recombinant Plasmids
These have all been described in a recent publication (17). The Ad-12
DNA contained in each recombinant plasmid is illustrated in Chart 1.
Tumorigenicity Studies
Single cell suspensions of each cell line were prepared in Ham's F-10
medium (serum free) and either inoculated s.c. into either conventionally
housed 3-week-old athymic nude mice (1 x 107 cells/mouse) or 1-day-
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Ad-12
ONCOGENESIS
AND
TRANSFORMATION
..
Chart 1. The Ad-12 E1 gene region (0 to
11.2 m.u.) showing the coding regions for the
E1A and E1B proteins (•)and the Ad-12
genomic sequences contained in the recom
binant DNA plasmids used in this study (ar
rows at bottom). Center right shows the sizes
of E1A and E1B mRNAs and the respective
protein sizes by polyacrylamide gel electrophoresis (our data) and by amino acid num
ber. The numbers below the Ad-12 genome
are nucleotide sequence numbers with the
dashes below the genome indicating impor
tant landmarks, i.e., E1A initiation codon (nu
cleotide 502), E1A termination codon (nucleo
tide 1374), E1B M, 18,000 initiation codon
(nucleotide 1541 ), and termination codon (nu
cleotide 2030) and the E1B M, 52,000 protein
initiation codon (nucleotide 1846) and termi
nation codon (nucleotide 3292). pA, polyadenylation sites. This figure is compiled from data
contained in Refs. 16,17, and 53.
E1B7\pAah1
E1A\
~0I502^•Tmm^A12E1K/
— ~
:>-tel•«•)VPA^2
' '
100..
mapunits
'IIff
mBNAE1A
0-9KbE1A
•/E1B
VOKb
p235'WMILY
41K
26618Kp
22KbPROTEINV
52K
E1B 1-OKb
PAsc
p163
n482165
p163
V7
pAsc 6-6
pAsc 10-3
PAsc 2
old HL rats (2 x 106 cells/rat). Autopsies were carried out on all experi
Preparation of Cellular DMA and DNA Hybridization Procedures
mental animals. Tumors and tissues were fixed in formaldehyde/acetic
acid/methanol (1/1/8). Histology sections (5 ^m) were stained with
hematoxylin/eosin.
Cytogenetic Studies
Metaphase preparations were made as previously described (22),
except that colchicine was added for 1 h only. Initial cytogenetic analysis
was carried out using acetic/orcein-stained chromosome preparations.
More detailed analysis was determined from Giemsa-banded prepara
tions (22).
Ad-12 Protein Studies
Immunoprecipitation.
Ad-12 proteins were detected by a modification
of the method of Paraskeva ef a/. (7). The immunoprecipitation reaction
was carried out in the presence of 1.5% (w/v) BSA to reduce nonspecific
interactions. Immunoprecipitated proteins were dissolved in 9 M urea/50
HIM Tris-HCI (pH 7.7)/150 mw 0-mercaptoethanol (25 ¿¿I)
and electrophoresed on 13% polyacrylamide gels run in the presence of 0.1 M Tris/O.1
M Bicine (pH 8.3)/0.1% SDS. Radioactive proteins were detected by
fluorography.
Western Blotting. Ad-12 proteins were also detected using a modifi
cation of the procedure previously described by Towbin et al. (23). Two
x 107 cells/ml were solubilized in 8 M urea/50 ITIMTris-HCI (pH 7.6)/0.15
M /tf-mercaptoethanol/1 % SDS and sonicated for 1 min. Fifteen-Ã-ilaliquots
were electrophoresed in 13% polyacrylamide gels containing 0.1 M Tris,
0.1 M Bicine, and 0.1% SDS (pH 8.3) at 60 ma for 3 h. The proteins
were then electrophoretically transferred to cellulose nitrate filters (0.45
um) using a Bio-Rad Transblot apparatus run for 16 h at 30 V and 100
ma. Filters were incubated in 0.85% saline/10 HIM Tris-HCI (pH 7.3)
containing 3% (w/v) BSA for 1 h at 40°C and then in Ad-12 rat tumor
bearer serum or preimmune rat serum appropriately diluted (usually 1 in
200) in 0.85% saline/Tris/BSA at 30°C. After 2 h, filters were washed
extensively in Tris/0.85% saline containing 0.05% Nonidet P-40 and then
incubated in Tris/0.85% saline/BSA containing 125l-labeled sheep anti-rat
IgG (50,000 cpm/sq cm of cellulose nitrate filter) (2 h at 30°C). After
washing extensively in Tris/0.85% saline containing 0.1% SDS, 0.5%
Triton X-100, and 0.1% EDTA, the filters were dried and autoradiographed for 1 to 2 days. Ad-12 tumor bearer serum has been described
previously (7) and identified E1A and E1B proteins.
High-molecular-weight DNA was isolated from normal rat tissues and
from transformed cells using the procedure of Gross-Bellard ef a/. (24).
All DNAs were examined on 0.4% agarose gels and compared with
intact Ad-12 DNA to confirm that each sample contained high-molecularweight DNA. Five-fig samples of DNA were digested with restriction
endonucleases, and the DNA fragments were fractionated on 0.8%
agarose gels. The sizes of specific fragments were determined relative
to restriction endonuclease-digested Ad-12 DNA or Ad-12 plasmids. Gelseparated DNA fragments were depurinated, denatured, and transferred
to nitrocellulose filters as previously described (25). After transfer, the
filters were treated using the method of Kidd and Glover (26); 10%
dextran sulfate (25) was included in the hybridization solutions. Ad-12
DNA was used as the probe, and this was radiolabeled in vitro with [«^PjdCTP (27). After air-drying, the filters were exposed for 17 to 96 h
at -70°C to preflashed Kodak X-Omat RP film with a Fuji Machll
intensifying screen (28). The adenovirus DNA copy numbers referred to
in "Results" are estimates derived from reconstruction experiments.
RESULTS
Transformation of HL REB Cells. Initialtransformation exper
iments were carried out on secondary REB cells. Table 1 con
tains the data obtained from 4 transfection experiments using
either pAsc 2 or pAsc 6.8 DNAs. In Experiment 4 (Table 1), a
comparison of transforming activity was made between uncut
and restriction enzyme-cleaved (to release Ad-12 DNA from the
bacterial plasmid sequences) pAsc 2 and pAsc 6.8 DNAs. Re
leasing the adenovirus sequences from the plasmid sequences
did not enhance transformation (Table 1, Experiment 4). Table 3
shows that, when the means of the pAsc 6.8 data were com
pared with the means of the pAsc 2 data, pAsc 2 was between
2 and 11 times more efficient at transforming HL REB cells than
pAsc 6.8. The time of appearance of pAsc 2-transformed foci
and the focus morphology were indistinguishable from Ad-12
virus-induced foci. Although pAsc 6.8-induced foci characteristi
cally had a less-defined and more migratory border compared to
the pAsc 2-transformed colonies, they appeared at the same
time as Ad-12 virus- or pAsc 2-induced foci, both microscopically
(2 to 3 weeks) and macroscopically (3 to 4 weeks). Of the 4
pAsc 6.8-transformed HL REB foci picked, 2 developed into cell
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Ad-12 ONCOGENESIS
AND TRANSFORMATION
Table 1
Ad-12 plasmiti transformationof HL REBcultures
PlasmidExperi
ants/fig
DMA
no. of cells
no. of
génome
fragmentEcoRICEcoRICEcoRICEcoRICEcoRICH/ndMIGH/ndlllGH/noïlIGEcoRICEcoRICEcoRICEcoRICEcoRICEco
exposed1
transformants11(4)"6(4)13(3)8(3)4(3)5(3)3(3)1(3)18(4)10(4)6(4)23(4
équivalent0.3740.4080.2940.3620.3620.1450.17
X10"1
ment
Designation1
22
pAsc
23
pAsc
10s1.5
x
10"1.5
x
061.5
x 1
1061.5X
x
10e1.5X
10e1.5X
1068X1068X1058X1058x10*8X1058
a2pAsc2pAsc2b
pAsc
pAsc
6.8pAsc
6.8pAsc
6.84
a2pAsc2pAsc2b
pAsc
2pAsc2pAsc2C
pAsc
1058x
x
1058x
1058X1058x
pAsc
6.8pAsc
6.8pAsc
6.8d
10=8x
pAsc
6.8pAsc
10s8x
6.8pAsc
105Total
6.8fig/dish2a1«4a2a1*2e1e0.5e3a1a0.33a310.333e1e0.33e310.33Ad-12
Plasmid DMAcut to completion with EcoRI.
" Numbers in parentheses, number of 5-cm dishes.
c Plasmid DNA cut to completion with EcoRI and H/ndlll.
lines without cessation of growth, while the other 2 isolates went
through one crisis period before establishment. Five independent
pAsc 2-induced foci developed into cell lines without cessation
of growth, and at tissue culture passage 2, they produced
progressively growing anaplastic tumors in 100% of inoculated
nude mice and rats with a tumor latent period of 35 ±15 (SE)
days. Two pAsc 6.8-transformed cell lines, designated Ad-12
H/ndlllG/HL REB 1 and 2, were tested for tumor-inducing poten
tial. Ad-12 H/ndlllG/HL REB 1, a crisis-free cell line, was not
tumorigenic in either rats or nude mice. A higher cell dose (5 x
107 s.c.) and a different route of inoculation (intracerebrally, 2 x
106 cells/mouse) also failed to produce tumors in nude mice. Ad12 H/ndlllG/HL REB 2, a cell line which had traversed a crisis
period during isolation, was tumorigenic in both nude mice and
syngeneic rats (100% tumor incidence), but the tumor latent
period was 2 to 4 times longer (110 ±40 days) than pAsc 2 cell
line-induced tumors. Ad-1 2 H/'ndlllG/HL REB 1 cells only express
Ad-12 E1A Mr 41,000 protein, whereas Ad-12 H/ndlllG/HL REB
2 cells express both the M, 41 ,000 and E1 B M, 18,000 proteins.5
These data imply that the E1 B M, 52,000 protein is not obligatory
for either transformation or for the production of malignant cell
lines.
Transformation of HL BRK Cells. In order that our studies
could be directly compared with previously published work (6,
10, 14, 15), we decided to repeat the transformation experiments
with pAsc 2 and pAsc 6.8 on BRK cells and to extend the study
by including pAsc 10.3 and pAsc 4.7. The experiments were
carried out with intact plasmids, because we did not find an
enhancement of transformation using restriction enzyme-cleaved
plasmid DNA (see Table 1, Experiment 4). Preliminary investi
gations carried out with pAsc 4.7 indicated that DNA concentra
tions below 5 MQ/dish did not transform BRK cells. Table 2
5K. W. Brown and P. H. Gallimore,unpublished results.
contains the data for 4 transformation experiments. Experiments
6(a) and 6(b) were set up at the same time, but utilized different
batches of HL BRK cultures, while Experiments 5 and 7 were
set up independently of Experiment 6 and of each other. In all of
these experiments, the number of transformed foci observed per
u.g of genome equivalent increased with increasing size of the
Ad-12 DNA fragment; i.e., the Ad-12 EcoC fragment (0 to 16.5
m.u.) containing plasmid pAsc 2 was about 30 times more
efficient at transforming BRK cells than the pAsc 4.7 plasmid
carrying the Ad-12 Acc\H fragment (0 to 4.7 m.u.). Table 3
summarizes the transformation data for each Ad-12 plasmid and
also provides a numerical comparison. All transformation exper
iments were compared with BRK cultures transfected with 10
^g of pAT153 per dish (bacterial plasmid without adenovirus
sequences; 5 dishes/experiment) and additional cultures ex
posed to calcium phosphate precipitate [4-(2-hydroxyethyl)-1 piperazineethanesulfonic acid buffer/0.85% saline with CaCI2]
without DNA (5 to 10 dishes/experiment). At the end of the
experimental period, a few overgrowing fibroblastic colonies
were identified on these control dishes (as well as on Ad-12
plasmid-transfected dishes) which rapidly senesced either di
rectly on isolation or after one or 2 tissue culture passages. We
concluded that these fibroblastic foci, which were Ad-12 Tantigen negative, were composed of normal rat fibroblasts which
grew out of the BRK cultures as the epithelial kidney cells
terminally differentiated. Foci induced by all the cloned Ad-12
DNA fragments were easily identified and were composed of
small epithelioid cells. pAsc 2- and pAsc 10.3-transformed foci
were indistinguishable from those induced by purified Ad-12
virus. The periphery of foci induced by pAsc 6.8 and 4.7 was
less well defined than the Ad-12 virus-induced foci, with trans
formed cells haphazardly migrating away from the leading edges
of these foci.
Five pAsc 2- and 3 pAsc 10.3-transformed foci (each repre-
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Ad-12 ONCOGENESIS
AND TRANSFORMATION
Table 2
Ad-12 plasmiti transformationof HL BRK cells
PlasmidExpenDesignation5
ment
pAsc2pAsc10.3pAsc
6.8pAsc
4.76
a
DNA
no. of cells
no. of
¿ig
genome
fragmentEcorneSaneHindmeAcc\HEcorneHindlHGAcctHEcomcHindWGAcc\HEcoR\CSa/ICH/ndïllGAcclHTo
transformants14(11)"9(11)3(11)1(11)7(4)6(12)7(12)9(4)8(12)11(12)21(10)1
exposed8.8
equivalent0.06920.03120.00950.00200.23780.01740.0
10e8.8
x
1068.8
x
10"8.8
x
10*3.2
x
10«9.6
x
10"9.6
x
10"1.6X
x
10e4.8
10"4.8
x
10s4x10«4x
x
pAsc
2pAsc
6.8pAsc
4.7b
pAsc
2pAsc
6.8pAsc
4.77
pAsc2pAsc
10.3pAsc
6.8pAsc
4.7jig/dish5S5S251025105555Ad-12
Numbers in parentheses, number of 5-cm dishes.
fragmentExperi
ment34
567Cell
typeREB
REB
BRKBRK
Tabte3
Summary table: numerical differences in transforming frequencies
Ad-12 genomic
(pAsc
4.7)NDa
6.8)1
(pAsc
ND
1(0.0020)"1
(0.145)
1
(0.069)
4.75C(0.0095)
(0.0082)
BRKAcclH
10e4x
10"4x
10"Total
1 (0.0044)HindmG
1
2.48
1
5.55
1
(pAsc10.3)ND
(0.0095)
(0.0203)
(0.0203)
(0.0244)
(0.0244)Sa/IC
2)2.34
(pAsc
(0.339)
ND
11.32(0.781)
15.60(0.0312)ND13.02(0.0573)EcoRIC
34.60 (0.0692)
7.28 (0.0692)
33.15(0.2718)
13.39(0.2718)
25.93(0.1141)
4.68(0.1141)
8 ND, not determined.
Numbers in parentheses, mean of the transformants/Mggenome equivalents derived from the data shown in Tables
1 and 2.
c The transformation efficiencies were calculated by dividing the number of transformants/iig genome equivalents for
each plasmid construct by that obtained for the smallest plasmid (either pAsc 4.7 or pAsc 6.8). For simplicity, the
transforming activity of the smallest plasmid was then given the arbitrary value of 1.
senting a unique transformation event) established into cell lines
without difficulty. In comparison with the above 8 foci, the initial
growth rate of 6 foci picked from pAsc 6.8-transformed HL BRK
dishes were discernibly slower. This slower growth rate was
entirely due to a proportion of pAsc 6.8-transformed cells (as
high as 80%) flattening, then stretching, and finally failing to
divide. At the first subculture of these foci, this nondividing
population was lost and not replaced at subsequent passages.
Six of 7 pAsc 4.7-induced foci each required 3 attempts at
isolation before confluent cultures of transformed cells were
obtained. Initial attempts to grow these 6 pAsc 4.7 foci failed,
because the isolated foci aborted rapidly. Between each isolation
attempt, the foci on the original transfected dishes were allowed
to completely reform. At the third picking, these pAsc 4.7 foci
established slowly into cell lines. Although the focus giving rise
to the seventh pAsc 4.7 cell line (ACC.H5; Fig. 1) grew slowly in
the first instance, it developed into a cell line from the first
picking. Morphologically, the pAsc 2- and pAsc 10.3-transformed
BRK cell lines were indistinguishable from Ad-12 virion-transformed cell lines. The pAsc 6.8- and 4.7-transformed cell lines,
although clearly epithelioid, had a more discrete cytoplasmic
border, often displaying multiple ruffling edges. Representative
phase-contrast photomicrographs are shown in Fig. 1.
Properties
of Ad-12 Recombinant
Plasmid-transformed
BRK Cell Lines. Table 4 contains a summary of the in vitro
growth properties and cytogenetic analysis for 5 pAsc 2, 3 pAsc
10.3, 6 pAsc 6.8, and 7 pAsc 4.7 cell lines. Where possible,
these studies were carried out at the same passage level as the
cells used in animal experiments. Ten to 28% of cells plated (200
cells/5-cm dish) in medium supplemented with 10% FCS formed
colonies. For this growth property, no obvious difference be
tween cell lines transformed by the different plasmids was noted.
An attempt to compare transformed cell lines for colony formation
using medium supplemented with 1% FCS was unsuccessful in
that no cell line produced colonies at this low FCS concentration
in the 3-week incubation period. Dramatic differences in plating
efficiency were, however, observed in experiments carried out
in semisolid DME/methylcellulose medium (supplemented with
10% FCS). Plating efficiencies of greater than 1% were observed
for 4 of 5 pAsc 2, 2 of 3 pAsc 10.3, and 1 of 6 pAsc 6.8 cell
lines. Another pAsc 6.8 cell line (HIN.G5), although plating less
than 1%, produced large, visible colonies. Although the 4 re
maining pAsc 6.8 and the 7 pAsc 4.7 cell lines did not produce
large colonies (those containing 200 cells or more), microscopic
colonies of between 8 and 32 cells per colony were observed
for all cell lines.
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Ad-12 ONCOGENESIS
cyiogeneticsCell
AND TRANSFORMATION
Table 4
Plating efficiencies and
on plastic. OME
(10% PCS),200 cells
plated"%201216142524281822101018242810122024181814Saturation
inmethylcellulose/DME(10%FCSf
of plating
densi
chromosomeno.42.XX
"26.44.83.42.20.80.63.411.26.2<0.001<0.0010.9<0.001<0.001<0.001<0.001<0.001<0.001<0.001<0.001<0.001Plating
karyotypescND"42,XXt(12:?),t(13:?)ND42.XX;
linesECO.C1ECO.C2ECO.C3ECO.C4ECO.C5SAL.C1SAL.C2SAL.C3HIN.G1HIN.G2HIN.G3HIN.G4HIN.G5HIN.G6ACC.H1ACC.H2ACC.H3ACC.H4ACC.H5ACC.H
ties cm)5.5
(cells/sq
1053.5
x
10s4.2
x
1054.5
x
105NO3.1
x
10s5.2
x
X1055.4
1053.2
x
X1053.5
X105NO4.9
1051.1
x
10"6.2
x
1055.6
x
10sND1.5
x
1053.2
x
1055.2
x
X1054.4
10s4.8
x
x 105Modal
(92)"42.XX
(80)42.XY
(78)'42.XX
(58)42.XY
36%42.XY
43.XX + 13,del(9)q36
(56)75.XXXX
pseudotetraploidsND42.XX42.XY;
plus 30%
(90)42.XX
(100)42.XY
(62)43.XX
34%43.XX
84.XXYY
(56)42.XY
36%42.XYND42.XY;
+ 12 (or 12 like); 42.XX
(84)42.XX
(62f42.XY
(48)42.XY
40%42.XY43.XY
43.XY + 12 (or 12 like)
(94)43.XY
(78)43.XY
22%43.XY
+ 12 (or 12 like); 42.XY
1qter)41+ 4,t(1:8) (8pter-»8qter::1q35—
(100)41.XY
3qter->cent-mter)42.XY;
,XY
t(3:13)
(1
(100)42.XY
(66)84.XXYY
24%84.XXYY;
84.XXYY
(60)42.XY
32%42.XYND42.XY
42.XY
(94)42.XX
(96)42.XY
(80)CytogeneticsGiemsa-banded
3 Carried out as described by Gallimoreef al. (21).
" Means of duplicate experiments, 4 dishes/group.
°Karyotypesdescribed as per International System for Human Cytogenetic Nomenclature (51) and Levan (52). Ten to 30 metaphase cells
examined per cell line.
d Numbers in parentheses, percentage of total population.
* ND, not done.
' Twenty-two % of cells pseudodiploid with fragments and dicentnc chromosomes.
s Twelve % of cells with 43 chromosomes and a metacentric marker.
G-banding studies (Table 4) revealed that 8 of the 16 lines
examined were karyotypically normal, 7 lines being diploid and
one line being predominantly tetraploid (ACC.H4). Of the remain
ing 8 lines, one line, ECO.C2, was 80% diploid {42 chromo
somes), but these cells contained 2 marker chromosomes pro
duced by addition of chromosome material of unknown origin
onto the long arms of autosome 12 and autosome 13. Cell line
ACC.H2 was hypodiploid, and all cells contained a marker chro
mosome that was produced by centromeric fusion of autosomes
3 and 13. The hyperdiploid line ACC.H1 was trisomie for autosome 4, had a deleted autosome 1, and had a marker chromo
some which was shown to be composed of a translocation
between the long-arm telomeres of autosome 8 with the longarm material deleted from autosome 1. Three cell lines were
composed of both normal diploid cells and hyperdiploid cells (43
chromosomes per cell). By G-banding, these 3 lines (HIN.G 1,4,
and 6) were shown to contain an additional chromosome in the
hyperdiploid cells that was indistinguishable from autosome 12.
Cell line HIN.G1 was entirely composed of female cells, whereas
lines HIN.G 4 and 6 were male. ECO.C4 was predominantly
normal diploid, but a minor hyperdiploid clone in this cell line was
trisomie for chromosome 13, and one autosome 9 was deleted
at Band q36. Finally, ECO.C5 was predominantly diploid (56%)
but also contained pseudotetraploid cells (30%).
Southern Blotting Analysis of Ad-12 BRK Cell Lines. South
ern blotting analysis (29) was used to demonstrate the presence
of Ad-12 DMA sequences in cells transformed by pAsc 2 (2 lines),
pAsc 10.3 (2 lines), pAsc 6.8 (5 lines), and pAsc 4.7 (5 lines).
Bands equivalent to the pAsc 2 Pvull 1716, 996, 873, and 507
bp fragments were identified in Pvull-digested Eco.d cell DNA,
indicating that the Ad-12 sequences from nucleotides 398 to
CANCER RESEARCH
3617 and 3727 to approximately 4600 were represented in this
cell line; the 507 and 110 bp pAsc 2 fragments were run off the
gel in the Pvull digests shown in the composite in Fig. 2. In
contrast to the high copy level of pAsc 2 sequences in ECO.C1
cells (~33 copies/cell), ECO.C5 cells were found to contain only
4 to 5 copies/cell of the recombinant plasmid. All pAsc 2 Pvull
fragments were identified in ECO.C5 cell DNA, with the exception
of the 110 bp fragment (nucleotide 3619 to 3729). Since only
one "off-sized" fragment was detected, it is likely that this cell
line contains tandemly repeated complete pAsc 2 molecules
integrated at a single chromosomal site. This analysis demon
strated that the Ad-12 sequences encoding the entire E1 region
were present in ECO.C1 and ECO.C5 cells.
Digestion of SAL.C1 cell DNA with Hind\\\, an enzyme that
cleaves pAsc 10.3 at one site, produced 4 Ad-12-specific bands
(Fig. 2). None of these bands comigrated with linear pAsc 10.3,
suggesting that the 2 copies of plasmid retained in this cell line
were probably integrated at different sites. The pattern of hybrid
ization to H/ndlll-digested SAL.C2 cell DNA suggested that this
cell line most probably contains one complete and one incom
plete copy of pAsc 10.3 integrated at the same site.
Analysis of pAsc 6.8-transformed cell DNA digested with Kpn]
(Fig. 2) and SamHI (not shown), both single-cut enzymes, dem
onstrated that HIN.G1, 3, 4, 5, and 6 contained few copies (one
to 4 per cell) of the plasmid. HIN.G6 was unique in that bands
equivalent to linear pAsc 6.8 were produced by both Kpn\ and
SamHI (data not shown). The only evidence of tandemly dupli
cated plasmid sequences in the other cell lines was the presence
of a 5.9 kbp band in Kpnl-digested HIN.G5 DNA.
The pAsc 4.7-transformed cell lines retained between 6 copies
(ACC.H4) and 60 copies (ACC.H7) of the recombinant plasmid.
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Ad-12 ONCOGENESIS
AND TRANSFORMATION
The hybridization patterns in Pvull-digested ACC.H2, 4, and 7
Å“il DNA indicated that most copies of the plasmid DNA were
tandemly repeated in these cell lines. Consistent with this con
clusion was the identification of linear pAsc 4.7 bands in DNA
from each cell line, following Kpn\ or EcoRI digestion (data not
shown). The complex hybridization patterns in Pvull-digested
ACC.H3 and ACC.H5 cell DNA indicated that pAsc 4.7 se
quences were integrated at many sites in both cell lines. How
ever, Kpn\ and EcoRI digestion showed that approximately 4 to
6 copies of pAsc 4.7 were organized in a tandem array in ACC.H3
cell DNA (data not shown). This analysis and additional Southern
blotting analysis using the restriction enzyme Tha\ (data not
shown) demonstrated that all segments of the E1A region were
represented in these Ad-12 AcclH-transformed cell lines.
Detection of Ad-12 Proteins. Two techniques were used for
this study, namely, immunoprecipitation and Western blotting. It
is our experience that the former technique identified the Ad-12
E1B M, 18,000 (163 amino acid protein) protein efficiently,
whereas this protein is poorly transferred by electroblotting. The
major advantage of Western blotting was that the Ad-12 M,
41,000 E1A (235 and 266 amino acid proteins) and the E1B M,
52,000 (482 amino acid protein) proteins could be identified in
the absence of host proteins, which are often seen as contami
nating bands in immunoprecipitation experiments. Table 5 con
tains a summary of the Ad-12 proteins found in the 21 cell lines
examined. All 5 pAsc 2- and 3 pAsc 10.3-transformed cell lines
expressed all the Ad-12 E1 proteins with molecular weights of
41,000, 52,000, and 18,000. As expected, the 7 pAsc 4.7transformed cell lines expressed only the Ad-12 E1A M, 41,000
protein as did HIN.G3. Three pAsc 6.8-transformed
lines
(HIN.G1, HIN.G5, and HIN.G6) in 3 immunoprecipitation experi
ments produced low levels of Ad-12 M, 18,000 protein. These
cell lines, like the remaining 2 pAsc 6.8 lines (HIN.G2 and 4)
which expressed normal levels of M, 18,000 protein, also ex
pressed Mr 41,000 protein. Fig. 3 shows immunoprecipitation
experiments carried out on cell lines ECO.C1 (Lanes 8 and 9),
ECO.C3 (Lanes 12 and 13), HIN.G3 (Lanes 4 and 5), HIN.G4
(Lanes 10 and 11), SAL.C1 (Lanes 6 and 7), and ACC.H5 (Lanes
2 and 3), using preimmune rat serum (Lanes 2, 4, 6, 8, 10, and
72) and ECO.C1 tumor bearer serum (Lanes 3, 5, 7, 9, 11, and
73), which detects Ad-12 M, 41,000,18,000, and 52,000 proteins
in Ad-12 virus-transformed cells. It can be seen that ECO.C1
and SAL.C1 express M, 41,000, 18,000, and 52,000 proteins;
HIN.G4 expresses M, 41,000 and 18,000 proteins; and ACC.H1
and HIN.G3 express only Mr 41,000 protein.
Western blotting was also used to compare the level of expres
sion of the E1A M, 41,000 protein in 4 pAsc 2 (ECO.C1, 2, 3,
and 4), 4 pAsc 6.8 (HIN.G1,4,5, and 6), and 3 pAsc 4.7 (ACC.H5,
6, and 7) cell lines. Total cell protein (initially 3 x 107 cells/ml)
was extracted from each cell line, serially diluted, run out on
polyacrylamide gels, blotted, reacted with specific antiserum,
and further processed as described in "Materials and Methods."
Autoradiographs were examined after 3 days of exposure, and
for all 11 cell lines, M, 41,000 protein titrated out with an end
point equivalent to 6 x 104 cells per sample. Fig. 4 shows the
data obtained for 2 pAsc 2 (ECO.C 3 and 4), 2 pAsc 6.8 (HIN.G
1 and 4), and 2 pAsc 4.7 (ACC.H 6 and 7) cell lines.
Tumorigenicity Studies. All 21 cell lines contained in Table 5
were examined for tumorigenic potential in athymic nude mice
and 1-day-old rats. Five pAsc 2, 3 pAsc 10.3, and 2 pAsc 6.8
(HIN.G2 and HIN.G5) transformed cell lines produced tumors
when inoculated s.c. (1 x 107 cells/mouse, 6 mice/group) into 3week-old athymic nude mice. A 100% tumor incidence was
obtained with a latent perod of 31 ±9 (SE) days, except for
mice given injections of HIN.G2 and HIN.G5. Three of 6 mice
(HIN.G2) and 2 of 6 mice (HIN.G5) developed tumors after long
latent periods (93 ±5 days). The 4 remaining pAsc 6.8 and 7
pAsc 4.7 cell lines failed to produce tumors when inoculated at
TabteS
Tumorigenicity in syngeneic rats and Ad-12 protein content of BRK cell lines transformed by the Ad-12-transforming
gene region E1 and subunits of this region
2 proteins identified by
plasmid used to produce
latent
time of death
immunoprecipitationand
linesAd-12EcoRI-C/HLBRK1Ad-12EcoRI-C/HLBRK2Ad-12£coRI-C/HLBRK3Ad-12EcoRI-C/HLBRK4Ad-12£coRI-C/HLBRK5Ad-12Sa/IC/HLBRK1Ad-12Sa/IC/HLBRK2Ad-12Sa/IC/HLBRK3Ad-12
Cell
(%)pAsc2(0-16.5m.u.)
the cell line
Tumor positive
period
(days)303233364341512915477>36515312014385>365167>365127>36592Mean
(days)62(12/12)"79(10/10)82(12/12)62(14/14)68(12/12)78(6/6)83(5/5
(M,)52.000,41,000,18,00052,000,41,000,18,0
Western blotting
100(12/12)"100(10/10)100(12/12)100(14/14)100(12/12)pAsc
18,00052,000,41,000,18,00052,000,41,000,1
(6/6)100(5/5)100(12/12)pAsc
10.3 (0-1 0.3 m.u.)
100
(6/7)100(13/13)0(0/10)100(12/12)100(7/7)100(7/7)pAsc
6.8 (0-6.8 m.u.)
85.7
1Ad-12H/ndlllG/HLBRK2Ad-12H/ndlllG/HLBRK3Ad-12
H/m/IIIG/HL BRK
18,000C41,000,18,00041,00041,000,
4Ad-12Hmc(IIIG/HLBRK5Ad-12H/ncflllG/HLBRK6Ad-12AcclH/HLBRK1Ad-12AcclH/HLBRK2Ad-12AcclH/HLBRK3Ad-12AcclH/HLBRK4Ad-12AcclH/HLBRK5Ad-12/UxlH/HLBRK6Ad-12AcclH/HLBRK7
Hind\\tG/HL BRK
18,00041,000,18,000°41,000,18,000°41,0
4.7 (0-4.7 m.u.)
100(13/13)0(0/12)80(8/10)0(0/11)100(10/10)0(0/8)100(11/11)Mean
(8/8)>365225(10/10)>365192(11/11)Ad-1
,00041,00041,00041,000
* Number of tumor-positive rats per number of rats inoculated s.c. with 2x10" cells.
6 Number of tumor-positive rats per number of dead or killed as tumor positives.
°Low level detected.
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Ad-12 ONCOGENESIS
AND TRANSFORMATION
the same cell dose (a total of 12 mice per cell line) and observed
for 150 days. Inoculation of 1-day-old syngeneic HL rats with 2
x 106 cells s.c. produced similar results (Table 5) for those lines
already shown to be tumorigenic in nude mice. However, addi
tional cell lines were found to be malignant in the natural host.
Lines HIN.G1, 4, and 6 and ACC.H1, 3, 5, and 7 produced
tumors in all or a high proportion of rats after a long latent period
(85 to 167 days). Rats inoculated with 2 x 106 HIN.G3, ACC.H2,
ACC.H4, or ACC.H6 cells did not develop tumors during the 1year experimental period (Table 5).
(Refs. 6, 10, 14, and 15; Table 3) have shown that products of
adenovirus E1B DNA play a significant role in transformation.
The higher transformation frequencies obtained with pAsc 2 and
pAsc 10.3, compared to pAsc 4.7 and 6.8, suggest that the E1B
region provides a function(s) which either increases the fre
quency of E1A-induced transformation or stabilizes E1A trans
formation once it has been initiated.
The cytogenetic findings (Table 4) clearly demonstrate that
adenovirus transformation can occur without visible disruption
of the normal rat karyotype; in that 8 of the 16 G-banded cell
lines examined were karyotypically normal. However, one-third
of our Ad-12 DNA transformed BRK cell lines were either entirely
DISCUSSION
composed of or contained a high proportion of abnormal clones.
Trisomy 12 (or 12-like; this autosome has few distinguishing
Transformation of normal BRK cells by DMA equivalent to the
Ad-5 E1A region was originally reported by van der Eb et al. (6).
These transformants were described as incomplete transformants with a fibroblastic morphology clearly distinguishable from
BRK cells transformed by Ad-5 virus or Ad-5 DNA encoding both
E1A and E1B transcription units (6, 10). Later attempts to
transform BRK cells with Ad-12 E1A DNA were unsuccessful
(12) as were similar experiments carried out using Ad-5 E1A on
landmarks) was observed in 3 lines (HIN.G1, 4, and 6), and a
marker chromosome involving autosome 12 was seen in 80% of
ECO.C2 cells. The latter clone was also marked with an abnormal
autosome 13. This chromosome was observed to be trisomie in
a clone found in ECO.C4 and also involved in a rearrangement
(Robertsonian fusion) with autosome 3 in cell line ACC.H2. We
previously reported (38) that 3 of 6 Ad-12 virus transformed REB
cell lines were normal diploid but that one cell line had a minor
clone which was trisomie for autosome 1. A study carried out on
Ad-12 DNA transformed 3Y1 cells (39) showed that 2 lines (WY3
and CY1) contained clones marked with a metacentric chromo
some, an isochromosome of the long arms of 2 chromosomes
3, and one line (GY1 ) which was hyperdiploid and marked with
a large metacentric chromosome which was an isochromosome
of autosomes 1. Clearly, no consistent aberration has been
identified in adenovirus DNA transformed rat cells. One interpre
tation of our data would be that the involvement of autosomes
3, 12, and 13 in rearrangements retains the nucleolar organizer
in a diploid state; we have shown by silver staining that, for the
HL rat strain, these autosomes make up the nucleolar organizer
component.8 The precise significance of Trisomies 4, 12, and 13
hamster kidney cells (30). We have been able to transform normal
rat cells with the Ad-12 E1A region, and although we showed
subsequently that the pAsc 4.7 transformants retained several
copies of pAsc 4.7 (Fig. 2) and expressed the E1A M, 41,000
protein (Figs. 3 and 4), these rare epithelioid colonies were
difficult to isolate. Nevertheless, with careful handling, all 7 of
the pAsc 4.7 foci that were picked developed into established
cell lines, and over one-half of the lines were tumorigenic when
inoculated into newborn syngeneic rats (Table 5). Other workers
have also encountered some problems when isolating Ad-5 E1A
transformed foci in that a significant number of these foci failed
to develop into cell lines.6 Evidence to date suggests that the
maintenance and expression of E1A can extend the culture
lifetime of a rare cell from a large population of normal cells, i.e.,
approximately one transformation event in 5 x 10s cells. It would
appear that immortality is achieved during the establishment
phase with the selection of a cell type that predominantly repli
cates without differentiation. The cellular events that determine
this phenotype are unknown, but they could include one or more
of the following: (a) change in protooncogene expression; (ti)
growth in the absence of tissue culture medium modification or
escape from a "feeder effect"; and (c) stabilization of E1A gene
expression.
Our data (Fig. 4) strongly suggest that the amount of E1A
protein in established transformed rat cells is tightly and surpris
ingly uniformly controlled. The transformation events produced
by the entire E1 region are already in a rapid cell-cycling mode
at the time of focus isolation. Could it be that E1A in the presence
of E1B proteins returned the cells to a more primitive state?
Some evidence to support this comes from Ad-12 tumor histopathology in that most tumors are composed of primitive, undifferentiated cells (31), and although diagnosed as sarcomas, they
lack connective tissue markers.7 The precise mechanism or
genetic events that determine immortalization in this system
require further investigation.
Genetic evidence (5, 32-37) and DNA transfection studies
and the translocations seen in this and other cytogenetic studies
may be resolved when a more detailed gene location map is
constructed for the rat.
The biological properties of cell lines that express E1A M,
41,000 and E1B M, 18,000 proteins, but not the E1B M, 52,000
protein, were clearly distinguishable from the pAsc 2- and pAsc
10.3-transformed cell lines which expressed all the Ad-12 E1
proteins (Tables 4 and 5). In contrast to the findings of Jochemsen ef al. (15), a proportion of our Ad-12 H;'ndlllG transformants
(those transformed by pAsc 6.8) were tumorigenic in athymic
nude mice. More significantly, 5 of 6 of the pAsc 6.8-transformed
BRK cell lines reported here produced tumors when inoculated
into newborn syngeneic rats (Table 5). These findings are similar
to those of Rowe and Graham (40), who reported that baby
hamster kidney cells transformed using DNA from an Ad-5 Group
II hr mutant [these mutants map to E1B (32, 36)] were fully
transformed and produced tumors in hamsters. Another impor
tant observation from our study was that the tumor latent period
and time of death were significantly shorter for those rats inoc
ulated with cell lines expressing all the Ad-12 E1 proteins (Table
5). Our tumorigenicity findings with pAsc 2- and pAsc 10.3transformed cell lines are indistinguishable from similar studies
carried out with Ad-12 virus-transformed REB cell lines (38). The
' A. van der Eb, personal communication.
7 P. H. Gallimore, unpublished results.
8 P. H. Gallimore, unpublished data.
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Ad-12 ONCOGENESIS
AND TRANSFORMATION
implication of these results is that, while the large E1B protein is
not absolutely required for malignant transformation of rat cells
by naked Ad-12 DNA, its presence in addition to the M, 41,000
and 18,000 proteins either increases the cell survival or growth
rate of such Ad-12-transformed cells in vivo. In contrast to our
findings, Rowe ef a/. (30) have reported that the large E1B
protein of Ad-5 does not contribute to the malignant phenotype
in hamster cell lines.
It has recently been suggested (12) that E1B products regulate
the level of E1A transcriptions. However, quantitation, by West
ern blotting, of the amounts of E1A protein produced by 11 Ad12-transformed BRK cell lines (including 4 pAsc 2 and 3 pAsc
4.7 lines) revealed that each line produced an equivalent amount
of E1A product. This finding suggests that E1B did not influence
the amount of E1A protein produced nor did the copy number
of E1A DNA carried by each cell line (Fig. 2) and implies that the
level of expression of E1A in this system is tightly controlled (Fig.
4).
From all of the studies carried out on Ad-12 transformation, it
can be concluded that bona-fide transformants (indistinguishable
virus/host protein interactions that are manifested as malignant
transformation.
from virion transformants) are obtained only if the entire E1
region is expressed. Recently, we have reported the isolation of
3 Ad-12 E1A and 2 Ad-12 E1B hr mutants (37). Four of these
mutants were completely defective for both transformation and
tumor induction. However, one of the E1A mutants, H12 hr 700,
had some transforming capability and produced tumors in rats,
albeit at a low frequency. Similarly, Ad-12 E1B cytolytic mutants
(5, 41, 42) which map in the M, 18,000 E1B gene are frequently
defective for both transformation and tumorigenicity (5, 41, 42).
These observations strongly suggest that expression of the full
complement of E1 genes is required for Ad-12 virus oncogenesis.
The recent reports that Ad-12-transformed rat cells express
5. Mak, S., Mak, I., Smiley, J. R., and Graham, F. L. Tumorigenicity and viral
gene expression in rat cells transformed by Ad 12 virions or by the £coRIC
fragment of Ad 12 DNA. Virology, 98: 456-460,1979.
6. van der Eb, A. J., van Ormondt, J., Schrier, P. I., Lupker, J. H., Jochemsen,
H., van den Eisen, P. J., DeLeys, R. J., Maat, J., van Beveren, C. P., Dijkema,
R., and de Waard, A. Structure and function of the transforming genes of
human adenoviruses and SV40. Cold Spring Harbor Symp. Quant. Biol., 44:
383-399, 1979.
7. Paraskeva, C., Brown, K. W., Dunn, A. R., and Gallimore, P. H. Adenovirus
type 12-transformed rat embryo brain and rat liver epithelial cell lines: adeno
virus type 12 genome content and viral protein expression. J. Viral., 44: 759764,1982.
8. Byrd, P. J., Brown, K. W., and Gallimore, P. H. Malignant transformation of
human embryo retinoblasts by cloned adenovirus 12 DNA. Nature (Lond.),
298:69-71,1982.
9. Whittaker, J. L., Byrd, P. J., Grand, R. J. A., and Gallimore, P. H. The isolation
and characterization of four adenovirus type 12 human embryo kidney cell
lines. J. Mol. Cell. Biol., 4:110-116,1984.
10. Houweling, A., van den Elsen, P. J., and van der Eb, A. J. Partial transformation
of primary rat cells by the left most 4.5% fragment of adenovirus 5 DNA.
Virology, 705: 537-550, 1980.
11. Ruley, H. E. Adenovirus early region 1A enables viral and cellular transforming
genes to transform primary cells in culture. Nature (Lond.), 304: 602-606,
1983.
12. van den Elsen, P. J., Houweling, A., and van der Eb, A. J. Morphological
transformation of human adenoviruses is determined to a large extent by gene
products of region E1A. Virology, 737: 242-246, 1983.
13. Shiroki, K., Shimojo, H., Sawada, Y., Uemizu, Y., and Fujinaga, K. Incomplete
transformation of rat cells by a small fragment of adenovirus 12 DNA. Virology,
95:127-136,1979.
14. van den Elsen, P. J., de Pater, S., Houweling, A., van der Veer, J., and van
der Eb, A. J. The relationship between region E1a and E1b of human adeno
viruses in cell transformation. Gene, 78:175-185,1982.
15. Jochemsen, H., Daniels, G. S. G., Hertoghs, J. J. L., Schrier. P. I., van den
Elsen, P. J., and van der Eb, A. J. Identification of adenovirus-type 12 gene
products involved in transformation and oncogenesis. Virology, 722: 15-28,
1982.
16. Bos, J. L., Polder, L. J., Bernards, R., Schrier, P., van den Elsen, P. J., van
der Eb, A. J., and van Ormondt, H. The 2.2 kb mRNA of the E1b region of
human adenovirus types 12 and 5 directs the synthesis of two major tumor
antigens from different AUG triplets. Cell, 72: 721 -732,1981.
17. Byrd, P. J., Chia, W., Rigby, P. W. J., and Gallimore, P. H. Cloning of DNA
fragments from the left end of the adenovirus type 12 genome: transformation
by cloned early region 1. J. Gen. Virol., 60: 279-293,1982.
18. Gallimore, P. H. Interactions of adenovirus type 2 with rat embryo cells.
Permissiveness, transformation, and in vitro characteristics of adenovirustransformed rat embryo cells. J. Gen. Virol., 25: 263-273, 1974.
19. Frost, E., and Williams, J. Mapping temperature-sensitive and host-range
mutations of adenovirus type 5 by marker rescue. Virology, 97: 39-50,1978.
20. Graham, F. L., and van der Eb, A. J. A new technique for the assay of infectivity
of human adenovirus 5 DNA. Virology, 52: 456-467. 1973.
21. Gallimore, P. H., McDougall, J. K., and Chen, L. B. In vitro traits of adenovirustransformed cell lines and their relevance to tumourigenicity in nude mice. Cell,
70:669-678,1977.
22. Gallimore, P. H., and Richardson, C. R. An improved banding technique
lower levels of the Class I major histocompatibility antigen than
Ad-5 transformants and are more resistant to the cytolytic activity
of allogeneic cytotoxic T-cells (43, 44) led these authors to
postulate that their findings offered an explanation for Ad-12
oncogenesis. While reductions in the level of the Class I major
histocompatibility antigen are also a feature of some of the cell
lines described here, they are nevertheless susceptible to allo
geneic, cytotoxic, T-cell-induced cytolysis (45). It is important to
point out that Ad-12 virus is only tumorigenic when inoculated
into newborn or very young, immunologically immature rodents
(46). It therefore seems likely that the rejection of adenovirustransformed cells in vivo is a complex cell-mediated response in
which cytotoxic T-cells (43, 44), NK cells (47, 48), and activated
macrophages (49) have all been implicated. In vivo transformation
events which lead to tumor formation must evade the hosts
developing cell-mediated response, and the precise phenotype
that determines this has still to be resolved.
At present, our understanding of the coding potential of the
Ad-12 E1 region would appear to be complete. However, al
though we have developed a series of mutants affecting trans
formation and tumorigenicity, no defined biochemical activity has
been ascribed to any of the E1 proteins. The recent reports that
Ad-12 E1A is distantly related to myc and myb (50) and that
members of the ras gene family can complement E1A for trans
formation of BRK cells (11) in the absence of E1B have fired
speculation that different virus-transforming proteins have re
lated functions. With this exciting prospect in mind, it must be a
primary goal of researchers in this field to unravel the complex
ACKNOWLEDGMENTS
The following are acknowledged for the technical skills shown throughout this
study: Paul Biggs; Peter Grabham; Ann Maguire; Val Nash; Paul Reeve; Carl
Roberts; and Liz Withington. The authors would like to thank Professor A. B.
Rickinson and Dr. A. M. R. Taylor for constructive criticism of the manuscript and
Professor D. G. Hamden for his encouragement throughout this study. Excellent
secretarial assistance was provided by Debbie Williams.
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Fig. 1. Phase-contrast photomicrographs of Ad-12-transformed HL-BRK cell lines, a, Ad-12 virus-transformed cell line, passage 4; o, ECO.C2, passage 5; C, SAL.C1,
passage 5; d, HIN.G3, passage 3; e, ACC.H3, passage 4; and t, ACC.H5, passage 5. x 280.
Fig. 2. Southern blotting analysis of the Ad-12 HL BRK cell lines. Each cell line is identified above its lane. In control lanes, the amount of pAsc 2, pAsc 10.3, pAsc
6.8, and pAsc 4.7 that was mixed with normal rat cell DNA was equivalent to 4.8, 3.4, 3.7, and 6.0 copies per cell, respectively. DNA samples were digested with the
enzyme indicated above the horizontal lines. The lanes containing HIN.G1 to G5 DNA were overexposed to high-light weak bands. The identities of specific plasmid
fragments indicated on the right are linear pAsc 10.3, 6.5 kbp; linear pAsc 6.8, 5.9 kbp; pAsc 2 Pvull fragments, 5.0,1.7, 0.99, and 0.87 kbp; pAsc 4.7 Pvull-A, 3.2 kbp.
The sizes and mobilities (•)of the Ad-12 EcoRI fragments are also indicated on the right. The enzymes used in this analysis cleave the plasmids within the Ad-12
sequences only. The Pvull sites in pAsc 2 are 398, 905,1901, 3617,3727, and approximately 4600 nucleotides from the left end of Ad-12. The Hind\H site in pAsc 10.3
is at nucleotide 2318, and the Kpnl site in pAsc 6.8 is at nucleotide 588; these plasmids are converted to linears using these enzymes. pAsc 4.7 contains only the Pvull
sites at nucleotides 398 and 905. The 507-bp and 110-bp fragments contained between the Pvull sites at 398 and 905, 3617 and 3727, respectively, were run off the
gel in the Pvull digests shown in this figure. The left-hand 16.5% of the Ad-12 genome is represented at the bottom. The segment of the E1 region cloned in each plasmid
is shown above the line; e.g., pAsc 4.7 extends from nucleotides 1 to 1594. The sites for the restriction enzymes used in the Southern blotting analysis are shown below
the line. P, Pvull; K, Kpnl; and H, Hind\\\.
Fig. 3. Immunoprecipitation of proteins expressed in Ad-12-transformed rat cell lines. Rat cells were labeled with ["SJmethionine, and the labeled proteins were
immunoprecipitated and subjected to SDS/polyacrylamide gel electrophoresis as described in "Materials and Methods." Lanes 2 and 3, ACC.H5; Lanes 4 and 5, HIN.G3;
Lanes 6 and 7, SAL.C1 ; Lanes 8 and 9, ECO.C1; Lanes 70 and 77, HIN.G4; and Lanes 72 and 73, ECO.C3. Lanes 2.4, 6, 8,10, and 12 were immunoprecipitated using
preimmune rat serum, and Lanes 3, 5, 7, 9, 11, and 13, using ECO.C1 tumor bearer serum. Lane 1 contains the following "C-labeled molecular weight markers:
phosphorylase D, 93,000; bovine serum albumin, 66,000; ovalbumin, 45,000; a-chymotrypsinogen, 26,000; /Mactoglobulin, 18,000; and cytochrome c, 12,000. Lanes 1
to 9 and Lanes 10 to 13 are taken from different gels.
Fig. 4. Relative levels of E1A M, 42,000 and E1B M, 52,000 proteins in Ad-12 DNA-transformed rat cell lines. The SDS-soluble proteins extracted from 1 x 107 cells
per cell line were serially diluted and electrophoresed upon polyacrylamide gels in the presence of SDS as described in "Materials and Methods." Total proteins were
then transferred to nitrocellulose filters, reacted with Ad-12 tumor bearer serum, and further processed as described in "Materials and Methods." The cell line being
examined is indicated over each block of lanes. The number of cells extracted per lane was as follows: Lane 7, 5.0 x 105; Lane 2, 2.5 x 10s; Lane 3, 6.0 x 10*; and
Lane 4, 1.5 x 10*. The positions of molecular weight standards are shown. Blocks a tod were run on a different gel to Blocks e and f. The Ad-12 E1B M, 18,000 protein
does not efficiently etectroblot.
CANCER RESEARCH VOL. 45 JUNE 1985
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Ad-12 ONCOGENESIS
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VOL. 45 JUNE 1985
2679
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VOL. 45 JUNE 1985
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Properties of Rat Cells Transformed by DNA Plasmids
Containing Adenovirus Type 12 E1 DNA or Specific Fragments
of the E1 Region: Comparison of Transforming Frequencies
Phillip H. Gallimore, Philip J. Byrd, Joanne L. Whittaker, et al.
Cancer Res 1985;45:2670-2680.
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