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Transforming Potential Is Detectable in
Arteriosclerotic Plaques of Young Animals
Arthur Penn, Frank C. Hubbard Jr., and Joan Lee Parkes
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The carcinogen-treated cockerel is a model for studying the early stages of arteriosclerotic
plaque development. Carcinogen administration accelerates arteriosclerotic plaque development in cockerels, and transforming elements are present in DNA from advanced human
plaques. In this study, we asked whether transforming elements could also be detected at early
stages of plaque development in cockerels. NIH3T3 cells were transfected with DNA from
plaques isolated from carcinogen-treated cockerels and from the healthy arterial wall underlying the plaques. Approximately 5 x 106 cells from each group were injected into nude mice.
Tumors appeared in five of five mice in the plaque DNA group; no tumors appeared in mice
from the healthy arterial wall group. All five plaque DNA-associated tumors hybridized to a
cockerel genomic probe. Eight cockerel-specific bands were identified in EcoRl digests of firstround (primary) tumors. DNA from a primary tumor was tested in a second round of
transfection. Five of five mice developed tumors after injection with these secondary transformants. All second-round tumors contained cockerel DNA, and a prominent cockerel-specific
band (>28 kb) was seen in£coRI digests of all second-round tumors. In addition, a 5.2 -kb band
appeared prominently in one of five second-round tumors. No evidence was found for activation
of the oncogenes Ha-ras, Ki-ras, src, or myc in the plaque-associated tumors. Similarly, DNA
from plaque-associated tumors did not hybridize to probes for Marek disease virus, herpes
simplex virus 1, or reverse transcriptase, suggesting that neither herpesviruses nor retroviruses
are involved in the transforming activity of plaque DNA. These results indicate that transforming elements are a general property of arteriosclerotic plaques and are detectable in plaques of
young animals. (Arteriosclerosis and Thrombosis 1991;ll:1053-1058)
T
he proliferation of smooth muscle cells
(SMCs), a rare event in the normal adult
arterial intima, is thought to be critical to the
development of arteriosclerotic plaques. Most theories of plaque development have regarded SMC
proliferation as a reactive process, occurring in response to such stimuli as injury1 or inflammation.2
Under these conditions the SMCs involved in plaque
formation are regarded as indistinguishable (except
for receipt of stimuli that cause proliferation) from
the rest of the arterial wall SMCs. According to this
view, it is the availability of mitogens or other modulators of cell division rather than any unique property of the responding arterial SMCs that determines
whether intimal SMC proliferation and subsequent
plaque development will occur.
From the Institute of Environmental Medicine, New York
University Medical Center, N.Y.
Supported by National Institute of Environmental Health Sciences grant Nos. 02143 and 00260, American Heart Association
grant-in-aid No. 87-993, and the Council for Tobacco Research.
Address for reprints: Arthur Penn, PhD, Institute of Environmental Medicine, NYU Medical Center, New York, NY 10016.
Received September 20, 1990; revision accepted March 21,
1991.
A fundamentally different view is presented by the
monoclonal hypothesis.3 This proposes that the proliferating SMCs are derived from a stably transformed and thus permanently altered cell population
that is distinct from the bulk of arterial SMCs. One
inference from this hypothesis is that there should be
demonstrable similarities, on the molecular level,
between events associated with the development of
both plaques and tumors. Indeed, the prediction has
been made that mutagens and viruses should play a
role in plaque formation comparable to the role that
they play in tumor formation. Studies including those
using cockerels as an animal model have provided
indirect support for this view. Weekly injections of
polynuclear aromatic hydrocarbon carcinogens
(PAHCs) at nontumorigenic doses into cockerels
markedly accelerate plaque development.4-7 The inducible enzyme systems responsible for the metabolism of PAHCs have been identified in the artery wall
of both cockerels and mammals.8"10 The PAHC
7,12-dimethylbenz[a]anthracene (DMBA), administered as an "initiator" followed by weekly injections
of the a-adrenergic agonist methoxamine (as a promoter) yielded microscopic aortic plaques in cocker-
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Arteriosclerosis and Thrombosis Vol 11, No 4 July/August 1991
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els.11 A series of studies during the past decade have
also suggested a role for herpesviruses in plaque
formation.12-20 Two recent sets of in vitro studies
have provided evidence for possible mechanisms of
the interaction between viruses and SMCs. In the
first of these studies fibroblast growth factor receptor
was identified as the site of herpesvirus entry into
mammalian cells, including SMCs.21 In the second
study rabbit aortic SMCs were transformed by the
early region of SV40 DNA.22
Carcinogen and virus studies have thus far provided
only indirect support for the predictions of the monoclonal hypothesis. Direct support requires demonstration of molecular alterations in plaque cells comparable to those observed in tumor cells. Earlier, we
reported that human coronary artery plaque DNA
contained transforming potential and that mouse fibroblasts (NIH3T3 cells) transfected with human coronary artery plaque DNA gave rise to tumors after
subcutaneous injection into nude mice.23 The protocols followed in these studies were originally developed to demonstrate the presence of dominant transforming genes (oncogenes) in tumors and transformed
cell lines.24"26
One limitation associated with these human studies is that the plaques were obtained from older
patients in whom atherosclerosis developed over a
period of decades. Thus, it could be argued that
acquisition of transforming potential is a late-stage
event in atherosclerosis and is only indirectly associated with plaque etiology.
An advantage of using the carcinogen-treated cockerel as a model for plaque development is that large
plaques can be generated in the abdominal aortas of
these young animals after carcinogen treatment.
These plaques are fibromuscular, with limited lipid
involvement and without apparent calcification or
necrosis. We have shown that in this system carcinogen treatment acts primarily to accelerate plaque
development rather than to initiate it.4"7 Morphological and ultrastructural similarities between fibromuscular abdominal aortic plaques in cockerels and
coronary artery plaques in humans have been documented.27 The objective of the present studies was to
determine whether molecular alterations exist in
carcinogen-associated cockerel plaques similar to
those previously identified in human plaques.23
In this article we report that NIH3T3 cells are
transformed by carcinogen-associated cockerel
plaque DNA and that injection of these transformed
cells into nude mice elicits development of tumors
that contain cockerel genomic DNA. Moreover, this
plaque transforming potential is transmitted serially.
Thus, there are molecular alterations in the DNA of
plaques from diverse sources (human and avian) and
from young and old subjects that are similar to
alterations that have been identified in tumor DNA.
This suggests that somatic cell mutations play a role
in plaque etiology.
Methods
Animals
Four-week-old white leghorn cockerels (Kerr
Hatcheries, Hightstown, N. J.) were housed in stainless
steel cages. Water and standard mash (Purina Co., St.
Louis, Mo.) were available ad libitum. All animal
handling and treatment procedures were conducted
according to New York University Medical Center
guidelines. From 6-22 weeks of age the cockerels
received weekly injections of DMBA (20 mg/kg body
wt, Sigma Chemical Co., St. Louis, Mo.) into the
pectoral muscle. The DMBA was dissolved in dimethyl sulfoxide (Fisher Chemical Co., Valley Forge, Pa.).
After the cockerels were humanely killed at 23 weeks
of age, plaques and healthy arterial wall underlying
the plaques were removed under sterile conditions
and frozen immediately in liquid N2.
Transfections
All tissue samples were pulverized in liquid N2.23
DNA extraction was performed via standard phenol/
CHCyisoamyl alcohol procedures.28 DNA from four
separate sources was tested in the transfection assay: 1)
cockerel aortic plaque, 2) healthy cockerel arterial wall
that had underlain the plaques, 3) T24 human bladder
carcinoma cell line (positive transfection control), and
4) NIH3T3 cells (negative transfection control). The
DNAs were cotransfected with pSV^eo*29 the hybrid
plasmid vector conferring antibiotic resistance, by the
standard Ca3(PO4)2 transfection protocol.30 For each
sample 40 fig genomic DNA was transfected along with
4 fig pSV2neo into three T-25 flasks, each containing
approximately 0.5 x 106 NIH3T3 cells grown in Dulbecco's modified Eagle medium (DMEM) with 10% fetal
bovine serum. Twenty-four hours later cells were split
1:3. Transfected cells were grown in the presence of
gentamicin (G418).
Nude Mouse Assay
Three weeks after cotransfection the colonies were
collected, and 4-5 xlO6 cells from each group were
injected subcutaneously into the scapular area of five
4-5-week-old athymic (nu+/nu+) female mice (Harlan Sprague-Dawley, Indianapolis, Ind.). Animals
were checked for the presence of tumors three times
per week. The mice were killed when the tumors
were at least 15 mm in diameter, and samples of
tumors were taken for histological observation, DNA
isolation, and growth in culture.
Southern Blot Hybridization
DNA was digested with the restriction enzymes
EcoRl and BamHI according to the manufacturers'
instructions, subjected to electrophoresis overnight in
0.7% agarose (Bio-Rad, Richmond, Calif.) at 20 V, 15
mA per gel, and blotted onto nitrocellulose31 (Schleicher and Schuell, Keene, N.H.). pSV2n<:o was provided
by E. Newcomb, New York University Medical Center. The probes Ha-ras, Ki-ras, src, and myc, consisting
Penn et al
Transforming Potential in Plaques
TABLE 1. Tumorigenic Efficiency of Plaque DNA-Transfected Cells
No.
DNA source
Arteriosclerotic plaques
from DMBA-treated
cockerels
Healthy arterial wall
(underlying plaque)
from DMBA-treated
cockerels
T24 cells (human bladder
carcinoma cell line)
NIH3T3 cells
Plaque-associated primary
tumor CT1-3 (from first
source above)
T24 cells
NIH3T3 cells
tumors/No.
mice
Tumor
latency
(days)
5/5
33-45
1 2
3
4
5
1055
6
0/5
7-10
5/5
0/5
2.32.0-
5/5
5/5
0/5
'
24-42
19-23
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For each group, ~5xl0 6 cells were injected subcutaneously in
the dorsal surface of five female nu+/nu+ mice.
DMBA, dimethylbenz/ayanthracene.
of inserts cut out and purified from plasmids, were
purchased from Oncor, Gaithersburg, Md.; the reverse transcriptase (RT) probe, contained in plasmid
pRT 432-1, No. 31990, was purchased from the American Type Culture Collection, Rockville, Md. The
herpes simplex virus 1 (HSV-1) probe was a gift of
David Hajjar, Cornell University Medical Center,
New York. The Marek disease virus (MDV) probe
(BamHI-H insert), which does not hybridize to DNA
from uninfected chicken cells, was provided by K.
Schat, Cornell University, Ithaca, N.Y. The cockerel
genomic probe was prepared from cockerel heart
DNA. All the probes were labeled with phosphorus32-labeled cytidine triphosphate (New England Nuclear, Boston, Mass.) by nick translation.32
Hybridization conditions were as follows: for Ha-ras,
myc, src, and RT, 40% formamide, 40°C, 18 hours; for
Ki-ras, 45% formamide, 43°C, 18 hours; for HSV-1 and
cockerel probe, 50% formamide, 43°C, 18 hours; and
for MDV, 50% formamide at 51°C, 30 minutes, and
then at 42°C, 24 hours. Washes and exposures were
performed by use of standard procedures.28
Results
Cockerels were injected weekly with DMBA from
6-22 weeks of age and were killed at 23 weeks of age.
Four of five DMBA-injected cockerels displayed
grossly visible plaques (12x3 mm) in the distal third
of the abdominal aorta. These plaques were similar
to DMBA-associated plaques that we have described
previously4-7; they werefibromuscularwith little lipid
involvement. DNA samples obtained from the
pooled plaques, healthy arterial wall underlying the
plaques, T24 human bladder carcinoma cell line, and
NIH3T3 cells were used to cotransfect host NIH3T3
cells. Results of the transfection-nude mouse study
are presented in Table 1. Tumors developed in all
FlGURE 1. Hybridization of plaque-associated tumor DNA
to a cockerel genomic probe. EcoRI-digested DNA samples
(20 ugllane) were Southern blotted and hybridized to a
phosphorus-32-labeled probe prepared from cockerel heart
DNA. Hybridizations were performed under high stringency
conditions (50% formamide, 43°C.) Sources of DNA were
lane 1, T24-associated nude mouse tumor; lane 2, NIH3T3
cells; lane 3, first-round cockerel plaque DNA-associated
nude mouse tumor (CT1-3); lane 4, CT1-3 primary explant;
lane 5, second-round nude mouse tumor (CT2-3); lane 6,
second-round nude mouse tumor (CT2-4). Note that faint
bands present in the T24-derived tumor and in the NIH3T3
cells (lanes 1 and 2) are most likely due to cross hybridization
of the cockerel probe to mammalian repetitive sequences with
which cockerel repetitive sequences share homology.33-35
Numbers at left are in kb.
five mice injected with plaque DNA-transfected
cells. The tumor latency period was 33-45 days. In
contrast, no tumors appeared in any mice injected
with cells transfected with DNA from normal arterial
wall or from untreated NIH3T3 cells. All five mice in
the T24 carcinoma cell line group developed tumors,
with a latency period of 7-10 days. Tumors were
excised when they reached 15-20 mm in diameter.
All tumors had the appearance of poorly differentiatedfibrosarcomas.DNA from all first-round plaqueassociated tumors hybridized to a cockerel genomic
probe. At least eight distinct cockerel-specific bands
were visible in first-round plaque-associated tumors
(see Figure 1).
DNA extracted from one of the plaque DNAassociated tumors (CT1-3) was tested in a second
round of transfection. All five mice injected with the
second-round transfectants developed tumors. Latency periods ranged from 24 to 42 days (Table 1).
The positive control group in this study displayed
tumors (five offivemice) after 19-23 days. Again, no
tumors appeared in the negative controls.
DNAs isolated from the secondary tumors were
digested with EcoKl and hybridized to a cockerel
genomic probe. All secondary tumors contained
cockerel genomic DNA. A prominent band (>28 kb)
was identified in all secondary tumors. EcoRl digests
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Arteriosclerosis and Thrombosis
Vol 11, No 4 July/August 1991
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9.46.6-
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FIGURE 2. Absence of cockerel myc sequences in NIH3T3
transformants is shown by Southern-blotted BamHI-digested
DNA hybridized to a phosphorus-32-labeled v-myc probe.
Hybridizations were performed in the presence of 40% formamide at 40°C. Sources of DNA were lane 1, cockerel heart;
lanes 2 and 3, first-round plaque-associated nude mouse
tumors; lane 4, nude mouse tumor primary explant; lane 5,
NIH3T3 cells. Cockerel myc is represented by an intense band
(of >9.4 kb) in lane 1. A less intense marine myc signal
appears at —6.6 kb in lanes 2-5. The v-myc probe derived
from the avian MC29 virus has greater homology to cockerel
myc than it does to mouse myc and therefore hybridizes more
strongly to the cockerel proto-oncogene.
of two of these tumors are shown in Figure 1 (lanes
5 and 6). In addition, a 5.2-kb band appeared prominently in one of five second-round tumors (Figure 1,
lane 6). As expected, neither DNA from the T24associated tumors nor that from NIH3T3 cells hybridized to the cockerel genomic probe (Figure 1,
lanes 1 and 2).
DNA from the first round of plaque-associated
tumors was tested for the presence of activated
oncogenes. Ki-ras, Ha-ras, myc, and src probes were
used to screen Ba/nHI-digested DNA from plaqueassociated tumors. None of these was responsible for
the transforming potential of cockerel arteriosclerotic plaque DNA (Figure 2 and data not shown). In
Figure 2 the intense band (>9.4 kb) in lane 1
(cockerel heart DNA) is cockerel myc; the less
intense bands of approximately 6.6 kb in lanes 2-5
are murine myc. The absence of a >9.4-kb cockerel
myc signal in the plaque-associated nude mouse
tumors indicates that myc is not the cockerel plaquetransforming gene.
We also tested the possibility that retroviral infection plays a role in cockerel arteriosclerotic plaque
development. DNA from plaque-associated tumors
was hybridized to an RT probe. No RT-specific bands
were identified (data not presented).
Because a single injection of the avian herpes virus
MDV to 4-day-old cockerels results in focal plaque
formation,12 we screened the plaque-associated tumor
DNA with MDV. This probe failed to hybridize to
plaque-associated rumor DNA (data not presented).
Finally, because herpesviruses have been implicated as
etiologic factors in the pathogenesis of human arteriosclerosis and human HSV-1 has been shown to influence lipid accumulation and metabolism in both human
and bovine arterial SMCs,14 we also screened the
plaque-associated tumor DNAs with an HSV-1 probe.
This, too, was negative, suggesting that herpes viral
sequences are not associated with the transforming
element(s) in cockerel plaque DNA.
Discussion
Previously, we have shown that carcinogen treatment accelerates plaque development in cockerels.4-7
We have also demonstrated, via the transfectionnude mouse tumor assay, that human coronary artery
plaque DNA has transforming potential.23 Recently,
we reported that DNA from SMC strains derived
from human aortic plaques is also positive in the
transfection-nude mouse tumor assay.36
The data presented here show that cockerel plaque
DNA, like human plaque DNA, contains transforming element(s). NIH3T3 cells transfected with cockerel plaque DNA gave rise to nude mouse tumors in
five of five injected mice. The tumor latency period
(33-45 days) was significantly shorter than that for
human coronary artery plaque-associated tumors
(50-112 days)23 but longer than that for the T24associated tumors (7-10 days), which contain an
activated Ha-ras oncogene. As is the case with human
coronary artery plaque DNA, cockerel plaque DNA
transforming elements are serially transmitted, with
DNA from primary plaque-associated tumors giving
rise to secondary plaque-associated tumors (Table 1).
DNA from both rounds of tumors hybridized to a
cockerel genomic probe. A number of cockerelpositive bands were present in ZscoRI-digested DNA
from plaque-associated first-round tumors and their
primary explants (Figure 1). When DNA from one of
the plaque-associated tumors was tested in a second
round of transfection, a prominent cockerel-positive
band (>28 kb) was retained in all the resulting
tumors (Figure 1). In addition, a 5.2-kb band was
present in one of the second-round tumors. The
retention of cockerel-positive bands in the secondround tumors suggests that the putative transforming
gene is associated with these sequences. We are
currently investigating this phenomenon.
One approach to identifying the transforming gene
in cockerel plaque DNA is to screen the plaqueassociated tumor DNA with oncogene probes. We
tested for the presence of an activated cockerel
Ha-ras gene because mutations in codons 12 and 61
of Ha-ras have been reported in skin cells transformed by the PAHCs benzo[a]pyrene (B[a]P) and
DMBA, respectively,37 and because B[a]P and
DMBA markedly accelerate plaque development in
cockerels.7 However, we found no evidence of cockerel Ha-ras in any of the plaque-associated tumors
even though the plaques were obtained from DMBAtreated cockerels. Similarly, we found no evidence
for the presence of exogenous Ki-ras, src (not shown),
or myc (Figure 2) in any of the cockerel plaqueassociated nude mouse tumors. Ki-ras is one of the
most commonly activated dominant transforming
genes that has been identified in human tumors.38
Activation of src, which is responsible for the transforming activity of Rous sarcoma virus, results in
malignant transformation of avian cells.39 myc was
Penn et al
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first identified as the transforming gene of avian
myelocytomatosis virus.40 In humans, enhanced
expression of myc has been noted in cases of leukemia, lymphoma, and carcinoma of the breast and
prostate. A modest elevation of myc expression was
also observed in SMCs of spontaneously hypertensive
rats compared with normotensive rats.41 We have
recently described a twofold to sixfold enhancement
of myc expression in SMC strains derived from
human abdominal aortic plaques compared with
healthy human aortic SMCs, although myc was not
responsible for the transforming potential of the
DNA from these cell strains.36
Finally, the absence of RT, MDV, and HSV-1 sequences in the plaque-associated nude mouse tumors
suggests that these viruses are not involved in the
etiology of these cockerel plaques. Although one recent
report failed to identify transforming activity in human
carotid plaques,42 other investigators have confirmed
that serially transmitted transforming activity is present
in human arteriosclerotic plaques (Reference 43 and
R.M.L. Zwijsen, personal communication).
The results described herein confirm our previous findings with plaque samples of human origin23
and demonstrate that the presence of transforming
elements is a general characteristic of plaque DNA.
Furthermore, the fact that these activated elements exist in animals only 6 months old demonstrates that transforming potential is detectable in
plaques of young animals.
Acknowledgments
We thank Tom Schat for performing the hybridization with the MDV probe; Alan Bowers and
Dale Hubbard for excellent technical assistance;
and Sy Garte and Carroll Snyder for critically
reviewing this manuscript.
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43. Ahmed AJ, O'Malley BW, Yatsu FM: Presence of a putative
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Arteriosclerosis 1990;10:755a
KEY WORDS • arteriosclerotic plaque • transforming genes •
cockerel • carcinogens • tumors • transfection
Transforming potential is detectable in arteriosclerotic plaques of young animals.
A Penn, F C Hubbard, Jr and J L Parkes
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Arterioscler Thromb Vasc Biol. 1991;11:1053-1058
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