(CANCER RESEARCH 57. 5542-5549.
December 15. 1997]
A Transgenic Mouse Model with Cyclin Dl Overexpression Results in Cell Cycle,
Epidermal Growth Factor Receptor, and p53 Abnormalities1
Annegret Mueller, Robert Odze, Timothy D. Jenkins, Ali Shahsesfaei,
Anil K. Rustgi2
Hiroshi Nakagawa, Takuya liminolo, and
Gastrointestinal Unit ¡A.M.. T. D. J., H. N.. T. I., A. K. R.¡.Hematology-Oncology Unit ¡A.K. K.¡.Massachusetts
Women's Hospital /K. O.. A. S.I. Hazard Medical Schon/. Boston. Massachusetts 02114
General Hospital, unii Department of Pathology. Brigluim and
(18, 19). The oncogenic properties of cyclin Dl have been demon
strated in vitro by its ability to cooperate with ra.v (20) and its ability
to complement a detective adenovirus Eia (21) in cultured cells.
Transgenic mouse models have involved the targeting of cyclin Dl to
breast tissue, resulting in mammary hyperplasia and carcinomas in
lactating mice (22). More recently, we have developed a transgenic
mouse model in which the EBV ED-L2 promoter when fused to the
human cyclin Dl cDNA results in dysplasia restricted to the oral
cavity and esophagus, both sharing a stratified squamous epithelium
(23). This tissue specificity is attributable to the interaction of squa
mous epithelial nuclear transcriptional factors with the EBV ED-L2
ABSTRACT
The cyclin DI oncogene is critical in the progression of the cell cycle
through the (ì,phase. It is frequently overexpressed in squamous cell
carcinomas originating from the head/neck and esophagus. Yet, the func
tional consequences of aberrant cyclin 1)1 overexpression are not entirely
understood apart from increased cell proliferation. To address this ques
tion, we have developed a transgenic mouse model in which the EBV
1 l)-l 2 promoter targets cyclin DI to the stratified squamous epithelium
in a tissue-specific fashion to the tongue and esophagus, thereby resulting
in a dysplastic phenotype. We now demonstrate that the dysplastic phenotype is associated with increased cell proliferation based on proliferat
ing cell nuclear antigen overexpression and abnormalities in cyclin-dependent kinase 4, epidermal growth factor receptor, and p53. In
aggregate, these studies suggest that alterations in certain oncogenes and
tumor suppressor genes occur early during head/neck and esophageal
carcinogenesis.
INTRODUCTION
Cyclin Dl is a key protein that contributes to cell cycle progression
through the G, phase. It associates with cdks.3 preferentially cdk4 or
cdk6. and PCNA ( 1). A consequence of the association between
cyclin Dl and cdk4 or cdk6 is the phosphorylation of the retinoblastoma tumor suppressor gene product (pRh. Ret. 2). Hyperphosphorylation of pRb leads to its dissociation from transcriptional factors,
such as E2F. that transcriptionally regulate growth-promoting genes.
Although pRb remains a key target of cyclin Dl/cdk4 or cdko, other
proteins have been postulated to be putative targets, for example the
myb-like protein (3).
Cyclin Dl overexpression is associated with a number of malig
nancies. One prominent pattern is that of cyclin Dl overexpression
due to gene amplification in squamous cell cancers, originating from
the head/neck (4-11) and esophagus (12-14). Alternatively, chromo
somal translocation is responsible for cyclin Dl overexpression in
parathyroid adenomas (15). centrocytic lymphomas (16), and some
breast cancers, although gene amplification is a major factor in breast
cancers (17). Insights into how cyclin Dl contributes to malignant
transformation have been gained through in vitro and HI vivo ap
proaches. Overexpression of cyclin Dl in cultured cells leads to a
more rapid traversion through the G, phase and entry into the S phase
Received 4/14/97; accepted 10/15/97.
The costs of publication of this article were defrayed in part hy the payment of page
charges. This article must therefore he hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported hy the ADHF/AGA Fiterniun and Funderburg Awards (to
A. K. R.I. an American Cancer Society Jr. Faculty Research Award JFRA-649 (to
A. K. R.). NIH Grants DK4056I and DK53377 do A. K. R.l. and Department of Knergy
DE-FG-2-9I-ER61228
Award (to H. N. and A. K. R.(. A. M. was supported by the
Deutsche Forschungsgemeinschaft
<1304-1-1). R. O. was supported by the Stanley L.
Rohhins Research F'und from the Department of Pathology at Brigham and Women's
Hospital. T. J. was supported by the Glaxo Wellcome Institute for Digestive Health
Award.
: To whom requests for reprints should be addressed, at Gastrointestinal and
Hematology-Oncology Units. Jackson 904, Massachusetts General Hospital. 50 Blossom
Street, Boston. MA 02114. Phone: (617)724-3740;
Fax: (617)726-3673; E-mail:
rustgi(a: helix, mgh.harvard.edu.
' The abbreviations used are: cdk. cyclin-dependeni kinase; PCNA. proliferating cell
promoter (24).
One of the inherent advantages of the dysplastic phenotype result
ing from the targeting of cyclin Dl to the tongue and esophagus is the
molecular dissection of the dysplastic tissues to assess whether other
genetic alterations are acquired as a function of cyclin Dl overex
pression. Thus, a multistage model of oral-esophageal carcinogenesis
can be developed with the correlation of key genetic alterations with
dysplasia. We show that cyclin Dl overexpression is associated with
increased cell cycle proliferation as interred from PCNA overexpres
sion, enhanced cdk4 overexpression. and increased EGFR overexpres
sion. as well as p53 abnormalities.
MATERIALS
AND METHODS
Generation of Cyclin Dl Transgenic Mice. We employed the EBV
ED-L2 promoter and fused it Io the human cyclin Dl cDNA to create a
transgene from which founder lines were generated and maintained in the
FVB/N genetic background. The cyclin DI transgene is expressed in a tissuespecific fashion with targeting in the tongue, esophagus, and forestomach (23).
The cyclin Dl transgenic mice and age-matched wild-type mice were sacri
ficed at 1.9. and 20 months for tissue processing and examination. There were
15 cyclin Dl transgenic mice from two different founder lines analy/ed each
at 9 and 20 months: these time points demonstrate dysplasia in the tongue and
the esophagus. Five transgenic mice from two different founder lines with
normal tongue and esophageal histology were analy/ed at age I month.
Because the wild-type mice did not show any tongue or esophageal histológ
ica! changes at the three different time points, a total of live wild-type mice
(ages 1. 9. and 20 months) were considered in aggregate as one group for
statistical analysis.
Immunohistochemistry.
All comparisons were made between agematched (1.9. and 20 months) wild-type and cyclin Dl transgenic mice tongue
and esophageal tissue sections. Tissues from the tongue and esophagus were
extracted, fixed in 10% formalin, embedded in paraffin, and processed for
H&E staining. Immunohistochemical
staining of the tongue and esophagus
was performed in the following manner with modifications as outlined for
individual antibodies. Tongue (encompassing dorsal and ventral surfaces) and
esophageal (proximal, mid. and distal) tissue sections were cut 3-5 jim thick,
and slides were dried at room temperature for 2 days. Then, the sections were
baked at 60°Cin a microwave oven for 20 min. deparaffini/ed in xylene. and
rehydrated in alcohol and water. For EGFR and cyclin Dl.
treated with 3% H2O2 in absolute methanol for 5 min to
peroxidase activity. All slides were then microwave treated
sodium citrate (pH 6.0) at 93°Cfor 30 min. The slides were
nuclear antigen: ECiFR. epidermal growth factor receptor.
slides were further
block endogenous
(800 W) in 10 HIM
then cooled for 15
min and transferred to PBS.
After blocking with 1.5% goat serum (blocking step for EGFR. cyclin Dl.
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TRANSCÃŒKNICMOUSE MODEL WITH CYCLIN
intmimohislocheinislr\Antibodyanli-PCNA(PC
Table 1 Antibodies for
DI OVKRIAI'KI
SSION
respect to dysplastic cells and expressed in the following categories: none, no
nuclei stained: rare. <30% of viable nuclei stained positive; moderate. 30-
clonalityMonoclonalPolyclonal(affinity time(h)1121llCompanyPharmingen(San
10)anti-cyclin
CA)Gift
Diego.
Dl(#19)anli-cdk4(sc-260)anti-EGFR(sc-003)anti-p53(#1801)Antibody
HarlowSanta
of E.
purified)Polyclonal(affinity
:8(X)1:2001:1500IncubationCru/(Santa
purified)PolyclonalMonoclonalDilution1:6001:15001 ÇA)Santa
Cru/..
CruzI
CA)BioGenex(San
Santa Cruz.
60% of viable nuclei stained positive; and strong. >60% of viable nuclei
stained positive (27). Of note, the p53 antibody recognized a broad spectrum
of mutant p53 proteins as well as wild-type p53. All imnumohistochemical
changes were assessed and scored independently in a blinded fashion by three
different observers (A. M.. R. O.. and A. K. R.l.
Statistical Analysis. Statistical comparisons between different age groups
of mice were performed with the use of the one-way ANOVA lest and
Duncan's multiple range test. P < 0.05 was considered statistically significant.
Ramon, CAI
RESULTS
and cdk4) or with 1.5% horse serum (blocking step for PCNA and p53) in PBS
for 15 min. the slides were incubated with the primary antibody at room
temperature, except for cyclin Dl at 4°C(Table 1). After washing with PBS.
slides were incubated lor 30 min with a 1:2(K) dilution of the appropriate
biotinylated secondary antibody, washed with PBS. and then incubated for 40
min with the avidin and biotinylated peroxidase complex (Vectastain Elite
ABC kit: Vector Laboratory. Burlingame. CA) at room temperature. After
washing with PBS. the colorimetrie reaction was performed with 3.3'-diaminoben/.idine (Sigma Chemical Co., St. Louis, MO) and 0.1% H2O2 in 0.5 M
TrÃ-s-HClbutter (pH 7.6). The sections were counterstained with hematoxylin.
dehydrated, and mounted in Permount (Fisher Scientific. Pittsburgh. PA).
Human tissue sections served as positive controls for the primary antibodies:
tonsil (PCNA). and breast cancer (EGFR. cyclin Dl. cdk4. and p53). Normal
goat or horse serum was used as the primary antibody to serve as negative
controls.
For determination of PCNA. cyclin Dl. and cdk4 nuclear staining, the
average percentage positively was determined by counting the number of
positively stained cells of 500 cells at X400. EGFR cytoplasmic and mem
branous staining was assessed in a semiquantitative fashion: grade 0. no
staining; grade 1+, less than 25% of cells stained positive; grade 2 + , 25-49%:
grade 3 + , 50-74%; and grade 4+, greater than or equal to 75% (25. 26).
Scoring of p53 staining was based upon the percentage of positive nuclei with
We have previously demonstrated that an EBV ED-L2 promoterdirected cyclin DI transgene leads to a oral-esophageal tissue-specific
dysplastic phenotype in mice (23), an important precursor to cancer.
This is initially evident by 6-9 months in the form of nuclear atypia
and progresses to greater dysplasia by 16-20 months (Fig. 1). Such
changes are not evident in age-matched wild-type mice in the same
FVB/N background or in young (1-month) transgenic mice, the latter
likely reflecting the luck of selection of cyclin Dl-overexpressing
cells. Overall, this model provides a unique opportunity to determine
not only whether cell cycle partners of cyclin Dl are aberrantly
expressed but also to determine whether genetic alterations frequently
described in human oral-esophageal carcinogenesis are associated
with dysplasia. In this context, EGFR overexpression and p53 muta
tions have been described in human oral and esophageal dysplasia and
squamous cancers.
PCNA and cdk4 Expression Are Increased in the Tongue and
Esophageal Tissues of the Cyclin Dl Transgenic Mice. The in
creased cyclin Dl expression was evident in the 9- and 20-month-old
transgenic mice tongue and esophageal tissues in comparison to
age-matched wild-type and 1-month-old transgenic mice, as apparent
Fig. 1. H&E staining (X400) of wild-type mouse
(age 16 months) longue (A) and esophagus (O and
cyclin Dl transgenic mouse (age 16 months) tongue
(ß)and esophagus (/)). Note the nuclear atypia, in
creased nuclear size, nuclear hyperchromasia. all indi
cated by medium-sized arrows in B and D. Also, there
is loss of polarity in the transgenic mouse tongue and
esophageal epithelium. A mitolic figure is observed in
the dysplastic tongue in B (small arrow). In addition,
the tongue has elongation and bridging of epithelial
pegs (large open arrow}. [Modified from Nakagawa el
al. (23).]
* H *
»
£»+
*'
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TRANSGENIC MOUSE MODEL WITH CYCLIN DI OVEREXPRESSION
Fig. 2. Cyclin Dl immunohistochemical stain
ing (X400) in 20-month-old wild-type mouse
tongue (A) and esophagus (C) and 20-month-old
transgenic mouse tongue (B) and esophagus (D).
Note the brawn cyclin Dl nuclear staining in ßand
D (also indicated by arrows).
M
Fig. 4. PCNA immunohistochemical
staining
(X400) in 20-month-old wild-type mouse tongue
(Ai and esophagus (C) and 20-month-old trans
genic mouse tongue (ßland esophagus (D). Note
the dark PCNA nuclear staining in B and D (also
indicated by arrows).
in the immunohistochemical staining in Fig. 2 and the quantitative
scoring in Fig. 3. The vast majority of cyclin Dl staining is nuclear,
although rare cytoplasmic staining is noted. We then determined
whether the cyclin Dl overexpression resulted in corresponding in
creases in PCNA and cdk4 expression. The 9- and 20-month-old
transgenic mice demonstrated an increase in nuclear PCNA expres
sion (Fig. 4) in the tongue and esophageal basal and intermediate
layers in comparison to the 1-month-old transgenic mice and all the
age-matched wild-type mice. Nearly 60% of cells counted in the
tongue epithelium were positive for PCNA staining in the 9-monthold transgenic mice, which increased to approximately 70% of cells in
the 20-month-old transgenic mice, representing statistically signifi
cant increases when compared to the 1-month-old transgenic mice and
all of the age-matched wild-type mice (Fig. 5). Similar numbers of
positive cells for PCNA staining were observed in the esophageal
epithelia of the 9- and 20-month-old transgenic mice (Fig. 5), also
statistically significant. Additionally, there was increased cdk4 ex
pression in the tongue and esophageal epithelial layers in the same age
groups (9- and 20-month-old) of transgenic mice when compared to
the 1-month-old transgenic mice and all of the age-matched wild-type
mice (Fig. 6). Again, the number of cells staining positive for cdk4 in
the basal and intermediate layers of the 9- and 20-month-old trans
genic mice was greater than those in the 1-month old transgenic and
all the age-matched wild-type mice (Fig. 7) in a statistically signifi-
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TRANSGENIC
MOUSE MODEL WITH CYCLIN
DI OVEREXPRESSION
100
TONGUE (Cyclin D1)
«ESOPHAGUS (Cyclin 01)
Fig. 3. Quantitative scoring of cyclin DI immunohistochemical staining in the tongue and esoph
agus. Wi. wild-type mice; 1-, 9-, and 20-month old
groups are transgenic mice. + + , age-matched
wild-type mice compared lo all transgenic mice
groups: P < 0.05 for tongue and esophagus. »,
I-month old transgenic mice compared to 9-rnonthold transgenic mice and 1-month-old transgenic
mice compared to 20-month old transgenic mice:
P < 0.05 for tongue and esophagus. **. 9-monthold transgenic mice compared to 20-monlh-old
transgenic mice: P < 0.05 for longue and esopha
gus. »**,comparisons as noted above.
Wild Type
1 month
9 months
20 months
GROUPS
100
•¿
TONGUE (PCNA)
90
•¿
ESOPHAGUS (PCNA)
80
Fig. 5. Quantitative scoring of PCNA immunohistocheniic.il
staining in the tongue and
esophagus. Wi. wild-lype mice; I-, 9-. and 20month old groups are transgenic mice. ++. agemalched wild-type mice compared lo all trans
genic mice groups: P < 0.05 for tongue and
esophagus. *. I-month-old transgenic mice com
pared to 9-month-old
transgenic mice and
I-month-old transgenic mice compared to 20month old transgenic mice: P < 0.05 for tongue
and esophagus. »*.9-month-old transgenic mice
compared to 20-monlh-old
transgenic mice:
P < 0.05 for tongue and P > 0.05 for esophagus.
***. comparisons as noted above.
10
Wild Type
1 month
9 months
20 months
GROUPS
cant fashion. Because cdk4 is one of the major catalytic subunit
may be present in the cyclin Dl transgenic mice dysplastic oral-
partners of cyclin Dl, to our knowledge, this is the first in vivo
demonstration of parallel increases in cyclin Dl and cdk4 expression
in the setting of dysplasia.
Genetic Abnormalities Are Acquired in Cyclin Dl Transgenic
Mice. Given the high frequency of ECFR oncogene and p53 tumor
suppressor abnormalities in human head/neck (28-34) and esophageal
(35-40) squamous cell cancers, we determined whether such changes
esophageal tissues,
In the wild-type and the 1-month-old cyclin Dl transgenic mice,
focally weak EGFR cytoplasmic staining was detectable only in
the basal layer consistent with grade 1+ staining. However, all
9-month and 20-month cyclin Dl transgenic mice revealed an
increase in the intensity and degree of cytoplasmic and membranous EGFR staining in the basal and intermediate layers. This was
5545
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TRANSGENIC
MOUSE MODEL WITH CYCLIN
DI OVÃœREXPRESSION
Fig. 6. cdk4 immunohistochemical
staining
(X400) in 20-monlh-old wild-type mouse tongue
</t) and esophagus (C) and 20-month-old transgenie mouse tongue (H) and esophagus (O). Note
the brown edk4 nuclear staining in fì
and D (also
indicated by arrows).
Fig. 8. HGFR immunohistochemical
staining
(X400) in 20-month-old wild-type mouse tongue
(A) and esophagus <C")and 20-month-old transgenie mouse tongue (B) and esophagus (D). Note
the cytoplasmic and membranous EGFR staining in
the basal and intermediate layers of the transgenic
mice tongue and esophageal epithelia in B and D
(also indicated by arrows).
a
r
uniformly grade 2+ in the 9-month old mice and grade 3+ in the
proliferation observed in the dysplastic tongue epithelium might be
20-month-old
mice (Fig. 8). Thus, EGFR overexpression
was
responsible for the increased p53 staining, which may represent de
associated with dysplasia induced by the targeting of cyclin Dl to
novo p53 mutations.
the tongue and esophageal epithelia.
Regardless of the age of the wild-type mice. p53 staining was
DISCUSSION
not detected in the wild-type mouse tongue and esophageal layers,
consistent with the notion that p53 mutations did not arise in these
Cyclin Dl associates with cdks (cdk4 or cdk6) and PCNA in the G,
mice. This was also the case for the 1- and 9-month-old cyclin Dl
phase of the cell cycle. The cyclin Dl/cdk4 or cdko complex phostransgenic mice. In contrast, all 20-month-old cyclin DI transgenic
phorylates the retinoblastoma tumor suppressor gene product (2). The
mice showed rare p53 staining (5-10% of cells) in the dysplastic
hyperphosphorylation of pRb leads to its dissociation from key transcriptionul factors, such as E2F, which allows for these factors to
tongue epithelium but not in the dysplastic esophageal epithelium
activate growth-promoting genes. It is within the realm of possibility
(data not shown). It is possible that the greater degree of cell
5546
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TRANSÕU NIC MOrSi.
M()I)I-;l. WITH CYO.IN
1)1 OVI HI M'KI SSK IN
100
90
Fig. 7. Quantitative scoring of cdk4 mimmi. >
histochemical staining in the tongue and esoph
agus. Wt. wild-type mice; I-, <)-. and 20-monthold groups are transgenie miee. + +, agematched wild-type mice compared to all
transgenic mice groups: P < 0.05 for tongue and
esophagus. *. I-month-old transgenie mice com
pared to 9-month-old
transgenic mice and
I-month-old transgenic mice compared to 20month-old transgenic mice: P < 0.05 tor tongue
and esophagus. **, 9-monlh old Iransgenic mice
compared to 20-month old transgenic mice:
P < 0.05 for tongue and esophagus. ***, com
parisons as noted above.
Wild Type
1 month
9 months
20 months
GROUPS
that other substrates are targets for the cyclin Dl/cdk complex, as has
been postulated recently with the myb-like transcriptional factor (3).
Cyclin D l overexpression has been associated with a number of
cancers, especially those of squamous epithelial origin from the head/
neck and esophagus. In an attempt to understand the molecular mech
anisms that underlie the contribution of cyclin DI to malignant
transformation in this tissue and cell type, we have recently developed
a transgenic mouse model where cyclin Dl is targeted to these
epithelia using an EBV promoter (23).
A number of environmental and genetic factors are common to
head/neck and esophageal squamous cell cancers. Among the fre
quently associated genetic factors are oncogene activation, (cyclin D l
and EGFR) and tumor suppressor gene inactivation, (p53 and pl6
mutations) (41-45). Thus, we have used the cyclin Dl model to
tions correlate with cyclin Dl amplification in head and neck squa
mous cell carcinomas (49).
The link between EGFR and cyclin Dl in our transgenic mouse
model may be direct or indirect. It is possible that the EGFR promoter
is a potential target of cyclin Dl. resulting in increased EGFR tran
scription. Conceivably, cyclin Dl may activate other transcriptional
factors that modulate EGFR transcription. Alternatively, increased
EGFR expression may be the consequence of increased cell prolifer
ation. Nonetheless, the finding of increased EGFR expression in
dysplasia is important and implicates it as an early event in head and
neck as well as esophageal carcinogenesis.
p53 is a well-described tumor suppressor gene, the encoded
protein of which has several effects related to cell cycle regulation,
DNA replication and repair, transcriptional regulation of genes,
and apoptosis. p53 serves as a cell cycle checkpoint at the G,/S
transition, in part through the transcriptional activation of p21, a
general cdk inhibitor (50). The dysplastic phenotype in the cyclin
DI transgenic mice tongue epithelia was uniformly associated with
rare p53 staining, consistent with the notion, although not defini
tive, of p53 mutation and stabilization of the p53 protein. This
finding was restricted, interestingly, to the tongue epithelium of the
20-month-old transgenic mice, perhaps reflecting the increased
expression of cyclin Dl, cdk4, and PCNA in this particular age
group's tongue tissues. A possible, albeit speculative, link among
investigate whether other genetic alterations, either directly or indi
rectly as a consequence of cyclin D l overexpression. can be detected
in the dysplastic phenotype by using immunohistochemical staining
approaches.
One of the well-established aspects of cyclin Dl overexpression
in cultured cells is a shortened G, phase, resulting in a more rapid
entry into S phase and increased cell proliferation. We found that
cyclin Dl overexpression resulted in increased cell proliferation in
the tongue and esophageal epithelia of 9- and 20-month-old trans
genic mice, as inferred from increased PCNA staining in compar
ison to age-matched wild-type mice and the young (1-month)
these latter changes is that mutant p53 has been shown to activate
the PCNA (51) and EGFR (52) promoters. Thus, although cyclin
transgenic mice. This may potentially be a consequence of the
increased cdk4 and cyclin Dl overexpression observed in the same
Dl overexpression may be an initiating event, mutant p53 may
further augment cell proliferation, at least in the tongue, through
age groups of transgenic mice.
the ongoing activation of key genes, especially EGFR. However,
As noted, the cyclin Dl and cdk4 complex phosphorylates key
this is not an entirely complete explanation because EGFR overcellular targets. It is likely in this setting of increased cell proliferation
that other genetic alterations are acquired. Key abnormalities that have
expression preceded the p53 staining temporally and was also
evident in the esophagus. Nonetheless, this may be a contributing
been associated in head and neck as well as esophageal squamous cell
factor in the dysplastic tongue squamous epithelium.
carcinomas are EGFR overexpression and p53 mutations. EGFR
In aggregate, our cyclin Dl transgenic mouse model of oral-esophoverexpression and p53 mutations have also been observed in head
ageal dysplasia permits the unique development of a multistage model
and neck squamous epithelial dysplasia (46, 47). Interestingly enough,
of carcinogenesis (49, 53-55) in which cyclin Dl overexpression
p53 mutations have been found to correlate with EGFR overexpres
results in a dysplastic phenotype associated with EGFR and p53
sion in human esophageal squamous carcinomas (48). and p53 muta
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TRANSGENIC
MOUSE MODEL WITH CYCLIN
genetic alterations. Although not tested yet. other genetic changes
may be associated with dysplasia in this transgenic mouse model, such
as in other tumor suppressor genes, pl6 and pRb, or other oncogenes
and key enzymes (56) implicated in oral-esophageal carcinogenesis.
Mechanistically, pi6 mutation, deletion, or hypermethylation is likely
redundant with cyclin Dl overexpression in the cyclin Dl pathway.
Furthermore, actual pRb inactivation is infrequent in human head and
neck cancers (57) and has not been formally investigated in esophageal cancers, although loss of heterozygosity of the locus has been
implicated (58). The association of key genetic alterations with the
oral-esophageal dysplastic phenotype in the cyclin Dl transgenic
mouse model also lays the foundation for the application of diagnostic
strategies in the early premalignant lesions and the use of tissue-
21. Lovec. H.. Sewing. A.. Lucibello. F. C.. Muller. R., and Moroy. T. Oncogenic activity
of cyclin Dl revealed through cooperation with Ha-ras: link between cell cycle
control and malignant transformation. Oncogene. 9: 323-326. 1994.
Wang. T. C.. Cardiff. R. D., Zukerberg. L.. Lees, E., Arnold. A., and Schmidt, E. V.
Mammary hyperplasia and carcinoma in MMTV-cyclin Dl transgenic mice. Nature
(Lond.). 369: 669-671, 1994.
23. Nakagawa, H.. Wang. T. C.. Odze. R., Zukerberg, L., Togawa. K.. May. G. H. W.,
Wilson, J.. and Rustgi, A. K. The targeting of the cyclin DI oncogene by an
Epstein-Barr virus promoter in transgenic mice causes dysplasia in the tongue,
esophagus and forestomach. Oncogene. 14: 1185-1190. 1997.
Nakagawa. H.. Inomoto. T.. and Rustgi. A. K. A CACCC box like r/.ï-regulatory
element of the Epstein-Barr virus ED-L2 promoter interacts with a nuclear transcriptional factor in tissue-specific squamous epithelia. J. Biol. Chem.. 272: 16688-16699.
1997.
25. Taleishi. M.. Ishida, T.. Mitsudomi. T., Kaneko, S.. and Sugimachi. K. Immunohis26.
specific therapeutic approaches.
27.
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5549
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A Transgenic Mouse Model with Cyclin D1 Overexpression
Results in Cell Cycle, Epidermal Growth Factor Receptor, and
p53 Abnormalities
Annegret Mueller, Robert Odze, Timothy D. Jenkins, et al.
Cancer Res 1997;57:5542-5549.
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