Download Multiple genetic loci modify risk for retinoblastoma in

Multiple Genetic Loci Modify Risk for Retinoblastoma in
Transgenic Mice
Anne E. Griep,1 Jeff Krawcek,2 Denis Lee,2 Amy Liem,2 DanielM. Albert? Rey Carabeo2
Norman Drinkwater,2 Maureen McCall,4'5 Carol Sattler,2 Jacques G. H. Lasudry,5 and
Paul F. Lambert2
PURPOSE. Forty percent
of cases of retinoblastoma, a childhood malignancy of the retina, are linked
to the inheritance of a mutant allele of the retinoblastoma susceptibility gene Rbl. Tumor
penetrance varies among earners in different family pedigrees, indicating that other genetic factors
may modify risk for occurrence of retinoblastoma. This study was undertaken to determine
whether multiple genetic loci modify the risk for retinoblastoma in mice.
A line of aAcry-HPVl6E6/E7 transgenic mice expressing the human papillomavirus type
16 E6 and E7 oncogenes (HPV-16 E6 and ET) ectopically in the retina was characterized. E6 and
E7 proteins bind to and inactivate the cellular tumor suppressor proteins p53 and Rb, respectively.
METHODS.
Retinoblastomas developed rarely when the aAcry-HPVl6E6/E7 transgene was maintained on the FVB background, but tumors arose with high frequency on C57BL/6 X FVB and
C3H X FVB F, hybrid backgrounds. The incidence of retinoblastoma in the LHfi-TAG transgenic
mice, which express simian virus 40 large tumor antigen (SV40 T-ag), was also influenced by the
FVB and C57BL/6 backgrounds. Resistance of the aAcry-HPVl 6E6/E7 FVB mice to retinoblastoma
mapped in part to the retinal degeneration (rd) locus. However, multiple genetic experiments
indicate that resistance to retinoblastoma depends on additional loci in FVB mice.
RESULTS.
Multiple cellular genes can modify risk for retinoblastoma in mice. (Invest Ophthalmol Vis Sci. 1998;39:2723-2732)
CONCLUSIONS.
R
From the 'Department of Anatomy, the 2McArdle Laboratory for
Cancer Research and the Department of Oncology, the 'Department of
Ophthalmology and Visual Sciences, and the "Waisman Center, University of Wisconsin Medical School, Madison.
5
Present address: Departments of Psychology and Ophthalmology
and Visual Sciences, University of Louisville, 301 East Mohammed Ali
Boulevard, Louisville, KY 40202.
Supported in part by Grants EY09091, EY01917, EY01003,
CA22443, and CA07175 from the National Institutes of Health, Bethesda, Maryland; and Grants JFRA-393 and RPG-96-043-03-MGO from
the American Cancer Society, Atlanta, Georgia.
Submitted for publication February 26, 1998; revised June 12,
1998; accepted July 6, 1998.
Proprietary interest category: N.
Reprint requests: Anne E. Griep, Department of Anatomy, University of Wisconsin Medical School, 1300 University Avenue, Madison,
WI 53706.
generally estimated to be 80% to 95%, but in some pedigrees it
is as low as 20%.6'7 The absence of complete penetrance may
relate to differences in the severity of the defect in mutant Rbl
alleles.8 Alternatively, it may indicate that other cellular genes
can affect the risk in a person who carries a mutant Rbl allele
for development of retinoblastoma. Through the study of inbred strains of mice, genetic loci that affect the sensitivity or
resistance of an animal (that is, those that modify risk) to
specific cancers have been identified. For example, the mouse
mom locus modifies risk to intestinal tumors in mice that
inherit a defect in the mouse homologue to the human adenomatous polyposis coli gene,9 and multiple mouse loci modify
risk to liver tumors (for review, see Ref. 10). In this study, we
provide experimental evidence that multiple genes modify risk
to retinoblastoma in the mouse.
Genes in addition to Rbl are likely to contribute to the
development of retinoblastoma in mice. Mice carrying a germline mutation in one Rbl allele are not predisposed to retinoblastoma '' as are humans; nor are mouse chimeras that harbor
Rbl~'~ cells in their retinas predisposed to retinoblastoma. 1213 Yet transgenic mice expressing simian virus 40
(SV40) large-tumor antigen (T-ag) gene in the retina show
development of retinoblastoma.14"16 SV40 T-ag is a potent
oncoprotein that binds to and inactivates the Rb protein.17 In
addition to these direct effects on Rb, SV40 T-ag possesses
other activities that contribute to its transforming potential in
tissue culture18 and tumorigenic phenotype in vivo.l9'20 These
activities include its abilities to bind and inactivate Rb-like
proteins plO7 and pl30, 21 ' 22 to modulate function of the p300
protein,23 and to inactivate the tumor-suppressor protein
Investigative Ophthalmology & Visual Science, December 1998, Vol. 39, No. 13
Copyright © Association for Research in Vision and Ophthalmology
2723
etinoblastoma is a malignancy of the retina that occurs
in young children and is associated with the loss of
function of the retinoblastoma susceptibility gene
Rbl.1'3 Children who inherit one mutant allele of Rbl are
predisposed to bilateral, multifocal retinoblastoma and secondary tumors in numerous nonocular tissues. In these tumors,
loss of function of the remaining wild-type allele is found at the
Rbl locus. Nonhereditary, or sporadic, retinoblastoma is associated with somatic mutations in both alleles of Rbl and is
usually focal. As such, Rbl is representative of tumor-suppressor genes, the disruption of which is associated with cancer
development.4 Loss of function of both alleles of Rbl is the
cornerstone of the two-hit hypothesis of Knudsen5 for retinoblastoma development in humans. However, the penetrance of
retinoblastoma among human pedigrees is incomplete; it is
Downloaded From: http://iovs.arvojournals.org/ on 08/11/2017
2724
Griep et al.
p53 2 4 Of these activities, the inactivation of plO7 by T-ag may
contribute to the induction of retinoblastoma. Mice that are
plO7~/~;Rbl+/~ show retinal dysplasia, a potential precursor
to retinoblastoma.25 In additional, inactivation of p53 may
contribute to retinoblastoma formation in mice, because expression in the retina of the E7 oncogene from human papillomavirus (HPV) type 16 causes retinoblastoma only on a
p53~/~ background.26
Human papillomavirus E6 and E7 oncoproteins belong to
the same class of viral oncoproteins as does SV40 T-ag. E6 and
E7 are expressed in most HPV-associated cervical cancers27"29
and together are necessary and sufficient to transform cells in
tissue culture.30 Similar to T-ag, E6 inactivates p53, although
through a different mechanism, by targeting p53 for degradation by a ubiquitin-mediated proteolytic pathway.31'32 Similar
to T-ag, E7 binds to and sequesters Rb and Rb-like proteins33'34
leading to the deregulation of the E2F transcription factor
family. We have shown that expression of HPV-16 E6 and E7
oncoproteins in the ocular lens could induce proliferation and
inhibit differentiation in the lens in three independent lines of
transgenic mice.35 These lines of transgenic mice, referred to
as aAcry-HPVl6E6/E7 mice, were generated and maintained
on the FVB inbred mouse genetic background, which is homozygous for a recessive mutation at the retinal degeneration
(ret) locus. In the line of aAcry-HPVl6E6/E7 mice expressing
the highest levels of E6/E7 message, line 19, lens tumors
developed in approximately 40% of mice by 1 year of age.
Transgene expression in this line of mice was found in multiple
ectopic sites including the skin and extralenticular portions of
the eye. Expression of E6 and E7 in the skin correlated with the
high incidence of skin tumors.36 However, expression in other
tissues such as the extralenticular portions of the eye did not
correlate with tumor formation.
In this study we report that, when crossed with other
mouse genetic backgrounds such as C57BL/6 and C3H, the line
19 aAcry-HPVl6E6/E7 transgene confers high susceptibility to
retinoblastoma formation. Whereas C57BL/6 is wild type at the
ret locus, C3H is rd/rd. The retinoblastoma tumors expressed
the transgene, appeared to arise from the inner nuclear layer of
the retina, and sometimes developed into exophytic tumors
that invaded the brain through the optic nerve and metastasized to the cervical lymph nodes. Crosses of line 19 aAcryHPVl 6E6/E7 transgenic mice with transgenic mice of the
C57BL/6 genetic background in which photoreceptors have
been ablated (rdta [rhodopsin promoter driven diphtheria
toxin] transgenic mice37) also showed development of retinoblastoma. These results suggest that the FVB genetic background in which the transgene originally resides confers protection against retinoblastoma formation associated with
expression of E6 and E7. Backcross experiments have shown
that the FVB background can also confer resistance to retinoblastoma formation associated with expression of SV40 T-ag.
Taken together, these studies indicate that the protective effect
of the FVB genetic background is associated not only with the
rd/rd allele but also with additional loci.
METHODS
Mouse Strains
The generation, screening, and characterization of all transgenic mouse lines have been described. Line 19 aAcry-
Downloaded From: http://iovs.arvojournals.org/ on 08/11/2017
IOVS, December 1998, Vol. 39, No. 13
HPVl 6E6/E7 transgenic mice were generated and maintained
on the FVB genetic background.35 In these transgenic mice,
expression of the HPV-16 E6 and E7 genes is directed to the
lens using the aA crystallin promoter.35'38 The LH&-TAG transgenic mice express SV40 T-ag ectopically in the retina from the
luteinizing hormone promoter.15 These transgenic mice were
originally generated on the C57BL/6 X BALB/c hybrid background and subsequently maintained on the C57BL/6 inbred
background. The rdta transgenic mice express an attenuated
form of the diphtheria toxin A gene in rod photoreceptors by
virtue of the cell type specificity of the rhodopsin promoter
that drives its expression.37 These transgenic mice were generated originally on the FVB background, and subsequently,
the transgene was moved onto the C57BL/6 background
through backcrossing F, hybrids. All stock C3H/HeJ and
C57BL/6J mice were purchased from Jackson Laboratory (Bar
Harbor, ME), and FVB mice were purchased from Taconic
Farms (Tarrytown, NY). All experiments with animals were
conducted in accordance with the ARVO Statement for the Use
of Animals in Ophthalmic and Vision Research.
Genotyping
The aAcry-HPVl6E6/E7 transgenic mice were screened for
the presence of transgene by Southern blot or polymerase
chain reaction (PCR) analysis of DNA prepared from tail biopsy
specimens, as described previously.35 The LH(3-TAG transgenic
mice were screened for the presence of the transgene using
PCR analysis (Jolene J. Windle, personal communication). The
rdta transgenic mice were screened for the presence of the
transgene by PCR analysis as described.37 The rd status of F,
hybrid mice backcrossed with the FVB background was performed by PCR analysis. The murine-endogenous retrovirus
Xmv is integrated into the first intron of the rd allele of the /3-7
subunit of the phosphodiesterase gene, ((5-PDE^9). This retroviral integration event Xmv-28 is not present in the wild-type
allele of fi-PDE. The following oligonucleotides were generated to amplify the retrovirus-disrupted intron in the rd allele
or the intact intron of the wild-type allele of (i-PDE in ([FVB
line 19H X C57BL/6] X FVB) B, mice: rdl, 5'-ACCTGCATGTGAACCCAGTATTC-3'; rd2, 5'-GGGGAACCTGAAACTGAGGTGGG-3'; and rd3, 5'-CTCCTTTCTATTGCCCTGATCCACACC-3'. Oligo rdl is complementary to (i-PDE sequences in
intron 1 lying upstream of the Xmv-28 integration site, and rd2
is complementary to sequences within Xmv. The combination
of oligos rdl and rd2 selectively amplifies a region in the rd
allele, across the junction of die first intron and the integrated
retroviral element, generating a 470-bp fragment. Oligo rd3 is
complementary tofi-PDEsequences in intron 1 downstream of
rdl in the region disrupted by Xmv-28 in the rd allele. The
combination of rdl and rd3 selectively amplifies the intact first
intron of the wild-type allele resulting in a 370-bp fragment.
The PCR products were resolved on a 2% agarose gel. Animals
that were rd/rd at the locus would give rise only to the larger
of the two fragments, whereas animals that were rd/+ would
contain both fragment sizes in their products. The rdta and rd
status was verified phenotypically, when possible, by examining the retina microscopically for the presence or absence of
an outer nuclear layer.
IOVS, December 1998, Vol. 39, No. 13
Mapping aAcry-HPVl6E6/E7 Transgene
Integration Site
Genomic DNA samples were isolated by phenol-chloroform
extraction from skin tissues of 49 (FVB[line 19] X C57BL/6) X
C57BL/6 backcross animals. Each animal was genotyped for
simple sequence length polymorphism (SSLP) markers by PCR.
Briefly, 100 ng genomic DNA was used as a template in the PCR
reaction containing 0.13 mM forward and reverse SSLP primers
(Research Genetics, Birmingham, AL), 50 mM deoxyribonucleoside triphosphates, and Tag polymerase in IX PCR
buffer (10 mM Tris-HCl, 1.5 mM MgC12 and 50 mM KC1 [pH
8.3])- Amplification was performed in a GeneAmp PCR System
9600 (Perkin Elmer, Norwalk, CT) in an initial denaturing cycle
of 2 minutes at 94°C (1 cycle), 30 seconds at 94°C, 30 seconds
at 55°C, and 1 minute at 72°C (50 cycles), 7 minutes at 72°C (1
cycle), and a 4°C soak. PCR prodvicts were run on a 7%
polyacrylamide gel and visualized under UV illumination. Linkage analysis for the transgene was performed using the MAPMAKER program (Whitehead Institute Genome Center, Cambridge, MA).
Light Microscopy
Eyes were enucleated from mice, fixed in 10% buffered formalin overnight, dehydrated through a graded ethanol series, and
embedded in paraffin using an automatic tissue processor (Tissue-Tek, Miles Scientific, Naperville, IL). Sections (5 fxm) were
cut, mounted on glass slides, stained with hematoxylin and
eosin, protected with a coverslip, and examined. The intact
skull and brain were fixed overnight in 10% buffered formalin
and then decalcified in 5% nitric acid. Brain tissue was processed as described for ocular tissue and also examined by light
microscope.
Electron Microscopy
Eyes were fixed for 8 hours in Karnovsky's paraformaldehydeglutaraldehyde fixative40 prepared in cacodylate buffer immediately before use. Eyes were washed in 0.1 M cacodylate
buffer (pH 7.4) on ice for 1.5 hours and then washed in cold
ddH2O. After dehydration in a graded series of ethanol, the
eyes were infiltrated using a rotator with propylene oxide and
Eponate (Ted Pella, Redding, CA), beginning with a 2:1 ratio
and gradually increasing the proportion of Eponate over several hours until the eyes were in 100% Eponate. After overnight
infiltration with Eponate, each eye was embedded in freshly
made Eponate in individual flat-bottomed plastic vials. The
sections were cut on a microtome equipped with a diamond
knife (Ultracut E3; Reichert, Buffalo, NY), stained with uranyl
acetate and lead citrate, and examined by electron microscope
(H-7000; Hitachi, San Jose, CA) at 75 kV.
In Situ Hybridization
Eyes for in situ hybridization analysis were enucleated from
line 19 aAcry-HPVl6E6/E7 F, hybrid transgenic mice and
fixed in 4% paraformaldehyde overnight at 4°C, transferred to
phosphate-buffered saline (PBS), dehydrated through a graded
ethanol series, embedded in paraffin, and sectioned at 5-ju-m
thickness. For in situ hybridization, 5-ju,m paraffin sections
were deparaffinized in xylene, rehydrated through a graded
series of ethanol, and subjected to in situ hybridization, as
described previously,36'43 with the riboprobes described later.
Antisense and sense cRNA probes were synthesized in vitro
Downloaded From: http://iovs.arvojournals.org/ on 08/11/2017
Risk Factors for Retinoblastoma in Mice
2725
using a-35S-uridine triphosphate and T3, T7, or SP6 polymerase
and the following templates: For generation of the ah crystallin-specific probes, the clone pGEMaCrl containing a 900-bp
fragment of the mouse aA crystallin gene was used (obtained
from Kathleen Mahon, Baylor University, Houston, TX). For
generating the HPV-16 E6/E7-specific probes, the plasmids
pAB1.5 and pGEM/E6 containing the E7 and E6 open reading
frames of HPV-16, respectively, were used.36 Sections were
hybridized with either sense or antisense 35S-labeled riboprobes, washed to remove nonspecifically bound probe,
dipped in autoradiographic emulsion (NTB-2; Eastman Kodak,
Rochester, NY), and exposed in the dark at 4°C for 2 weeks.
After developing, dehydrating, and mounting, sections were
examined by dark-field microscopy. Three to four sections
from three tumors from three mice were examined.
RESULTS
Effect Of C57BL/6 Genetic Background on Tumor
Incidence in aAcry-HPVl6E6/E7 Mice
We have reported the generation and phenotypic characterization associated with expression of the HPV-16 E6 and E7
oncogenes in the mouse lens.35 In that study, three independent lines of aAcry-HPVl6E6/E7 mice, lines 4, 18, and 19,
were created on the FVB inbred genetic background. In these
mice, expression of the viral oncogenes was directed to the
lens by the murine aA crystallin promoter. Approximately 40%
of line 19 transgenic mice, which had the highest level of
transgene expression, showed development of lens tumors by
13 months of age. The transgenic mice on the FVB inbred
background carry a recessive mutation at the rd locus, which
in the homozygous condition leads to a complete loss of rod
photoreceptor cells during the first month of postnatal life.42
Because it was possible that the rc//rc/-induced defects in the
retinas of FVB/FVB mice could influence the fate of E6/E7expressing lens cells, homozygous aAcry-HPVl6E6/E7 mice
from line 1935 were crossed with the C57BL/6 inbred strain of
mice, which is homozygous for the wild-type rd allele, and the
F, transgenic progeny were characterized. No significant difference in the lens phenotypes was observed in perinatal and
young adult aAcry-HPVl 6E6/E7 FVB X C57BL/6 F, mice compared with that of their inbred FVB transgenic parents (data
not shown). However, the eye tumor incidence in line 19
aAcry-HPVl 6E6/E7 mice increased substantially in these F,
animals (Fig. 1). Less than 10% of line 19 mice on the inbred
FVB background had tumors by 10 months. In comparison, by
10 months of age 90% of the F, line 19 aAcry-HPVl6E6/E7
mice showed tumor development. These tumors began to arise
in mice as early as 3 months of age. The high incidence of eye
tumor formation seen in the F, mice was retained when the
line 19 aAcry-HPVl6E6/E7 transgene was backcrossed onto
the C57BL/6 background to generation N10 (data not shown).
A significant proportion of the line 19 aAcry-HPVl 6E6/E7 F,
mice had tumors develop in both eyes. Thus, genetic background influences the incidence of tumor formation in aAcryHPVl6E6/E7 mice.
Development of Retinoblastomas Rather than
Lens Tumors in Line 19 aAcry-HPVl6E6/E7 Fx
Transgenic Mice
To determine the cellular origins of the eye tumors in the line
19 aAcry-HPVl6E6/E7 FVB X C57BL/6 F, mice, microscopic
2726
Griep et al.
IOVS, December 1998, Vol. 39, No. 13
100-
line 19 x C57B1/6 F,
90"
SO
3
H
(n=58)
80"
7060
5040"
3020-
4
6
8 10 12 14 16 18 20 22 24
Mouse Age at Tumor Detection
(months)
FIGURE 1. Eye tumor incidence as a function of age in aAcryHPV16E6/E7 transgenic mice on FVB ( • ) inbred versus with
C57BL/6 X FVB F, hybrid genetic backgrounds ( • ) . Line 19
aAcry-HPVl6E6/E7 FVB transgenic mice were crossed with
C57BL/6 nontransgenic mice. Transgenic mice maintained on
the FVB inbred background and Ft progeny were monitored
for the presence of overt eye tumors at the indicated ages and
killed when tumors were observed. Mice were genotyped by
polymerase chain reaction analysis (see the Materials and Methods section). Indicated are the percentages of mice showing
eye tumors at various ages up to 13 months. The number of
mice in each group is indicated in parentheses.
analyses were performed. Examples of the histology seen in
intermediate and advanced line 19 aAcry-HPVl6E6/E7 Fj eye
tumors are shown in Figures 2A and 2B, respectively. These
histologic features are representative of what was observed in
most of the eye tumors in the line 19 F, aAcry-HPVl6E6/E7
mice. The tumor in Figure 2A seemed to arise in the retina,
rather than the lens. A small cataractous lens located within the
anterior portion of the eye remained intact, that is, the lens
capsule was not broken. The lens is similarly intact in the
advanced tumor in Figure 2B. These features suggest that the
retina rather than the lens was the source of the tumors.
Homer-Wright rosettes, a hallmark of retinoblastoma, were
evident in the tumor masses by light and electron microscopic
analyses (Figs. 2A, 2B, 3Q. Ultrastructural analyses showed
that these tumors also possessed basal bodies and trilaminar
nuclear membrane (Figs. 3A, 3B, respectively), both characteristics of retinoblastoma in humans. Further histologic analyses
indicated that a minority of the retinoblastomas were exophytic, invading the optic nerve (Fig. 2C). Of these, a significant number established metastases within the brain and cervical lymph nodes (Fig. 2C). These metastases also displayed
histopathologic characteristics of retinoblastoma including
Homer-Wright rosettes (Fig. 2D). The tumors that developed
in line 19 aAcry-HPVl6E6/E7 F, transgenic mice resemble
those observed previously in a line of SV40 TAG transgenic
mice, LHfi-TAG, that expressed SV40 T-ag ectopically in the
retina15 and in IRBP-TAG transgenic mice expressing SV40
T-ag specifically in photoreceptors.16 In each of these trans-
Downloaded From: http://iovs.arvojournals.org/ on 08/11/2017
genie mouse models the retinal tumor bears significant resemblance to human retinoblastoma.
To assess further the cellular origin of the tumors in line
19 aAcry-HPVl 6E6/E7 F, transgenic mice, eyes with earlystage tumors were examined microscopically. In multiple examined eyes, dysplastic and hyperplastic regions were evident
in the retina, whereas the cataractous lens remained intact
(Figs. 4A, 4B, 4C). Early lesions arose within the inner nuclear
layer of the retina (Fig. 4B), similar to the apparent origin of
tumors in the LHfi-TAG transgenic mice15'43 Crystallin-specific
immunohistochemical and in situ hybridization analyses indicated that these lesions failed to express lens-specific crystallins (data not shown). This further suggests that the tumors
arising in line 19 aAcry-HPVl6E6/E7 F, mice are not lenticular
in origin. Characterization of more than 150 tumors arising in
line 19 aAcry-HPVl6E6/E7 F, mice indicate that more than
90% of the eye tumors were retinal in origin. Thesefindingsled
us to examine additional eye tumors from line 19 aAcryHPV16E6/E7 FVB mice. From more than 100 tumors examined, less than 1% of the tumors arising in the inbred FVB
background were retinoblastomas. Thus, the genetic makeup
of the mouse influences not only the frequency of tumor
development but also the type of eye tumor in the line 19
aAcry-HPVl6E6/E7 mice.
Heightened Expression of the E6 and E7 Genes in
the Retinoblastomas in Line 19 otAcry-HPVl 6E6/
El Fx Mice
Previously, we reported that mRNA specific for the HPV-16 E6
and E7 genes was detected in extralenticular portions of the
eye of line 19 aAcry-HPVl6E6/E7 mice35 using the sensitive
method of reverse transcription (RT)-PCR analysis. We performed in situ hybridization to assess whether viral transgenes
were expressed in the retina and in the retinal tumors. Highlevel expression of the viral transgenes was detected not only
in the lens but also within the retinal tumor mass of this and
other aAcry-HPVl6E6/E7 F, mouse eyes (Fig. 4D). Lower-level
expression of the transgene was noted within the microscopic,
dysplastic lesions arising within the inner nuclear layer of the
retina. Expression in the unaffected regions of the retina was at
or below the limit of detection by in situ hybridization, as was
the case in the retina of aAcry-HPVl6E6/E7 FVB mice (data
not shown). This finding suggests that the basal level of transgene expression was not influenced by the mouse's genetic
background. Thus, the level of expression of the viral transgenes seems to be augmented specifically in the retinal tumors
of line 19 aAcry-HPVl 6E6/E7 F, mice. Similar increased levels
of E6/E7 expression have been noted in squamous cell carcinomas arising in line 19 transgenic mice.36
Chromosomal Site of Integration of the aAcryHPVl6E6/E7 Transgene in Line 19 Mice
Retinoblastoma in humans is associated with mutations at the
Rbl locus. We therefore determined whether the high incidence of retinoblastoma in line 19 Ft aAcry-HPVl 6E6/E7 mice
was a consequence of the transgene insertion into and disruption of the Rbl locus in the FVB genome. An insertion of this
kind would lead to an Rbl+/~ genotype in the FVB X C57BL/6
F, animals. The chromosomal location of the transgene was
mapped in the (FVB X C57BL/6) X C57BL/6 backcross (B,)
progeny using SSLP analysis.44 Briefly, linkage of 17 SSLP mark-
IOVS, December 1998, Vol. 39, No. 13
Risk Factors for Retinoblastoma in Mice
2727
\
FIGURE 2. Eye tumor histology in aAcry-HPVl6E6/E7 line 19 C57BL/6 X FVB F, hybrid mice. (A, B) Hematoxylin and
eosin-stained 5-/m.m paraffin sections from representative intermediate (A) and advanced (B) eye tumors in the line 19 F, hybrid
mice. Note the small cataractoiis lens characteristic of line 19 mice and large retinoblastoma with characteristic rosettes. (C) Low
magnification of head and neck from a line 19 F, hybrid mouse shows the large retinoblastoma in the eye (E), the invasion of the
tumor into the brain (arrow), and the metastasis to the cervical lymph node (LN)- (D) High magnification of a brain tumor from
another line 19 FT hybrid mouse shows the characteristic rosette appearance {arrow). L, lens.
ers to the transgene was analyzed using the Fisher's exact f-test.
A marker on chromosome 12, D12MU34, yielded the best
linkage significance, with P = 4.1 X 10~13 (Table 1). SSLP
markers that flank the tumor suppressor genes, Rbl (Dl4Mit7,
P = 0.07) and Trp53, (D11MU19, P = 1.0; D11MU14, P= 1.0)
did not show linkage to the transgene. Based on our genotype
data from the backcross, the transgene was positioned 2.1
centimorgans (cM) proximal to D12MU34, using the MAPMAKER program (data not shown). The location of line 19
transgene on chromosome 12 discounts the possibility diat its
position causes the disruption of the Rbl or Trp53 loci, which
are located on chromosomes 14 and 11, respectively. Thus, a
direct effect of the transgene's insertion does not account for
the high incidence of retinoblastoma in line 19 F, aAcryHPVJ6E6/E7 mice.
Resistance to Retinoblastoma in an SV40 T
Antigen Transgenic Mouse line on the FVB
Background
Alternatively, the high incidence of retinoblastoma in line 19 F,
aAcry-HPVl 6E6/E7 transgenic mice may reflect an influence
of genetic background on transgene expression, a consequence of the location of the transgene integration in the
mouse genome. To investigate this possibility, studies were
performed on another line of transgenic mice in which retinoblastomas develop15. This particular strain of mice carries an
LHji-TAG transgene whose integration site is on chromosome
4. In these mice SV40 large-tumor antigen (SV40 TAG), which
Downloaded From: http://iovs.arvojournals.org/ on 08/11/2017
is under the transcriptional control of the luteinizing hormone
(LH£) promoter, also is expressed ectopically in the retina.
These transgenic mice were originally generated on a C57BI7
6 X BALB/c hybrid background15 and subsequently maintained
on the C57BL/6 background in our laboratories. To examine
the influence of genetic background on SV40 T-ag-induced
retinoblastoma, we crossed the C57BL76 mice harboring the
LHfi-TAG transgene with FVB mice, and the F, hybrids were
then backcrossed for two generations onto the FVB background. Tumor incidence in the LH(5-TAG transgenic B2 generation decreased to approximately 75% of that seen on the
inbred C57BL/6 background (Fig. 5). That the FVB genetic
background seems to confer resistance to retinoblastoma induced by a second viral oncoprotein, SV40 T-ag, indicates that
the genetic influence does not result from specific effects on
the HPV transgene in line 19 aAcry-HPVl6E6/E7 mice or from
the site of transgene integration.
Involvement of the rd Locus in Retinoblastoma
Formation
The C57BL/6 and FVB inbred strains are highly polymorphic.
Therefore, the difference in susceptibility to retinoblastoma
between these mouse strains may be influenced by many
genes. We suspected diat one allele in particular may influence
retinoblastoma incidence-that is, the rd allele that is present
on the FVB genetic background but not on the C57BL/6 genetic background. The rd allele present in FVB 42 is a recessive
mutant allele of the gene that encodes the /3 subunit of cGMP
2728
Grlep et al.
IOVS, December 1998, Vol. 39, No. 13
FIGURE 3.
Ultrastructural analysis of retinoblastoma in line 19 aAcry-HPVl6E6/E7 C57BI/6 X
FVB F, hybrid mice. Electron micrographs show characteristic ultrastmctural features of
retinoblastoma in an eye tumor from a line 19 F, hybrid mouse. (A) Basal body located in
center of field; (B) trilaminar nuclear membrane on nucleus in upper left; and (C) HomerWright rosette. Magnification: (A) X 19,500; (B) X31,200; (C) X3,200.
phosphodiesterase gene 0-PDE?9*). In rd/rd mice (such as
inbred FVB), the rod photoreceptors in the outer nuclear layer
of the retina degenerate during the first month of life, and
subsequently, the cone photoreceptors degenerate as well. We
reasoned that the absence of the photoreceptor cells in the
retina may directly or indirectly influence the incidence of
retinoblastomas. The degeneration of the photoreceptor cells
Downloaded From: http://iovs.arvojournals.org/ on 08/11/2017
observed in rd/rd mice can be experimentally recapitulated by
directing expression of a diphtheria toxin transgene to the rod
photoreceptor cells using the rhodopsin promoter.37 We
therefore generated FVB X C57BL/6 F, animals that were
cotransgenic for line 19 aAcry-HPVl6E6/E7 and rdta transgenes. In these mice, the percentage of eyes in which retinoblastomas developed was less than half that seen in F, litter-
Risk Factors for Retinoblastoma in Mice
IOVS, December 1998, Vol. 39, No. 13
2729
FIGURE 4.
Microscopic and in situ hybridization analyses of early retinoblastomas in line 19
aAcry-HPVJ 6E6/E7 C57BL/6 X FVB F, hybrid mice. (A, B, C) Hematoxylin arid eosin-stained
paraffin section from a representative early retinoblastoma in a line 19 aAcry-HPVJ6E6/E7 F,
hybrid mouse. (A, C) Low and high magnification shows tumor in peripheral retina and (B)
high magnification of central retina. Note the clysplastic foci (arrows) in the inner nuclear layer
in (B). (D) In situ hybridization analysis for E6/E7 transgene expression. A sequential slide was
hybridized to an 35S-labeled antisense E6 and E7 probe. The peripheral retina, corresponding
to (C), is shown. Hybridization was also observed in the central retina corresponding to the
region shown in (B). No hybridization was observed in the retina when sense E6/E7 or
antisense ctA crystallin probes were used (data not shown). INL, inner nuclear layer; ONL,
outer nuclear layer; L, lens; R, retina.
mates transgenic only for the line 19 aAcry-HPVJ6E6/E7
transgene (Fig. 6). This result indicates that the degeneration of
photoreceptor cells in line 19 aAcry-HPVl6E6/E7 FVB mice
that resulted from the rcl/rd genotype probably contributed in
part to the low incidence of retinoblastomas.
tomas by 10 months). Thus, there must be recessive alleles in
the FVB mice that, in addition to the rd allele, confer resistance
to retinoblastoma in the FVB background. To test this hypothesis, we scored the incidence of retinoblastomas in F, hybrid
mice generated by crossing the ctAcry-HPVl6E6/E7 mice (inbred FVB background) to the C3H inbred mouse strain (Fig. 7).
Involvement of FVB Alleles in Addition to rd in
C3H and FVB inbred strains are also highly polymorphic, but
Resistance to Retinoblastoma in Line 19 otAcryboth strains carry the rd/rd allele. The F, animals, all of which
HPV16E6/E7 Transgenic Mice
retain the rd/rd genotype, displayed a moderately high inciRetinoblastomas developed in line 19 aAcry-HPVJ'6E6/E7, dence of retinoblastoma (30%). This result supports the
premise that FVB mice possess recessive alleles in addition to
rdta cotransgenic FVB X C57BL/6 F, animals (Fig. 6) at a
rd that confer resistance to retinoblastoma.
frequency (35% had at least one retinoblastoma by 7.5 months
or age) far above that seen in FVB inbred mice carrying only
the line 19 aAcry-HPVl6E6/E7 transgene (<1% had retinoblasDISCUSSION
1. Significance of Linkage to the Line 19 aAcryHPV16E6/E7 Transgene of Different Mouse Markers
on Chromosome 12
TABLE
Markers
Position (cM)
Linkage Significance (P)
17.0
19.0
29.0
38.0
2.3 X 10~6
1.3 X 10~ri
4.1 X 10" 13
1.4 x 10" 9
DJ2MU46
DJ2MU2
DJ2MU34
DJ2MU5
cM, centimorgan.
Downloaded From: http://iovs.arvojournals.org/ on 08/11/2017
In this study we report that retinoblastomas arose in mice
transgenic for the HPV type 16 E6 and E7 oncogenes and that
the animal's genetic background strongly influenced the penetrance of this tumorigenic phenotype. Our data suggest that
recessive alleles at multiple loci in the FVB inbred mouse
contributed to the resistance of these mice to development of
retinoblastomas and that these loci also influenced the induction of retinoblastoma in mice transgenic for SV40 T-ag.
Role of Viral Oncogenes in Retinoblastoma
The induction of retinoblastoma in the aAcry-HPVJ6E6/E7
mice specifically occurred in the line that ectopically ex-
2730
Griep et al.
IOVS, December 1998, Vol. 39, No. 13
100"
LHp-TAG C57B1/6 Mice
(n=30)
90"
inactivation of the Trp53 gene.26 E7 induces apoptosis through
p53-dependent and p53-independent pathways. Of interest, E6
can inhibit both.41
80-
o
LHP-TAG B 2 Mice
70-
(n=18)
60OeS 5 0 40302010"
1
2
I
3
I
4
I
5
I
6
I
7
I
8
Mouse Age at Retinoblastoma
Detection(months)
FIGURE 5. Retinoblastoma incidence in LHfi-TAG C57BL/6 inbred (A) or FVB B2 transgenic (A) mice. LH&-TAG transgenic
mice maintained on the C57BL/6 background were crossed to
FVB mice, and the F, hybrids then were backcrossed to FVB
mice for two generations. Mice were genotyped by polymerase
chain reaction analysis (see the Materials and Methods section).
Transgenic mice on the C57BL/6 and B2 backgrounds were
monitored for the presence of overt eye tumors at the indicated ages and killed when tumors were observed. Tumors
were confirmed to be retinoblastomas by microscopic analysis.
Indicated are the percentages of mice in each group showing
retinoblastomas at ages up to 5 months for LHfi-TAG C57BL/6
mice and 7 months for LH(5-TAG B2 mice. The number of mice
in each group is indicated in parentheses.
Differential Sensitivity of Site-Specific Tumor
Induction in Inbred Strains of Mice
By crossing line 19 aAcry-HPVl6E6/E7 FVB transgenic mice
with C57BL/6, we observed that the ocular phenotype in this
transgenic line can be modified by genetic background, indicating FVB genotype confers a resistance to tumor formation in
the retina. However, FVB mice are not highly resistant to tumor
formation in all tissues. For example, FVB mice are highly
sensitive to squamous cell carcinoma associated with expression of HPV-16 E6 and E7 together,36 HPV-16 E7 alone,51, or
resulting from treatment with chemical carcinogens.52 Conversely, C57BL/6 mice are relatively resistant to skin tumor
induction.53 Such differential susceptibility of tissues in a given
inbred mouse strain to tumor induction is commonly observed.53 This property of inbred mouse strains makes them
a
O
3
o
a
&
pressed its transgenes in the retina (Fig. 2), implying that
expression of E6 and E7 in the retinal tissue is necessary for
tumor development. In HPV-positive cervical cancers, the Rbl
and p53 genes have been found primarily to be intact, whereas
they are mutant in the HPV-negative cervical cancers.45'46 This
finding supports the premise that the functional inactivation of
p53 and Rb proteins by E6 and E7, respectively, substitutes for
the selective advantage conferred by the mutational inactivation of these tumor suppressors. Mutational inactivation of Rbl
is a hallmark of human retinoblastoma. To assess whether
inactivation of Rbl and Trp53 had occurred in the retinoblastomas that arose in the FVB X C57BL/6 F, aAcry-HPVl 6E6/E7
mice, we performed SSLP analysis on the tumors. The Rbl and
Trp53 loci, located on chromosome 14 or 11, were found to be
intact (data not shown). Thus, as is true in HPV-positive cervical cancers, the presence of the dominantly acting viral transgenes obviates the need for mutational inactivation of the
genes encoding Rb or p53- That SV40 T-ag and E6/E7 each can
induce retinoblastoma in mice yet retinoblastoma does not
develop in mice carrying germline mutations in Rbl, Trp53, or
both47"50 indicates that additional activities shared by T-ag and
E6/E7 must be involved. E7 can modulate the activities of the
Rb-like proteins, plO7 and or pl30 34 ; inactivation of plO7, in
addition to Rb, may be required for tumorigenesis in the retina,
because retinal dysplasia is noted in Rb+/~;pl07~/~ mice.25
However, expression of E7 alone in the retina is insufficient to
induce development of retinoblastoma; rather, E7 efficiently
induces cell proliferation but also apoptosis of the photoreceptor cells. This apoptosis may be partially relieved by mutational
Downloaded From: http://iovs.arvojournals.org/ on 08/11/2017
10
12
Mouse Age at Retinoblastoma
Detection (months)
FIGURE 6. Retinoblastoma incidence in line 19 aAcryHPV16E6/E7, rdta C57BL/6 X FVB F, cotransgenic mice.
Homozygous line 19 aAcry-HPVl 6E6/E7 FVB transgenic mice
were crossed with heterozygous rdta C57BL/6 transgenic
mice, and F, hybrid progeny were monitored for the presence
of overt eye tumors at the indicated ages. Mice were genotyped
for the presence of the aAcry-HPVl6E6/E7 and rdta transgenes by polymerase chain reaction analysis (see the Materials
and Methods section). ( • ) Mice with rdta transgene; (•) mice
without rdta transgene. Tumors were confirmed to be retinoblastomas by microscopic analysis. Indicated are the percentages of mice in each group showing retinoblastomas at ages up
to 12 months. The number of eyes in each group is indicated
in parentheses.
Risk Factors for Retinoblastoma in Mice 2731
IOVS, December 1998, Vol. 39, No. 13
present in the inner nuclear layer to a tumor cell displaying
characteristics of photoreceptor cells. An alternative explanation is that retinoblastoma arises from an undifferentiated precursor cell, that is, a retinoblast, that is retained within the
inner nuclear layer. We know from cell fate experiments that
many cell types arise from a common progenitor cell. Therefore, differentiation of a retinoblast residing in the inner nuclear layer could result in a tumor displaying characteristics of
a photoreceptor cell.
90-
o
80"
bias
mas
100-1
70-
o
60"
5040"
line 19 x C3H Fj
(n=51)
3020-
10"
line 19
(n=50)
Mouse Age at Retinoblastoma
Detection (months)
Role of Alleles in Addition to rd in Modifying
Risk to Retinoblastoma
We observed a high incidence of retinoblastoma in line 19
aAcry-HPVl6E6/E7 FVB X C3H F, mice that are rd/rd. Thus,
recessive alleles on the FVB genetic background other than rd
must contribute to resistance to retinoblastoma. We have begun backcross experiments to determine whether homozygosity at other loci, in conjunction with homozygosity at rd,
correlates with resistance to retinoblastoma in the aActyHPV16E6/E7 (FVB X C57BL/6) X FVB B, mice. Preliminary
results indicate the existence of at least two alleles; homozygosity at either one decreases risk of development of retinoblastoma in rd/rd mice (data not shown). Confirmation of
these preliminary results and more refined mapping of these
loci are under way.
FIGURE 7. Retinoblastoma incidence in line 19 aAcryHPV16E6/E7 C3H X FVB F, mice. Homozygous line 19 aAcryHPV16E6/E7 FVB transgenic mice were crossed with nontransgenic C3H mice, and F, hybrid progeny were monitored
for the presence of overt eye tumors at the indicated ages. Mice
Acknowledgments
were genotyped by polymerase chain reaction analysis (see the
Materials and Methods section). Mice were killed when tumors
were observed. Tumors in the F, mice were confirmed to be The authors thank Angie Buehl for expert histotechnology, Henry Pi tot
for preliminary histopathology, Kathleen Mahon for the pGEMcvCrl
retinoblastomas by microscopic analysis. Indicated are the perplasmid, Nancy Robinson for editorial suggestions on the manuscript,
centages of mice showing retinoblastomas at various ages up to
and Jolene Windle and David Papermaster for sharing unpublished
14 months for F, mice and 13 months for FVB inbred mice. The
results.
number of mice in each group is indicated in parentheses.
References
useful for identifying strain-specific modifiers of tumor induction in a given tissue and may provide insight into the variable
penetrance of tumor development in humans.
Role of the Retinal Degeneration Phenotype in
Modifying Risk to Retinoblastoma
We think that the phenotype resulting from the rd allele in FVB
mice contributes in part to their resistance to retinoblastoma.
This is based primarily on our observations that ablation of the
photoreceptor cell layer by the directed expression of a diphtheria toxin transgene caused a twofold decrease in the incidence of retinoblastoma in line 19 cxAcry-HPVl6E6/E7 FVB X
C57BL/6 F, transgenic mice. How the physical ablation of the
photoreceptor cell layer in rd/rd mutant and rdta transgenic
mice contributed to the reduced incidence of retinoblastoma
remains unclear. The retinoblastomas that arose in the HPV-16
transgenic mice clearly possessed histopathologic and ultrastructural characteristics of photoreceptor cells. The early dysplastic lesions that arose in the retinas of these mice and are the
presumed precursors of the retinoblastomas were located
within the inner nuclear layer of the retina, where bipolar,
amacrine, and horizontal cells reside (Fig. 4). Similarly, the
retinoblastomas that develop in the LHfi-TAG mice arise within
the inner nuclear layer.15 However, photoreceptor cells normally reside in the outer nuclear layer. One possible explanation is that retinoblastoma in these animals results from metaplasia, that is, the conversion of a differentiated cell type
Downloaded From: http://iovs.arvojournals.org/ on 08/11/2017
1. Friend SH, Bernards R, Rogelj S, et al. A human DNA segment with
properties of the gene that predisposes to retinoblastoma and
osteosarcoma. Nature. 1986;323:643-646.
2. Fung Y-KT, Murphree AL, T'Ang A, et al. Structural evidence for
the authenticity of the human retinoblastoma gene. Science. 1987;
236:1657-1661.
3. Lee W-H, Bookstein R, Hong F, et al. Human retinoblastoma
susceptibility gene: cloning, identification and sequence. Science.
1987;235:1394-1399.
4. Cavenee WK, Hansen MF, Nordenskjold M, et al. Genetic origins of
mutations predisposing to retinoblastoma. Science. 1985:228:501503.
5. Knudson AG Jr. Mutation and cancer: statistical study of retinoblastoma. Proc Natl Acad Sci USA. 1971;68:820-823.
6. Matsunaga E. Hereditary retinoblastoma: delayed mutation of host
resistance? Am JHum Genet. 1978;30:406-424.
7. Bonaiti-Pellie C, Clerget-Darpoux F, Babron M-C. Hereditary
retinoblastoma: can balanced insertion entirely explain the differences of expressivity among families? Hum Genet. 1990;86:203208.
8. Otterson GA, Chen W, Coxon AB, Khleif SN, Kaye FJ. Incomplete
penetrance of familial retinoblastoma linked to germ-line mutations that result in partial loss of RB function. Proc Natl Acad Sci
USA. 1997;94:12036-12040.
9. Dietrich WF, Lander ES, Smith JS, et al. Genetic identification of
Mom-1, a major modifier locus affecting Min-induced intestinal
neoplasia in the mouse. Cell. 1993;75:631-639.
1.0. Poole TM, Chiaverotti TA, Carabeo RA, Drinkwater NR. Genetic
analysis of multistage hepatocarcinogenesis. In: Walker C, Groopman J, Slaga T, Klein-Szanto A, eds. Genetics and Cancer
Susceptibility: Implications for Risk Assessment. New York:
Wiley-Liss; 1996:33-45.
2732
Griep et al.
11. Williams BO, Remington L, Albert DM, et al. Cooperative tumorigenic effects of germline mutations in Rb and p53. Nat Genet.
1994;7:480-484.
12. Maandag EC, van der Valk M, Vlaar M, et al. Developmental rescue
of an embryonic-lethal mutation in the retinoblastoma gene in
chimeric mice. EMBOJ. 1994; 13:4260-4268.
13- Williams BO, Schmitt EM, Remington L, et al. Extensive contribution of Rb-deficient cells to adult chimeric mice with limited
histopathological consequences. EMBOJ. 1994;13:4251-4259.
14. Hammang JP, Baetge EE, Behringer RR, et al. Immortalized retinal
neurons derived from SV40 T-antigen-induced tumors in transgenic mice. Neuron. 1990;4:775-782.
15- WindleJJ, Albert DM, O'Brien JM, et al. Retinoblastoma in transgenic mice. Nature. 199O;343:665-669.
16. Howes KA, LasudryJGH, Albert DM, WindleJJ. Photoreceptor cell
tumors in transgenic mice. Invest Ophtbalmol Vis Sci. 1994;35:
342-351.
17. DeCaprio JA, Ludlow JW, Figge J, et al. SV40 large tumor antigen
forms a specific complex with the product of the retinoblastoma
susceptibility gene. Cell. 1988;54:275-283.
18. Fanning E. Modulation of cellular growth control by SV40 large T
antigen. In: Doerfler W, Bohrn P, eds. Malignant Transformation
by DNA Viruses. Weinheim, Germany: VCH; 1992:1-19.
19. Chen J, Tobin G, Pipas JM, Van Dyke TA. T-antigen mutant activities in transgenic mice: roles of p53 and pRb binding in tumorigenesis of the choroid plexus. Oncogene. 1992;7:1167-1175.
20. Symonds HS, McCarthy SA, Chen J, Pipas JM, Van Dyke T. Use of
transgenic mice reveals cell specific transformation by a simian
virus T-antigen amino terminal mutant. Mol Cell Biol. 1993; 13:
3255-3265.
21. Zalvide J, DeCaprio JA. Role of pRb-related proteins in simian virus
large T-antigen-mediated transformation. Mol Cell Biol. 1995;15:
5800-5810.
22. Stubdal H, Zalvide J, DeCaprio JA. Simian vims 40 large T antigen
alters the phosphorylation state of Rb-related proteins pi30 and
p107./ Virol. 1996;70:2781-2788.
23. Yaciuk P, Carter MC, Pipas JM, Moran E. Simian virus large Tantigen expresses a biological activity complementary to the p300associated transforming function of the adenovirus El A gene products. Mol Cell Biol. 1991;ll:21l6-2124.
24. Perry ME, Levine AJ. Tumor suppressor p53 and the cell cycle.
CurrOpin Genet Dev. 1993;3:5O-54.
25. Lee M-H, Williams BO, Mulligan G, et al. Targeted disruption of
pi 07: functional overlap between plO7 and Rb. Genes Dev. 1996;
10:1621-1632.
26. Howes KA, Ransom N, Papermaster DS, et al. Apoptosis or
retinoblastoma: alternative fates of photoreceptors expressing the
HPV-16 E7 gene in the presence or absence of p53. Genes Dev.
1994;8:1300-!310.
27. Schwarz E, Freese UK, Gissman L, et al. Structure and transcription
of human papillomavirus sequences in cervical carcinoma cells.
Nature. 1985;3l4:ll 1-114.
28. Yee C, Krishnan HI, Baker CC, Schlegel R, Howley PM. Presence
and expression of human papillomavirus sequences in human
cervical carcinomas cell lines. Am J Pathol. 1985;! 19:361-366.
29- Smotkin D, Wettstein FO. Transcription of human papillomavirus
type 16 early genes in a cervical cancer and a cancer-derived cell
line and identification of the E7 protein. Proc Natl Acad Sci USA.
1986;83:4680-4684.
30. Munger K, Phelps WC, Bubb V, Howley PM, Schlegel R. The E6
and E7 genes of the human papillomavirus type 16 together are
necessary and sufficient for transformation of primary human keratinocytes./ Virol. 1989;63:44l7-4421.
31. Huibregtse JM, Scheffner M, Howley PM. A cellular protein mediates association of p53 with the E6 oncoprotein of human papillomavirus types 16 and 18. EMBOJ. 1991;10:4l29-4l35.
32. Huibregste JM, Scheffner M, Howley PM. Cloning and expression
of the cDNA for E6-AP, a protein that mediates the interaction of
the human papillomavirus E6 oncoprotein with p53- Mol Cell Biol.
1993;! 3:775-784.
Downloaded From: http://iovs.arvojournals.org/ on 08/11/2017
IOVS, December 1998, Vol. 39, No. 13
33. Dyson N, Howley P, Munger K, Harlow E. The human papilloma
virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene
product. Science. 1989;243:934-937.
34. Dyson N, Guida P, Munger K, Harlow E. Homologous sequences in
adenovirus E1A and human papillomavirus E7 proteins mediate
interaction with the same set of cellular proteins./ Virol. 1992;
66:6893-6902.
35. Griep AE, Herber R, Jeon S, et al. Tumorigenicity by human
papillomavirus type 16 E6 and E7 in transgenic mice correlates
with alterations in epithelial cell growth and differentiation. / Virol. 1993;67:1373-1384.
36. Lambert PF, Pan H, Pitot HC, et al. Epidermal cancer associated
with expression of human papillomavirus type 16 E6 and E7
oncogenes in the skin of transgenic mice. Proc Natl Acad Sci USA.
1993:90:5583-5587.
37. McCall MA, Gregg RG, Merriman K, et al. Morphological and
physiological consequences of the selective elimination of rod
photoreceptors in transgenic mice. Exp Eye Res. 1996;63:35-49.
38. Overbeek PA, Chepelinsky AB, Khillan JS, Piatigorsky J, Westphal
H. Lens-specific expression and developmental regulation of the
bacterial chloramphenicol acetyltransferase gene driven by the
murine alpha A-crystallin promoter in transgenic mice. Proc Natl
Acad Sci USA. 1985;82:7815-7819.
39- Bowes C, Li T, Danciger M, et al. Retinal degeneration in the rd
mouse is caused by a defect in the |3-subunit of rod cGMP-phosphodiesterase. Nature. 1990;347:677-680.
40. Hayat MA. Principles and Techniques of Electron Microscopy:
Biological Applications. New York: Van Nostrand Reinhold; 1970.
41. Pan H, Griep AE. Temporally distinct patterns of p53-dependent
and p53-independent apoptosis during mouse lens development.
Genes Dev. 1995;9:2157-2l69.
42. Taketo M, Schroeder AC, Mobraaten LE, et al. FVB/N: an inbred
mouse strain preferable for transgenic analyses. Proc Natl Acad Sci
USA. 1991;88:2065-2069.
43. Kivela T, Virtanen I, Marcus DM, et al. Neuronal and glial properties of a murine transgenic retinoblastoma model. Am J Pathol.
1991;138:1135-1148.
44. Dietrich WF, Miller JC, Steen RG, et al. A genetic map of the mouse
with 4006 simple sequence length polymorphisms. Nat Genet.
1994;7:220-245.
45. Scheffner M, Munger K, Byrne JC, Howley PM. The state of the p53
and retinoblastoma genes in human cervical carcinoma cell lines.
Proc Natl Acad Sci USA. 1991;88:5523-5527.
46. Crook T, Wrede D, Tidy J, et al. Status of c-myc, p53 and retinoblastoma genes in human papillomavirus positive and negative
squamous cell carcinomas of the anus. Oncogene. 1991 ;6:1251 1257.
47. Clarke AR, Maandag ER, van RM, et al. Requirement for a functional Rb-1 gene in murine development. Nature. 1992;359:328330.
48. Donehower LA, Harvey M, Slagle BL, et al. Mice deficient for p53
are developmentally normal but susceptible to spontaneous tumours. Nature. 1992;356:215-221.
49. Jacks T, Fazeli A, Schmitt EM, et al. Effects of an Rb mutation in the
mouse. Nature. 1992;359:295-3OO.
50. Lee EY, Chang CY, Hu N, et al. Mice deficient for Rb are nonviable
and show defects in neurogenesis and haematopoiesis. Nature.
1992;359:288-294.
51. Herber R, Liem A, Pitot H, Lambert PF. Squamous epithelial hyperplasia and carcinoma in mice transgenic for the human papillomavirus type 16 E7 oncogene./ Virol. 1996;70:1873-1881.
52. Hennings H, Click AB, Lowry DT, et al. FVB/n mice: an inbred
strain sensitive to the chemical induction of squamous cell carcinomas in the skin. Carcinogenesis. 1993;l4:2353-2358.
53. Drinkwater NR, Bennett LM. Genetic control of carcinogenesis. In:
Ito N, Sugano H, eds. Modification of Tumor Development in
Rodents. Basel, Switzerland: S. Karger; 1991 ;33:1-20.