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Molecular and Cellular Biology, September 2001, p. 6017-6030, Vol. 21, No. 17
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.17.6017-6030.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Selective Inactivation of p53 Facilitates Mouse
Epithelial Tumor Progression without Chromosomal Instability
Xiangdong
Lu,1
Gregg
Magrane,2
Chaoying
Yin,1
David N.
Louis,3
Joe
Gray,2 and
Terry
Van Dyke1,*
Department of Biochemistry and Biophysics,
University of North Carolina at Chapel Hill, Chapel Hill, North
Carolina 275991; Department of
Laboratory Medicine, University of California at San Francisco, San
Francisco, California 941432; and
Molecular Neuro-Oncology Laboratory, Massachusetts General
Hospital, Harvard Medical School, Charlestown, Massachusetts
021293
Received 3 April 2001/Returned for modification 22 May
2001/Accepted 4 June 2001
 |
ABSTRACT |
We examined the selective pressure for, and the impact of, p53
inactivation during epithelial tumor evolution in a transgenic brain
tumor model. In TgT121 mice, cell-specific inactivation of
the pRb pathway in brain choroid plexus epithelium initiates tumorigenesis and induces p53-dependent apoptosis. We previously showed
that p53 deficiency accelerates tumor growth due to diminished apoptosis. Here we show that in a p53+/
background,
slow-growing dysplastic tissue undergoes clonal progression to solid
angiogenic tumors in all animals. p53 is inactivated in all
progressed tumors, with loss of the wild-type allele occurring in 90%
of tumors. Moreover, similar progression occurs in 38% of
TgT121p53+/+ mice, also with loss of at least
one p53 allele and inactivation of p53. Thus, the selective pressure
for p53 inactivation, likely based on its apoptotic function, is high.
Yet, in all cases, p53 inactivation correlates with progression beyond
apoptosis reduction, from dysplasia to solid vascularized tumors.
Hence, p53 suppresses tumor progression in this tissue by multiple
mechanisms. Previous studies of fibroblasts and hematopoietic cells
show that p53 deficiency can be associated with chromosomal
instability, a mechanism that may drive tumor progression. To determine
whether genomic gains or losses are present in tumors that progress in
the absence of p53, we performed comparative genomic hybridization
analysis. Surprisingly, the only detectable chromosomal imbalance was
partial or complete loss of chromosome 11, which harbors the p53 gene and is thus the selected event. Flow cytometry confirmed that the
majority of tumor cells were diploid. These studies indicate that loss
of p53 function is frequent under natural selective pressures and
furthermore that p53 loss can facilitate epithelial tumor progression
by a mechanism in addition to apoptosis reduction and distinct from
chromosomal instability.
| |
INTRODUCTION |
The p53 gene is mutated in at least
50% of human cancers, including most tumor types (27, 35,
43). Although many possible mechanisms for p53 tumor suppression
have been defined for cultured cells, no mechanism has been fully
established in vivo. In cultured cells, p53 is activated in response to
a variety of cellular stress signals including DNA damage, aberrant
proliferation, hypoxia, and nucleotide deprivation (36, 50,
56). Activation of p53 leads to either growth arrest or
apoptosis, a decision that appears to depend on the cell type and
specific stimulus (2, 22, 36, 65, 67). Thus, the in vivo
signals for p53 tumor suppression and the mechanism by which p53
inactivation contributes to tumorigenesis are likely to vary depending
on the cell type and environment. Based on the known functions of p53,
there are several mechanisms by which p53 inactivation could contribute
to cancer. For example, p53 regulates a G1
checkpoint in fibroblasts in response to DNA damage, a response that
requires transcriptional activation of the p53 target gene p21
(7, 14). In addition, p53 can arrest cells in
G2, possibly via transcriptional activation of
14-3-3 
(25) and/or transcriptional repression of cdc2
and cyclin B1 (61). Importantly, Chk1 and Cds1/Chk2
kinases involved in replication and DNA damage-induced
G2 arrest have recently been shown to
phosphorylate p53 (9, 26, 58). The identification of a
Cds1/Chk2 mutation in a Li-Fraumeni syndrome family that lacks p53
mutation supports the idea that Cds1/Chk2 and p53 function in a pathway
for tumor suppression (3). Finally, p53 prevents DNA
endoreduplication in fibroblasts exposed to mitotic spindle inhibitors
(15) and has been implicated in centrosome
regulation (20). In the absence of p53, cultured
fibroblasts can be readily selected for gene amplification, indicating
a tendency for genetic instability upon p53 deficiency (38,
73). These observations led to the "guardian of the
genome" hypothesis for p53 tumor suppression (32), which suggests that p53 inactivation may contribute
to tumorigenesis by facilitating the propagation of genetically
defective cells as a result of checkpoint loss (23).
Secondary mutations or chromosomal changes that provide the cell a
selective advantage would thus facilitate tumorigenesis. Indeed,
hematopoietic tissues from p53-deficient mice contain a high percentage
of aneuploid cells (6, 21), the mice are predisposed to
thymic lymphoma (17, 30), and lymphomas are aneuploid
(37, 64). However, the direct contribution of
p53-deficiency-induced genetic instability to tumorigenesis has not
been demonstrated in vivo.
Another mechanism by which p53 can suppress tumorigenesis involves the
apoptotic response to aberrant proliferation induced by oncogene
expression or pRb inactivation. Many studies have demonstrated that
this response is p53 dependent (13, 28, 39, 41, 42, 47, 53,
68), indicating that developing tumors could activate p53, thus
establishing a selective pressure for p53 inactivation leading to tumor
cell survival. We previously demonstrated such a role for p53 in vivo
using a transgenic mouse model that undergoes epithelial cell
tumorigenesis in response to pRb pathway inactivation
(60). In the choroid plexus (CP) epithelium, p53
inactivation alone is inconsequential, as is the case for most mouse
epithelial cells (17, 30). However, cell-specific inactivation of the pRb pathway induces aberrant proliferation and
dysplastic growth, resulting in p53-dependent apoptosis (see Fig. 1). A
similar response occurs in the lens (46) and retina (28) and in several cell types during development
(41, 42). In the brain tumor model, germ line p53
deficiency leads to significant acceleration of dysplastic tissue
growth due to an 85% reduction in apoptosis (60).
Importantly, most human tumors harbor a defect in the pRb pathway
(70); thus, p53 and Rb pathway mutations frequently
coexist during the course of tumorigenesis. Hence, it is important to
understand the dynamic interplay between these aberrations during tumor
development. The dependency of tumor cell apoptosis on p53 function
suggests that selective pressure for inactivation of the p53 pathway
should be high in a developing tumor. Such direct selection for p53
inactivation is unlikely to result from a role in genome maintenance
alone. However, once a p53-deficient cell is selected based on
survival, its propagation in the absence of p53-dependent checkpoints
could result in genetic aberrations that may accelerate tumor
progression. By this scenario, p53 inactivation would contribute to
tumorigenesis by multiple mechanisms. In the present report, we test
this hypothesis by exploring the evolution of developing CP tumors in
p53 heterozygous and wild-type backgrounds. We assess the natural
selective pressure for p53 inactivation and the contribution of p53
inactivation to tumor progression. In particular, we explore the
possibility that chromosomal instability drives tumor progression in
tumors that evolve to a p53-deficient state. The contribution of p53 inactivation to tumor progression during the natural evolution of
epithelial tumors lacking the Rb pathway has not previously been explored.
| |
MATERIALS AND METHODS |
Mice.
Generation, screening, and characterization of
TgT121 transgenic mice (B6D2) were described
previously (54, 60). These mice harbor the
T121 mutant T-antigen gene under the control of the lymphotropic papovavirus transcriptional signals, resulting in
uniformly high levels of expression in the CP (10). The
T121 transgene encodes the first 121 amino acids
of simian virus 40 (SV40) T antigen and is capable of binding to the
pRb family proteins but not to p53.
TgT121p53+/ and
TgT121p53/ mice were
generated by crossing TgT121+/+
or TgT121+/ mice with
p53/ mice (C57BL6/J; Jackson Laboratories)
(30). Genotypes were identified by PCR analysis of tail
DNA as described previously (60). Mice were monitored
regularly for the outward signs of a brain tumor, which consist of
cranial bulging and decreased activity. Mice were then sacrificed, and
tumors were either frozen at 
80°C for nucleic acid analysis or
fixed in 10% formalin as described previously (54).
Histology and immunohistochemistry.
To analyze tumor
morphology and development, mouse brains were cut into halves, fixed in
10% formalin, embedded in paraffin, and sectioned for 10 successive
layers at 50-µm intervals. A 5-µm section from each layer was
stained with hematoxylin and eosin for morphological analysis
(60). To measure the tumor size, the slide with the
largest tumor cross section was chosen for each sample, and the tumor
area was measured using the PAXit image capture and analysis system
(MIS, Inc.). The tumor volume was then calculated with the assumption
that the tumor has the same volume as a sphere with an equivalent
cross-sectional area.
For CD31 immunostaining, sections were treated with 1 mg of trypsin/ml
in phosphate-buffered saline (PBS) for 10 min at 37°C and then
blocked in 5% normal rabbit serum (NRS) in PBS for 1 h at room
temperature (RT). The sections were incubated with anti-CD31 antibody
(1:50 in PBS containing 5% NRS; Pharmingen) overnight at 4°C. After
three washes in PBS, slides were incubated with biotin-conjugated
secondary antibody (1:100 in PBS containing 2% NRS) for 30 min at RT.
Slides were washed twice with TS buffer (50 mM Tris-HCl [pH 7.6], 150 mM NaCl, 0.1% Tween 20) and incubated with
avidin-biotin-peroxidase-alkaline phosphatase reagent (Vector Laboratories) for 30 min. After two washes with TS buffer, slides were
incubated with alkaline phosphate substrate (Vector Laboratories) for
10 min at RT, counterstained with methyl green, and dehydrated in two
changes of xylene. The blood vessel pattern of dysplastic CP and tumors
was evaluated by quantifying CD31-positive vessels. For each brain
sample, the numbers of blood vessels larger than 200 µm2 were counted in fields with abundant blood
vessels. Dysplastic CP and terminal tumors from seven
T121p53+/ mice each were
evaluated; the averages and the standard deviations of their blood
vessel counts are represented in Fig. 3.
Loss-of-heterozygosity (LOH) analysis.
To determine
the status of the wild-type p53 allele in terminal
TgT121p53+/ tumors,
semiquantitative PCR analysis was performed. The wild-type p53 allele
was amplified with primers directed against exon 6 (X6.5;
5'-ACAGCGTGGTGGTACCTTAT-3') and exon 7 (X7;
5'-TATACTCAGAGCCGGCCT-3'), while the p53 null allele was
amplified with primers directed against the neomycin gene contained
within the targeted locus (neo18;
5'-CTATCAGGACATAGCGTTGG-3') and p53 exon 7. The 25-µl PCR
mixture contained 0.2 mM concentrations of each of the four deoxynucleoside triphosphates, 2 µCi of
[
-32P]dCTP (3,000 Ci/mmol), 0.8 µM X6.5
and neo18, 1.6 µM X7, 0.5 U of Taq polymerase (Boehringer
Mannheim), and 100 ng of DNA template. PCR was performed for 25 cycles
using the following conditions: 1 min at 94°C, 2 min at 60°C, and 2 min at 72°C. PCR products were resolved on an 8% polyacrylamide gel
and quantified using a PhosphorImager (Molecular Dynamics). In
standardization assays, tail DNA from a p53+/
mouse and a p53/ mouse were mixed at ratios
ranging from 1:2 to 1:50. The ratio of the product intensity to the
template concentration was graphed and resulted in a linear
relationship. The ratio of wild-type allele intensity in the control
lane was normalized to 1.00. With the assumption that the tumor was not
likely to include more than 30% nontumor cells, an arbitrary 0.33 intensity ratio threshold was established for determining LOH. The
normalized ratio threshold is 0.20. If the ratio of the p53 wild-type
signal to p53 null signal was greater than 0.20, the p53 wild-type
allele was considered to be retained; a ratio of less than 0.20 indicated loss. Two independent PCR assays were performed for each sample.
To determine the status of the wild-type p53 allele in terminal
TgT
121p53
+/+ tumors,
quantitative real-time PCR analysis was performed. The
conditions for
analysis of the p53 locus were provided by Lynda
Chin and colleagues
(Harvard University). The primers for the
p53 allele were
5'-ATGGCCATCTACAAGAAGTCACAG-3' and
5'-ATCGGAGCAGCGCTCATG-3'.
The sequence of the p53 probe was
5'-ACATGACGGAGGTCGTGAGACGCTG-3'.
The primers for the
internal control
-actin gene were 5'-AAGAGCTATGAGCTGCCTGA-3'
and 5'-ACGGATGTCAACGTCACACT-3'. The sequence of the
-actin probe
was 5'-CACTATTGGCAACGAGCGGTTCCG-3'. Each
25-µl reaction mixture
contained 50 ng of DNA template, 18 nM p53
primers, 80 nM
-actin
primers, 8 nM probe, and 12.5 µl of TaqMan
Universal PCR Master
Mix (Applied Biosystems) containing AmpliTaq Gold
polymerase,
deoxynucleoside triphosphates, and PCR buffer. The cycling
conditions
were 50°C for 2 min and 95°C for 10 min for 1 cycle and
95°C for
15 s and 60°C for 1 min for 40 cycles. The reactions
were performed
using an ABI 7700 Sequence Detection system (Applied
Biosystems),
and the data were analyzed using Sequence Detector 1.7 (Applied
Biosystems) and standard protocols
(
http://www.appliedbiosystems.com).
The copy number of each sample
was determined by calculating
Ct
based on the formula
Ct = [sample Ct
(p53) sample
Ct
(
-actin)]
[p53
+/+ control Ct
(p53) p53
+/+ control
Ct
(-actin)], where Ct is the number of cycles
required
to reach a threshold based on linear amplification. Analyses
of
standard samples (L. Chin, Harvard University, personal
communication)
indicate that copy numbers of 2, 1, and 0 are indicated
by 2
Ctn values of >0.6, 0.15 to 0.6, and
<0.15, respectively. Standard
samples analyzed along with experimental
samples confirmed the
accuracy of these
assignments.
In situ RNA hybridization.
In situ hybridization was carried
out as described previously (48). For template
preparation, pBS-KSp21 was digested with EcoRI and
BamHI. The antisense probe was generated by T7 transcription of an EcoRI-linearized template, and the sense probe was
produced by T3 transcription of a BamHI-linearized template.
Probes were labeled with [-35S]UTP (5 × 104 cpm/µl) and hybridized to slides at
50°C overnight. Autoradiography was performed at 4°C for 3 days.
Sections were counterstained with 0.2% toluidine blue and observed and
photographed using dark-field microscopy.
CGH.
Comparative genomic hybridization (CGH) was performed
essentially as described elsewhere (16, 31). Tumor and
normal tail DNA were labeled by nick translation using
fluorescein-12-dUTP (NEN) and Alex-568-5-dUTP (Molecular
Probes), respectively. The optimum probe size was about 600 bp. Labeled
tumor and normal DNA (1 µg each) were coprecipitated and dissolved in
10 µl of hybridization solution to obtain a final composition of 50%
formamide, 10% dextran sulfate, and 2× SSC (1× SSC is 0.15 M NaCl
and 0.015 M sodium citrate [pH 7.0]). The mixture was heated to
70°C for 15 min to denature the DNA and was incubated at 37°C for
10 min. Normal mouse metaphase chromosomes prepared from mouse
embryonic fibroblasts were denatured at 70°C in 70% formamide-2×
SSC for 4 min and dehydrated through graded ethanols. The hybridization mixture was added to the slides, coverslips were sealed with rubber cement, and the slides were incubated at 37°C for 3 days. The slides
were washed in formamide and SSC as previously described (31) and stained with 0.1 µM
4',6'-diamino-2-phenylindole (DAPI). Digital images of each
fluorochrome were obtained using a fluorescence microscope and a
charge-coupled device camera. The profiles of different fluorescence
intensities and corresponding chromosomal losses and gains were
analyzed using Vysis software (Applied Images Inc.). The normalized
average ratio of green to red (tumor/normal ratio) is 1.0. The
threshold of variability using normal samples was 0.8 to 1.2.
Flow cytometry.
Brain tumor cells were isolated and stained
as described (52). Since normal CP is difficult to cleanly
dissect in sufficient quantity, normal spleen cells were used as a
diploid control. Glass slides and 70-µm-pore-size cell strainers
(Becton Dickinson) were used to dissociate tumor and spleen cells. The
cells were washed with PBS, fixed in 70% ethanol, and stained with 25 µg of propidium iodide/ml containing 0.1 mg of RNase A/ml. Flow
cytometry was carried out using a FACScan cytometer (Becton
Dickinson), and data were analyzed with Cyclops software (Cytomation).
| |
RESULTS |
High rates of tumor progression in
TgT121p53+/ and
TgT121p53+/+ mice.
In
TgT121 transgenic mice, pRb and related proteins
p107 and p130 (collectively referred to here as
pRbf) are inactivated in the CP by
tissue-specific expression of the truncated SV40 T antigen,
T121 (see Materials and Methods). Disruption of
the pRb pathway in CP induces slow-growing dysplastic masses, resulting in death of the mice at an average of 26 weeks (Fig.
1A) (60). Upon
pRbf inactivation, CP cells are induced to
proliferate aberrantly (54), resulting in the induction of
p53-dependent apoptosis (Fig. 1B) (60). In a
p53/ background, growth of the CP masses is
accelerated approximately sevenfold (50% survival time = 4 weeks [Fig. 1A]) due to an 85% reduction in cell death, although
masses remain morphologically dysplastic (60). We
previously showed that
TgT121p53+/ mice survive
to an intermediate age (50% survival time = 11 weeks [Fig. 1])
and show morphological signs of clonal tumor progression (Fig. 1B)
(60). As expected, apoptosis levels were significantly reduced in progressed tumors. This could occur by direct p53
inactivation or by inactivation of other genes in the relevant p53
pathway (if a linear pathway is involved) or in parallel pathways with similar function. To determine the genetic basis for tumor progression and the selective pressure for and the contribution of p53
inactivation, we carried out a comprehensive developmental analysis of
tumor evolution.
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FIG. 1.
A pathway to tumor development. (A) Time of survival
from brain tumors depends on p53 status. Mice were sacrificed when they
showed severely bulged crania and decreased activity. Histological
examination confirmed the presence of CP masses in all mice. (Data are
revised from those presented in reference 60.) (B) The
diagram depicts previously elucidated steps in T121-induced
development of CP tumors. T121 induces proliferation of
normally nondividing epithelial cells by inactivating the pRb family
proteins pRb, p107, and p130. p53-dependent apoptosis is then activated
by a process requiring E2F1 (48). As shown in the present
report, in a p53 heterozygous background, the selective pressure for
p53 inactivation is 100%, resulting in the focal development of solid
vascularized tumors.
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|
To more fully characterize tumor progression in
TgT
121p53
+/ and
TgT
121p53
+/+ mice, the
frequency, timing, and morphological characteristics
of tumor
progression were analyzed. Step sections were examined
at 50-µm
intervals for each brain. In 42 of 43 TgT
121p53
+/ mice analyzed
beyond 6 weeks of age, the CP showed evidence of
focal progression from
dysplasia to solid tumors (Fig.
2; see
also Fig.
4). Normal CP appears as frond-like papillary structures
extending from the walls of lateral ventricles and the roofs of
the
third and fourth ventricles. Each frond consists of a single
uniform
layer of differentiated epithelial cells (Fig.
2A).
TgT
121p53
+/ mice younger
than 6 weeks all showed only dysplastic changes
in CP (Fig.
2B). The
nuclei in these lesions were crowded and
elongated with a high
nuclear/cytoplasmic ratio, and the chromatin
was dense and coarse. The
single-layer structure was disrupted
with multilayer regions (Fig.
2B,
arrows), but the overall frond-like
structure of the CP was maintained.
No morphologically distinct
tumor foci were observed prior to 6 weeks
of age. In contrast,
TgT
121p53
+/ mice
sacrificed at 6 to 8 weeks of age showed emergence of focal
tumors in
addition to the preexisting dysplasia. The foci were
characterized by
nodules of solid tumor arising from the papillary,
dysplastic CP cells
(Fig.
2E). Tumors were relatively small at
this stage; 16 of 22 tumors
were smaller than 10 mm
3, with an average volume
of 1.1 ± 2.0 mm
3.
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FIG. 2.
Tumor progression in
T121p53+/ and
T121p53+/+ mice. Normal CP of a 3-month-old
nontransgenic mouse (br, brain tissue). Cells contain
regularly sized and shaped nuclei with significant cytoplasm in a
single layer forming a papillary architecture (A). Due to
T121 expression, the CP of young
T121p53+/ (3 weeks) (B) and
T121p53+/+ (5 weeks) (C) mice appears
dysplastic (dy) with nuclei of aberrant sizes and a high
nuclear/cytoplasmic ratio in most cells. The overall papillary
organization of the tissue is retained; however, multilayered regions
are present. The black arrow indicates dysplastic cells, and the pink
arrow indicates normal cells. Focal solid masses indicative of tumor
(tu) progression are detectable by 6 to 8 weeks in all
T121p53+/ mice (7-week-old mouse shown in
panel E) and at various times beyond 7 weeks in some
T121p53+/+ mice (7-week-old mouse shown in
panel F). These focal tumors arise among the background of dysplastic
CP. Solid vascularized tumors (designated type I) grow rapidly and
become life threatening by 12 weeks in all
T121p53+/ mice (12-week-old mouse shown in
panels D and H) and at 25 to 43 weeks in 38% of
T121p53+/+ mice (38-week-old mouse shown in
panel I). The red arrows in these figures indicate blood vessels.
Tumors of distinct morphology (type II) also arise in 59% of
T121p53+/+ mice (38-week-old mouse shown in
panel G). Loosely organized cells within these masses often contain
copious cytoplasm and grossly enlarged nuclei. These tumors appear to
grow slowly, since they do not reach the large sizes observed for type
I tumors. All slides were stained with hematoxylin and eosin. Each
white bar represents 50 µm, and each black bar represents 200 µm.
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From 9 to 14 weeks, all
TgT
121p53
+/ tumors became
life threatening. At this stage, tumor morphology was similar to the
previous
stage, but the tumors were much larger (Table
1). The large tumors
possessed an
extensive vasculature (Fig.
2D and H, e.g., arrows),
and necrotic foci
were sometimes present (data not shown). CD31
staining of blood vessels
showed that tumors contained approximately
five times the density of
enlarged vessels of that of dysplastic
CP (Fig.
3). Thus, tumor progression was
associated with promotion
of angiogenesis. In some regions, tumor cells
displayed a perivascular
orientation, and thus, the tumors resembled
poorly differentiated
papillary adenocarcinomas (Fig.
2D and H). In
some terminally
ill mice, tumors spread into the subarachnoid space
surrounding
the brain, but invasion into adjacent brain parenchyma was
rare
(2 of 21 mice). In summary, these studies showed that the
probability
of tumor progression in mice with a single functional p53
allele
was 100%. Furthermore, the event(s) responsible for tumor
progression
appeared to occur within a predictable time, with the
earliest
morphological signs of progression detectable at 6 to 8 postnatal
weeks (Fig.
4).
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FIG. 3.
Tumor progression is associated with angiogenesis.
Dysplastic CP (a) and tumor (b) sections from
T121p53+/ mice were immunostained using an
antibody specific for CD31 to detect blood vessels (A). Each white bar
represents 50 µm. Sections from brains of seven
T121p53+/ mice at 5 to 7 weeks were analyzed.
On the average, tumors contained larger vessels than did dysplastic
tissue. Since even normal CP contains numerous capillaries owing to its
function as a blood-cerebrospinal fluid barrier, vessels larger than
200 µm2 were counted for a quantitative comparison of the
dysplastic and tumor vasculatures (Materials and Methods). By this
analysis, tumors contained a significant increase in such vessels
(B).
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FIG. 4.
Frequency and timing of tumor progression (A) and p53
inactivation (B) in T121p53+/ and
T121p53+/+ mice. (A) Before 6 weeks of age, the
CP of all T121p53+/ mice was dysplastic with
no evidence of tumor progression. From 6 to 8 weeks, 95% of
T121p53+/ mice developed small focal tumors.
From 9 to 14 weeks, these solid tumors rapidly increased in size (up to
>100 mm3) and became life threatening. Tumor progression
occurred later and with a lower frequency in
T121p53+/+ mice. Only dysplasia was observed in
most mice younger than 15 weeks. Focal development of two distinct
morphologies could first be observed in mice at 6 to 14 weeks. Ten
percent of focal tumors were morphologically indistinguishable from
those developing in T121p53+/ mice (type I
tumors), while 25% of tumors appeared distinct (type II [Fig. 2]).
Beyond 25 weeks, 38% of mice had developed type I tumors and 59% had
developed type II tumors. (B) Inactivation of p53 was detected by in
situ detection of p21 transcripts and/or by gene loss using PCR
analysis as indicated (Fig. 5). More than 90% of
T121p53+/ terminal tumors showed loss of the
wild-type p53 allele. Terminal tumors that did not demonstrate p53 LOH
as well as early focal tumors (6 to 8 weeks) showed functional loss of
p53 based on loss of p21 expression. One-hundred-percent correlation of
p53 inactivation with tumor progression indicates that p53 inactivation
is likely the facilitating event. In T121p53+/+
mice, all type I tumors, but none of the type II tumors, showed loss of
p53 function. All three terminal type I tumors further analyzed by
quantitative PCR showed a corresponding loss of at least one p53
allele, while the three type II tumors analyzed retained both copies
(real-time PCR data are presented in Table 2 and are not depicted in
the histogram).
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Tumor progression also occurred with a high frequency in
TgT
121p53
+/+ mice, although
the timing was much less predictable (Fig.
4),
consistent with
the requirement for a minimum of two stochastic
genetic lesions to
inactivate p53. Emerging tumors could be classified
into two distinct
morphological types (arbitrarily referred to
as types I and II). Type I
tumors were identical to those arising
in
TgT
121p53
+/ mice (Fig.
2,
compare panels E and F and panels H and I). Focal
tumors with this
morphology were observed in 10% of mice 6 to
24 weeks old; at 25 to 43 weeks, 38% of mice harbored type I tumors
(Fig.
4). While
morphologically identical to
TgT
121p53
+/ terminal
tumors, these tumors were often smaller (Table
1),
possibly owing to
the older ages at which they developed. For
example, life-threatening
hydrocephalus, which was not present
in
TgT
121p53
+/ mice,
developed in 31% of the
TgT
121p53
+/+ mice with type
I
tumors.
Type II tumors were less compact and were characterized by considerable
nuclear polymorphism. Type II cells had a lower nuclear/cytoplasmic
ratio than did type I cells; the cytoplasm was copious, and nuclei
were
often grossly enlarged (Fig.
2G). Apoptotic cells were also
evident
(data not shown). While apoptosis in type I tumors had
dropped to 3.6% ± 0.2%, compared to 12.6% ± 2.8% in dysplastic
tissue, apoptosis
in type II tumors was at an intermediate level
(6.8% ± 1.8%). Type
II tumors were generally much smaller than
type I tumors, although a
subset of tumors containing type II
morphology grew to larger sizes
(Table
1). These larger tumors
were heterogeneous and contained regions
of densely packed cells
intermixed with typical type II morphology.
Type II foci were
first observed in 9-week-old mice and were present in
25% of mice
at 15 to 24 weeks and 56% of mice at 25 to 43 weeks.
Hydrocephalus
developed in 70% of mice with type II tumors and likely
accounts
for the life-threatening condition despite the relatively
smaller
tumor size. In general,
TgT
121p53
+/+ mice developed
either type I or type II tumors; of the 33 mice
analyzed with tumors,
only 2 harbored both tumor types. Of the
terminally ill mice, 41% had
type I tumors and 59% had type II
tumors.
p53 is inactivated in all type I tumors.
To determine
whether p53 inactivation was associated with tumor progression and
to assess the selective pressure for p53 inactivation, we examined
TgT121p53+/ tumors for
loss of the wild-type p53 allele. Semiquantitative PCR was used to
detect p53 wild-type and null alleles (Materials and Methods). DNA
isolated from terminal
TgT121p53+/ tumors was
compared with tail DNA from the same mice. Due to the presence of
stroma and vasculature in the tumor samples, a reduction of two-thirds
or more in the wild-type signal relative to the null signal was
considered a loss. A representative result is shown in Fig.
5A. Of 34 TgT121p53+/ tumors
analyzed, 32 showed convincing loss of the wild-type allele (Fig. 4B).
All tumors retained the null allele, indicating that gene loss was not
due to random genetic instability. Thus, the selective pressure for p53
loss during tumor progression was extremely high, with 94% of tumors
having specifically lost the functional p53 allele.
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|
FIG. 5.
p53 is inactivated in all
T121p53+/ and
T121p53/ type I tumors. (A) Genomic DNAs
from TgT121p53+/ terminal tumors (lanes 1 to
11) and TgT121p53+/ tail DNA ("C," lane
12) were analyzed by semiquantitative PCR to detect null (upper band)
and wild-type (WT; lower band) p53 alleles (see Materials and Methods).
Samples shown are representative of 34 tumors analyzed. The signal from
the wild-type p53 allele is selectively reduced in all but one tumor
(lane 9) relative to the upper band (the p53 null allele). The
normalized intensity ratios of wild-type to null alleles are listed
under corresponding lanes. L (loss) and R (retention) were determined
by using the arbitrary threshold (see Materials and Methods). A total
of 32 of 34 tumors analyzed in this fashion had lost the wild-type p53
allele (Fig. 4B). (B) Functional assessment of p53 by in situ
hybridization analysis of p21 transcript levels. p21 expression in CP
is an indicator of p53 function. p21 transcripts are undetectable in
nontransgenic CP (a). p21 is induced upon activation of p53 by
T121 (b). p21 expression is fully p53 dependent since it is
absent in TgT121p53/ CP (c). Functional
assessment of p53 in TgT121p53+/ (d) or
TgT121p53+/+ (f) CP tissue shows that p21
expression (p53 activity) is absent in type I tumor nodules but present
in surrounding dysplastic CP (e and g). Type II tumors (h) retain p21
expression (i). Subpanels d, f, and h show hematoxylin and eosin
staining as viewed by bright-field microscopy. Subpanels e, g, and i
show in situ hybridization with an antisense p21 RNA probe viewed by
dark-field microscopy. No signal was detectable using the sense probe
(data not shown). Each bar represents 200 µm. The frequency
and timing of p53 inactivation for all samples analyzed are graphed in
Fig. 4. non Tg, nontransgenic; br, brain tissue; tu, tumor; dy,
dysplasia.
|
|
To determine whether p53 function was also disrupted in the tumors that
retained the wild-type p53 allele and to determine
whether p53 loss
occurred concomitantly with tumor progression
rather than subsequently,
we assessed p53 activity in situ. As
described previously, the p53
target gene p21 is transcriptionally
activated in CP upon
T
121 expression (
48). In this
system, p21
expression is entirely dependent upon p53 activation, since
it
is undetectable in
TgT
121p53
/ CP
(
48) (Fig.
5B, subpanels a to c). Furthermore, we have
shown
elsewhere through genetic experiments that p21 is not required
for p53-dependent apoptosis in this system (C. Yin et al., unpublished
data). Thus, there should be no selective pressure for p21 loss
in the
absence of p53 loss. In situ hybridization was used to
detect p21
transcripts in
TgT
121p53
+/ CP in which
both dysplasia and a progressed tumor were present
(Fig.
5B, subpanel
d). p53 function, as indicated by the presence
of p21 transcripts, was
clearly absent from the progressed tumor
but was present in the
surrounding dysplastic regions (Fig.
5B,
subpanel e). In addition, five
of five terminal tumors that showed
p53 LOH had also lost p21
expression, indicating the consistency
of this assay in detecting p53
inactivation (Fig.
4B).
The two tumors that had retained the wild-type p53 allele by LOH
analysis showed loss of p21 transcripts, indicating that
the p53
pathway was functionally inactivated in 100% of progressed
tumors
(Fig.
4B). To assess the correlation between p53 inactivation
and tumor
progression, brains with the earliest detectable focal
lesions were
analyzed. Brains from all six
TgT
121p53
+/ mice analyzed
at 6 to 8 weeks old showed clear loss of p53 function
in focal tumors
with retention of function in dysplastic regions
(Fig.
4B). Thus, p53
inactivation was likely causal in the progression
of these
tumors.
To determine whether type I tumors in
TgT
121p53
+/+ mice showed
similar loss of p53 function, we examined 11 tumors at various
stages.
p21 in situ analysis again showed loss of p53 function
in all type I
tumors (Fig.
5B, subpanels f and g, and 4B). Consistent
with results
from TgT
121p53
+/ tumors,
p53 activity was lost at an early stage when tumor foci
were very small
(data not shown), indicating that loss of p53
is a rate-limiting event
in tumor progression. Real-time PCR analysis
showed that at least one
p53 allele was lost in three of three
tumors analyzed (Table
2), suggesting that p53, and not an
upstream
factor, was the target for inactivation. In contrast to type I
tumors, 11 of 11 TgT
121p53
+/+ type II tumors
analyzed at both early and late stages retained
p53 activity, and three
of these tumors analyzed for gene loss
retained both wild-type p53
alleles (Fig.
5B, subpanels h and
i; Table
2). These results indicate
that the genetic basis for
the differences between type I and type II
tumors includes the
absence and presence, respectively, of p53
activity. Thus, p53
loss is associated with progression to highly
vascularized aggressive
tumors. These results further emphasize that
the selective pressure
for p53 inactivation is high. Moreover, since
two functional alleles
of p53 are present in these mice, the strong
correlation between
p53 inactivation and type I tumor progression
indicates that loss
of the p53 pathway, likely of p53 itself, is the
major rate-limiting
step to tumor progression (see Discussion).
Furthermore, since
p53 inactivation is associated with tumor
progression rather than
simply faster-growing dysplastic masses, p53
inactivation must
contribute to tumorigenesis by mechanisms in addition
to apoptosis
modulation.
Does p53 inactivation during tumor progression cause genetic
instability?
A widely held hypothesis is that loss of p53 function
results in genomic instability and therefore could contribute to tumor progression by facilitating the propagation of genetic alterations. Although p53-deficient tumors and cell lines often contain genetic aberrations (almost exclusively aneuploidy), whether loss of p53 is a
direct cause of genetic instability is unknown (34). If inactivation of p53 function in TgT121p53+/
CP results in genetic instability that facilitates clonal tumor progression, such changes should be detectable in tumors. To determine whether this is the case, we analyzed
TgT121p53+/ terminal tumors by CGH. In CGH,
fluorescently labeled normal and tumor DNAs are competitively
hybridized to normal metaphase chromosomes to assess genome-wide copy
number changes. With a resolution of approximately 10 Mb, CGH readily
detects aneuploidy as well as partial chromosome gains and losses
(19).
Sixteen TgT
121p53
+/
terminal tumors were analyzed by CGH (Fig.
6A and B). Normal tail
DNA was used as a negative control, while
previously karyotyped
aneuploid thymic lymphoma DNA was used as
a positive control. A
sex-mismatched DNA mixture was also used
as an internal control for
copy number changes. As anticipated
based on the p53 LOH results
described above, most tumors (14
of 16) showed complete or partial loss
of a single copy of chromosome
11 (the location of the p53 gene) (Table
3). Surprisingly, chromosome
11 loss was
the only aberration detected in any of the 16 tumors
analyzed. We
also performed CGH analysis on six terminal
TgT
121p53
+/+
tumors. Four tumors harbored no detectable changes. These tumors
were
not characterized histologically and could have possessed
either type I
or type II characteristics. Two tumors showed loss
of chromosome 11 and
were presumably type I tumors based on the
association of p53
inactivation with these tumors as described
above. Both tumors also
harbored two to four additional changes
(Fig.
6B; Table
3). Since type
I TgT
121p53
+/+ tumors
appear after long latency likely due to the need to inactivate
two
wild-type p53 alleles, it is possible that events leading
to these
changes preceded p53 inactivation in these tumors. This
explanation would account for the difference in the level of
chromosome
changes between
TgT
121p53
+/+ and
TgT
121p53
+/ tumors that
have undergone p53 inactivation. Four
TgT
121p53
/ CP masses
were also analyzed and shown to contain no detectable
chromosomal
losses or gains (Table
3). A binomial statistical
test comparing these
results to previous CGH studies with p53
+/ and
p53
/ tumors from other models (
16,
64) indicates that the lack
of chromosomal instability in
TgT
121p53
/ CP is
statistically significant (
P < 0.01). As a positive
control
for CGH studies, thymic lymphomas known to be aneuploid by
karyotype
analysis (
37) were analyzed by CGH and shown to
contain numerous
copy number changes (Table
3). Since CGH detects only
unbalanced
chromosomal gains or losses, it was a formal possibility
that
tumors were polyploid. To test this possibility, tumor DNAs were
analyzed by flow cytometry. The majority of cells in all five
TgT
121p53
+/ tumors
analyzed were diploid (Fig.
6C). These results indicate
that, contrary
to popular hypothesis, p53 inactivation is not
sufficient to cause
chromosomal instability during tumor progression.
Furthermore, the lack
of chromosomal instability in these tumors
indicates that p53
inactivation contributes to tumor progression
by an as yet unidentified
mechanism(s).
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|
FIG. 6.
Chromosomal stability in p53-deficient tumors. (A)
DAPI-stained metaphase chromosomes from normal mouse embryonic
fibroblasts are shown, with chromosomes 11 and X indicated (left). A
composite digital image from CGH analysis of a representative
TgT121p53+/ terminal tumor is shown on the
right. Tumor DNA (fluorescein isothiocyanate, green) and normal tail
DNA (Alex-568, red) were hybridized to the metaphase spread shown in
the left panel. Relative green regions indicate increased copy numbers
in the tumor, while red regions indicate decreased copy numbers. The
centromeres appear blue because repeated satellite sequences were
blocked with unlabeled mouse Cot-1 DNA. This tumor sample shows loss of
chromosome 11. Apparent gain of the X chromosome serves as an internal
control; tumor DNA was derived from a female mouse, and normal DNA was
from a male mouse. (B) Summary of CGH analysis of 14 TgT121p53+/ and 6 TgT121p53+/+ tumors. Each bar to the left of a depicted chromosome represents DNA copy
number losses at the corresponding regions in a single tumor; no gains
were detected. Signal intensities were consistent with loss of a single
copy. Red bars represent TgT121p53+/ tumors,
and blue bars represent TgT121p53+/+ tumors.
Chromosomes were identified by DAPI banding. Black bands in the
chromosome diagrams represent the observed DAPI staining pattern. (C)
CP tumor cells from five TgT121 p53+/ mice
were analyzed by propidium iodide staining and fluorescence-activated
cell sorting analysis for DNA content. More than 90% of cells in all
five tumors are diploid.
|
|
| |
DISCUSSION |
High selection for p53 inactivation during tumor evolution.
The data presented here show that there is immense selective pressure
for p53 inactivation during the natural evolution of epithelial tumors
initiated by inactivation of the pRb pathway. Progression from
dysplasia to solid aggressive tumors occurs in 100% of
TgT121p53+/ mice, and all
of these tumors have inactivated p53. Even more indicative of the high
pressure for p53 inactivation was that 38% of
TgT121p53+/+ tumors
inactivated p53 function. Moreover, p53 was nonfunctional in 100%
of type I tumors arising in this background. In
TgT121p53+/ tissue,
where only a single allele loss is required for p53 inactivation, 95%
of the progressed tumors had selectively lost the wild-type p53 allele;
the remaining tumors inactivated p53 by some other mechanism. Tumors
that did not show p53 gene loss, and all type I tumors from
TgT121p53+/+ mice, had lost
p53-dependent activation of p21 transcription. Thus, since p21
activation is immediately downstream of p53, no tumors inactivated
events downstream of p53. Furthermore, real-time PCR analysis indicated
that even p53+/+ tumors had lost at least one p53
allele, suggesting that p53 is the target for inactivation and thus the
rate-limiting step in tumor progression. This indicates that there is
no single linear pathway upstream or downstream of p53 that is
responsible for tumor suppression. Consistent with this observation,
loss of p53 is associated with tumor progression, rather than just
rapid tissue growth, implying that multiple p53-dependent mechanisms
are responsible for overall tumor suppression. While p53-mediated
apoptosis significantly slows dysplastic tissue growth and likely
accounts for the high selective pressure for p53 inactivation, other
p53 functions appear to suppress tumor progression (see model in Fig.
1B).
By CGH analysis, we found that partial or complete loss of a single
copy of chromosome 11 occurred with a high frequency to
facilitate p53
inactivation in
TgT
121p53
+/ tumors. We
considered the possibility that hemizygosity of other
tumor suppressors
located on this chromosome could contribute
to tumor progression.
However, two observations indicate that
this is not the case. First,
some TgT
121p53
+/ tumors
with identical characteristics of progression did not
lose chromosome
11 (Tables
2 and
3). Second, we have induced
morphologically identical
tumors by focal transgenic expression
of full-length SV40 T antigen
(
11,
63). Since p53 is inactivated
by T antigen in this
case, there is no selective pressure for
p53 gene loss in these tumors.
Indeed, by CGH these tumors showed
no loss of chromosome 11 (data not
shown). Thus, p53 inactivation
appears to be the critical event causing
tumor progression
Together with previous studies of p53 LOH in mouse tumor models
(
40), the study presented here indicates that the
selective
pressure for p53 inactivation may vary with the cell type,
likely
reflecting distinct mechanisms of p53 tumor suppression or the
tissue-specific presence of alternate tumor suppression pathways.
Pituitary and thyroid tumors of
Rb
+/p53
+/ mice undergo
Rb loss at a high frequency but rarely lose the
wild-type p53 allele
(
71). In addition to the cell type difference
between
these studies, tumors in the present study were initiated
by
inactivation of all three pRb-related proteins. Thus, the difference
in
selective pressure for p53 inactivation could reflect cell
type or
mechanistic differences. In a study of lymphomas and sarcomas
developing in p53
+/ mice, the frequency of
p53 LOH was 15 to 50%, depending on the
age of tumor-bearing mice
(
64). In that study, p53 expressed
in tumors
without LOH appeared to be functional in that it was
inducible
by irradiation and was able to bind DNA specifically.
The
frequency of p53 LOH in mammary tumors arising in
Wnt-1p53
+/ mice was also about 50%
(
16), and in a chemically induced skin
tumor model, p53
LOH occurred in 64% of carcinomas (
8). A study
of
salivary and mammary tumors arising in the same
MMTV-ras/p53
+/ strain of mice offers a clear
demonstration of tissue-specific
p53 LOH (
29). While 10 of
10 salivary tumors lost the wild-type
p53 allele, all 7 mammary tumors
analyzed retained the locus.
Furthermore, by comparing this study of
mammary tumors (0% p53
LOH) to the study with Wnt-1 mice (50% p53
LOH), it is clear that
the selective pressure for p53 inactivation
appears to be influenced
by the preceding oncogenic event in addition
to the tissue type.
Indeed, the frequency of p53 LOH in mammary tumors
from Brca1
ko/cop53
+/ mice
was 80% (
72). With the exception of the analysis of
p53
+/ lymphomas and sarcomas, these studies did
not address whether
p53 function was intact in the tumors that retained
a wild-type
allele and thus represent a minimum estimate of p53
inactivation.
When evaluating the impact of p53 inactivation on tumor progression, it
is important to examine the morphological evolution
of the tumor and
its correlation with p53 inactivation. As we
observed with
TgT
121p53
+/+ mice, tumors
may progress to multiple grades within the same
genotype. In this
model, inactivation of p53 correlated precisely
with the
development of characteristic carcinomas, while slower-growing
tumors
of distinct morphology retained p53 function. In most previous
studies, the relationship between p53 status and histologic grade
was
not assessed. However, in the Wnt-1 mammary and MMTV-ras mammary
and
salivary tumors described above, p53 inactivation also correlated
with
a higher histologic
grade.
In our studies, the perfect correlation between p53 inactivation and
aggressive CP tumor progression indicates that p53 inactivation
is a
highly selected event and may be sufficient for progression.
In all
other models analyzed thus far, the basis of selective
pressure for p53
inactivation is unknown. In dysplastic CP, p53
is clearly required for
apoptosis, such that cells that inactivate
other tumor suppression
pathways but retain p53 would still undergo
cell death. Thus, if
apoptosis is not an element of p53 tumor
suppression in a given cell
type, a more random distribution of
p53 inactivation may be
observed.
p53 inactivation, genomic instability, and tumor progression.
A common hypothesis for p53 tumor suppression is that p53 prevents
genetic instability. Based on this idea, we tested whether CP tumor
progression after p53 inactivation was facilitated by the accumulation
of chromosomal abnormalities. Aneuploidy is observed in
p53/ hematopoietic cells (6,
21), in p53-deficient Li-Fraumeni syndrome (4) and
mouse (24, 62) fibroblasts, and in mouse thymic lymphomas
induced by p53 deficiency (37, 64). However, TgT121p53+/ CP tumors
were diploid and showed partial or complete loss of a single copy of
chromosome 11 as the only detectable chromosomal aberration. The mouse
p53 gene is located on chromosome 11, and thus, its loss represents the
selected causal event for tumor progression. Hence, in brain epithelium
p53 inactivation can contribute to tumor progression by mechanisms
other than the induction of chromosomal instability.
Although loss of p53 function has been associated with genetic
instability in numerous studies, there has been no direct proof
that
p53 inactivation is sufficient to cause genetic instability
or that
such a function facilitates tumorigenesis in the absence
of other
changes. In some mouse cancer models, tumors that develop
in the
absence of p53 become aneuploid. For example, CGH analysis
of
Wnt-1p53
+/ terminal mammary tumors showed that
tumors with p53 LOH contained
more chromosomal changes (4.3 changes per
tumor) than did those
without p53 LOH (1 change per tumor)
(
16). A similar observation
was made for
p53
+/ thymic lymphomas and sarcomas, where
tumors with p53 LOH contained
an average of 5.6 changes per tumor
compared to an average of
1 change without p53 LOH (
64).
Likewise, mammary and salivary
tumors induced by MMTV-ras were largely
diploid in the presence
of p53 but aneuploid in its absence
(
29). These studies indicate
that loss of p53 function in
tumors often correlates with chromosomal
instability.
Two alternative explanations could account for the apparent
differences in the level of genetic instability observed in
p53-deficient
mouse tumors. One possibility is that these differences
reflect
true tissue-specific differences in p53 tumor suppression
mechanisms.
A second possibility is that p53 deficiency is necessary
but not
sufficient to induce chromosomal instability. Indeed, some
studies
of cultured p53-deficient cells have indicated that p53
inactivation
is not sufficient to drive genetic instability (
18,
49). Furthermore,
in the study of Wnt-1-induced mammary tumors,
karyotype analysis
indicated that several p53-deficient tumors had
nearly diploid
profiles, similar to those of p53 wild-type tumors,
indicating
that p53 inactivation was insufficient to cause aneuploidy
(
16).
This notion is also supported by the observation
that the rapidly
dividing cells of p53-deficient embryos are diploid.
However,
combined deficiencies in Brca1 and p53 induce aneuploidy in
these
cells (
57). Thus, in previous studies, tumors that
harbored
numerous chromosomal changes may have undergone causal
stochastic
mutations in addition to p53 loss. In the previous studies,
tumors
were analyzed after substantial growth, during which such events
could have occurred. Although the
TgT
121p53
+/ CP tumors
were from terminally ill mice, brain tumors are limited
in the extent
of their growth due to adverse effects on the host.
To determine
whether p53 inactivation alone can facilitate genetic
instability and
to rule out the possibility that secondary changes
are responsible,
tumors must be analyzed for chromosomal instability
at early stages
post-p53 loss. It is possible that analysis of
the brain tumors in the
present study represents such an early
assessment of the impact of p53
inactivation.
Our study of p53 deficiency in an evolving tumor showed that genomic
instability resulting from p53 loss did not drive epithelial
tumor
progression. In a recent study, the frequency of epithelial
tumors was
significantly increased in p53-deficient mice that
had been propagated
through several generations in the absence
of telomerase function
(
1). Furthermore, these tumors show
chromosome instability
including end-to-end fusion and aneuploidy.
Since such epithelial
tumors are not observed in the absence of
telomere shortening, these
results indicate that p53 deficiency
is required to propagate
chromosomal instability when driven by
other mechanisms but is itself
not the driving force. This mechanism
also appears to be operative in
mouse lymphomas caused by a truncation
of Brca2 (
33). In
that study, p53 inactivation was shown to
be required to propagate the
Brca2 mutant cells due to severe
chromosomal aberrations caused by the
Brca2
defect.
If not chromosomal instability, what facilitates CP tumor progression?
Although aneuploidy is the most common genetic change
observed in
p53-deficient cells, it is possible that p53 inactivation
results in a
type of genetic instability that is undetectable
by CGH. Point
mutations, small deletions, inversions or amplifications,
balanced
translocations, and microsatellite instability would
all escape
detection by CGH. Currently, there is no convincing
precedent for the
association of p53 inactivation with such changes.
Although some
studies have suggested that p53 affects base (
45)
and
nucleotide (
69) excision repair activities in vitro, the
point mutation frequency is normal in p53-deficient cells, tissues,
and
thymic lymphomas (
44,
55). Future experiments to detect
the incidence of small deletions-amplifications-inversions in
progressing TgT
121p53
+/
tumors will be required to determine whether p53 inactivation
causes
any form of genomic
instability.
Another possibility is that yet other p53 functions suppress tumor
progression. For example, previous studies indicate that
p53 can
regulate the expression of angiogenesis factors. In cotransfection
experiments, p53 can induce transcription of the angiogenesis
inhibitor
thrombospondin 1 (
12) and other potential angiogenesis
inhibitors such as maspin (
75) and BAI1 (
59)
through p53-specific
DNA binding elements. In addition, the
angiogenesis activator
vascular endothelial growth factor is
induced in p53-deficient
cells by an unknown mechanism (
5,
51,
66,
74). Since
we show here that angiogenesis is a feature of CP
tumor progression
associated with p53 inactivation, we are currently
exploring the
possibility that p53 inactivation plays a direct role in
modulating
the expression of angiogenesis factors, thus facilitating
tumor
progression.
In summary, p53 appears to suppress tumorigenesis in a single tissue by
multiple mechanisms. When tumor growth is suppressed
by p53-dependent
apoptosis as in the CP tumors, there is a high
selective pressure
for p53 inactivation. Inactivation of p53 in
turn facilitates tumor
progression by a mechanism that, surprisingly,
does not include
the induction of chromosomal instability. These
studies underscore the
complexity of p53 tumor suppression and
the need to understand the
mechanisms in the context of natural
selective pressures occurring
during tumor evolution in
vivo.
| |
ACKNOWLEDGMENTS |
We thank Lynda Chin and colleagues (Harvard University) for
supplying real-time PCR conditions and Hua Wu (T.V. lab) for assistance in developing the assay. Robert Flandermyer (UCSF) and Le Zhang (T.V.
lab) provided excellent technical assistance. We acknowledge the UNC
Lineberger Comprehensive Cancer Center Histology Core for processing
tissues used in this study and the UNC Division of Laboratory Animals
for excellent animal care.
This work was supported by NCI grants 1 R01 CA46283 and 5 U01 CA84314.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry and Biophysics, University of North Carolina at Chapel
Hill, Chapel Hill, NC 27599. Phone: (919) 962-2145. Fax: (919)
962-4296. E-mail: tvdlab{at}med.unc.edu.
| |
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Molecular and Cellular Biology, September 2001, p. 6017-6030, Vol. 21, No. 17
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.17.6017-6030.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
