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1
* Program in Cell and Molecular Biology and
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA
1Correspondence: Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA. E-mail: jrosen{at}bcm.tmc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: mutant p53 • apoptosis • aneuploidy • centrosome abnormalities
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, responding to DNA damage or checkpoint failure by
either arresting cell cycle progression to facilitate DNA repair or
initiating an apoptotic pathway to remove damaged cells. Wild-type p53
is critical for the maintenance of genomic stability: aberrant ploidy,
gene amplification, increased recombination, and centrosomal
dysregulation have all been observed in cells lacking p53
(2)
. p63 and p73, two other proposed members of the p53
family, may compensate for wild-type p53 under some circumstances
(3)
.
Tumorigenesis is a multistage process involving multiple
genetic aberrations (4)
. p53 is the most commonly mutated
gene in human cancers, with 
40% of tumors displaying some genetic
alteration (5)
. Most p53 alterations are missense
mutations (6)
that may be accompanied by loss of the
remaining wild-type allele. The p53 175 Arg-His (R-H) mutation makes up
5% of all p53 mutations found in breast cancer and is the third
most frequent p53 mutation (ref 6
; and see web site at
http://perso.curie.fr/Thierry.Soussi/p53_databaseWh.htm).
Specific p53 mutations, including those at codon 175, have been
associated with poor prognosis in breast cancer patients and also with
primary resistance to chemotherapy (7)
.
Mutations in p53 may result in loss of wild-type function or generation
of dominant-negative and gain-of-function mutants (e.g., ref
8
). The 175R-H p53 protein is a dominant-negative mutant
that can interact with wild-type p53, but is no longer capable of
specific DNA binding (6
, 9
10
11)
and so loses many of the
direct transcriptional regulatory capabilities of wild-type p53. It
also appears to confer novel functions, indicating that it is a
gain-of-function mutant (see, for example, ref 12
).
However, these studies have been performed in either fibroblasts or
nonmammary epithelial cell lines usually containing multiple genetic
aberrations in addition to p53 loss or mutation. To better understand
the role of mutant p53 in mammary tumorigenesis, it is critical to use
the appropriate cell system (mammary epithelial cells) containing a
minimal number of other genetic aberrations and no competing wild-type
(or other forms of mutant) p53. A novel cell system meeting these
criteria has been developed and was used for the current experiments.
One of the gain-of-function phenotypes previously reported for the
172R-H mutant relates to centrosome number and ploidy. Primary skin
tumors from mice bearing a skin-targeted p53 172R-H mutation (murine
amino acid 172 is equivalent to human 175) display a much greater
degree of aberrant centrosomal duplication than do tumors from p53-null
mice (13)
. Normal nondividing cells have one centrosome,
and dividing cells two, which form the poles of the spindle
(14)
. Centrosomal amplification is implicated in two
processes adversely affecting prognosis in cancer patients:
1) loss of cell polarity and tissue organization, and
2) increased occurrence of multipolar mitoses, which
promotes unequal genomic segregation (15)
. A recent study
found that centrosomes in high-grade breast adenocarcinoma cells are
larger and more numerous than those from normal breast specimens
(14)
. Centrosome amplification induces chromosomal
instability (16)
, which is associated with tumor
aneuploidy (17)
.
A transgenic mouse model has previously been generated to explore the
role of the murine p53 172R-H protein in mammary tumorigenesis
(18)
. A genomic minigene construct containing this
mutation was targeted specifically to the murine mammary gland using a
whey acidic protein (WAP) promoter (19)
. Transgene
expression resulted in very few spontaneous tumors, but predisposed
mice to the development of mammary tumors once some initiating event
(e.g., carcinogen treatment or oncogene coexpression in the mammary
gland) had taken place (12
, 18
, 20
, 21)
. These tumors were
frequently aneuploid (12
, 18
, 20
, 21)
.
Studies were initiated using the unique p53-null mammary epithelial cell line in order to dissect the mechanism(s) by which the p53 172R-H mutant promotes aneuploidy and tumorigenesis in the mouse mammary gland. The results of these studies suggest that the p53 172R-H mutant may play a dual role in promoting mammary tumorigenesis by influencing genomic stability at the centrosome level as well as reducing both basal and DNA damage-induced apoptosis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. This line
was derived from p53-knockout mice generated by Dr. Larry Donehower
(Baylor College of Medicine) that were of mixed (129/Sv x
C57Bl/6) genetic background (23)
. Cells were maintained in
DMEM:F12 (Life Technologies, Inc., Grand Island, N.Y.) containing 2%
ABS, 5 µg/ml gentamicin, 10 µg/ml insulin, and 5 ng/ml EGF. Stable
cell lines were selected using this media plus 650 µg/ml G418. All
cells were used at the lowest possible passage numbers, typically 7–11
in transient transfection experiments and 12–16 in stable cell
experiments.
Plasmids
Transient transfections
The plasmid pBL120 contains a CMV-p53 172R-H minigene construct
in pBluescript SK (Stratagene, San Diego, Calif.), while the plasmid
pBL106B contains the wild-type p53 construct.
Stable transfections
The retroviral plasmid pL53NRNL (24)
, containing
p53 172R-H under the control of the Mo-MuLV LTR and a neo
selectable marker, was used in the generation of stable cell lines.
Control stables were generated using a similar plasmid lacking the p53
construct.
Transient adenoviral transfection
Cells were transiently transfected using a replication-deficient
adenovirus-mediated system. The adenovirus (25)
was
obtained from Dr. Nancy Weigel (Baylor College of Medicine). Cells were
at 50–80% confluency when infected. At the time of infection, media
were removed from the plates (10 cm dishes were used in these
experiments) and replaced with 2 ml of serum-free media.
Adenovirus–DNA complexes were prepared by incubating adenovirus with
plasmid DNA for 30 min at room temperature in the dark, followed by a
30 min incubation (as above) with polylysine at a molar concentration
equivalent to 125-fold the molar plasmid DNA concentration.
Adenovirus/DNA/lysine complexes were immediately added to the
serum-free media on the target cells and allowed to incubate for 2 h at 37°C. The viral solution was then removed and replaced with a
normal volume of growth medium. Cells were harvested for analysis at
the indicated number of days after transfection. Dishes (10 cm) were
transfected with a total of 1 µg of DNA each, which included
bax reporter plasmid (obtained from Dr. Moshe Oren, Weizmann
Institute, Israel) or p53 construct and internal control plasmids where
appropriate.
Stable transfection
293T retroviral packaging cells were transfected with pCL-Eco
(26)
and the appropriate retroviral plasmid using FuGene
(Boehringer Mannheim, Mannheim, Germany). After 2 days, the
virus-containing media were harvested and filtered (0.45 µm filter).
Polybrene (Sigma, St. Louis, Mo.) was added to a final concentration of
5 mg/ml and the media were placed on target cells. The plates of cells
were spun in a clinical centrifuge for 3 x 10 min at 1800 RPM,
rotating the plates 60° after each spin to ensure complete coverage
of the target cell plate. The medium was then removed and replaced with
normal growth medium. Two days after infection, the medium was replaced
with selective medium.
Western blotting
Western blots were performed in accordance with standard
protocols (27)
. Membranes were blocked in 5% nonfat dry
milk (NFDM)/TBST [Blotto] for 1 h, then incubated in either a
1:750 dilution of anti-p53 antibody (Santa Cruz, Santa Cruz, Calif.;
sc-6243) or a 1:200 dilution of anti-p73 antibody (Novus Biologicals,
NB 200–102) for 1 h at room temperature. After washing, the
membrane was incubated in a 1:2000 dilution (3% NFDM) of anti-rabbit
(or anti-sheep, respectively) biotinylated secondary antibody, followed
by incubation in a 1:2500 dilution (3% NFDM) of horseradish
peroxidase/streptavidin solution, treatment with ECL reagents, and
exposure to film. The pan-ERK mAb used to normalize for protein content
was obtained from Transduction Laboratories (Lexington, Ky.).
Immunocytochemistry
Cells to be immunostained were grown on lysine-coated circular
coverslips and fixed in methanol at -20°C for 10 min, then washed
and incubated in a solution of Blotto containing 1:50 polyclonal
anti-p53 antibody (Santa Cruz, catalog # sc-6243) and 1:400 monoclonal
anti-gamma-tubulin antibody (Sigma, catalog # T6557) for 3 h at
37°C. After washing with Blotto, they were further incubated with
1:500 Texas red-conjugated anti-rabbit secondary antibody and 1:200
FITC-conjugated anti-mouse secondary antibody (both from Molecular
Probes, Eugene, Oreg.) in Blotto in the dark at 37°C for 1 h.
The coverslips were then washed in TBST and mounted in DAPI solution
(Vector Labs, Burlingame, Calif.; catalog # H-1200). Cells for
p53/spindle double immunostaining were permeabilized in 0.5% Triton-X
in PEM/PEG (80 mM Pipes, 1 mM EGTA, 1 mM MgCl2,
(pH 6.9) + 4% [8000 MW] PEG) and then fixed in 3.7% formaldehyde
(all solutions in PEM/PEG) at room temperature for 15 min. After
washing in PEM/PEG, coverslips were blocked in 5% NGS and incubated in
1:15 (in 5% NGS) TU27B mouse monoclonal antibody (gift of Dr. B. R. Brinkley, Baylor College of Medicine) and 1:50 p53 antibody as above
for 1 h at 37°C. After washing in PEM/PEG, the cells were
incubated in 1:100 (in 5% NGS) FITC-conjugated goat anti-mouse
secondary antibody and 1:500 Texas red-conjugated goat anti-rabbit
secondary antibody for 1 h at 37°C, washed, and mounted as
above.
Centrosome analysis
Images from stained coverslips were captured with a Sony 3CCD
color video camera attached to a BX-50 Olympus microscope with Adobe
Photoshop software. All images were viewed using a 100x oil immersion
lens coupled with a 10x objective. The number of centrosomes in cells
with red nuclei (i.e., in cells expressing the transfected p53) was
observed and recorded. Centrosome numbers in apoptotic cells were
difficult to determine and therefore were not included in the analysis.
The number of centrosomes in nonpositive cells from the same coverslips
was recorded as an internal negative control. At least 1500 cells for
each transfected population were counted at various cell passage
numbers to ensure no passage number effect on centrosome results.
Statistical analysis (by t test) was performed using the JMP
Statistical Visualization software from SAS Institute, Inc (Cary, N.C.)
DNA damage protocols
Mitomycin C (MMC; Sigma) was applied to cells in media for the
indicated period of time at the indicated concentrations. UV
irradiation of cells was accomplished using a Stratalinker
(Stratagene). A Gammacell 1000 Irradiator was used for exposure of
cells to ionizing radiation. Doses of DNA-damaging agents and exposure
times were chosen in accordance with accepted ranges in the literature
(see, for example, ref 28
).
Caspase assays
Cells were seeded into 96-well dishes at 20,000 cells per
well and treated as appropriate for each experiment. Each experimental
point represents the average of at least four wells. At the time of
assay, the media were aspirated and 30 µl of lysis buffer (10 mM
Tris-Cl pH 7.5, 10 mM NaH2P04/NaHPO4 pH 7.5, 130
mM NaCl, 1% Triton-X 100, 10 mM NaPPi) was added to each well. The
plate was then incubated for 30 min on ice. After the incubation, a
mixture of 3.6 µl substrate (Ac-DEVD-AMC caspase-3 fluorogenic
substrate (PharMingen, San Diego, Calif.) and 216 µl of PAB (20 mM
HEPES pH 7.5, 10% glycerol, 2 mM DTT) was added to each well and the
plate was incubated in darkness at 37°C for 3–6 h. The quantity of
AMC liberated after cleavage of the substrate by activated caspase-3
was measured using a spectrofluorometer (excitation wavelength 380,
emission wavelength 430–460 nM). The protein content of each well was
determined after the assay, and the plate readout was normalized
accordingly to account for cell number variability and time of final
incubation. The results are presented as the relative caspase
activity/µg protein/h.
Colony assays
After irradiation, 500 cells were plated on each 10 cm dish and
allowed to form colonies. After 10–14 days, colonies were stained with
Coomassie blue; colony numbers (‘colony’ defined as >50 cells) were
counted and statistical analysis was performed as above.
Luciferase assays
Cell extracts were made in 1x CAT assay lysis buffer
(Boehringer Mannheim). Luciferase assays were performed as per
manufacturer’s instructions (PharMingen) using a Monolight 2010
luminometer (Analytical Luminescence Laboratory, Ann Arbor,
Mich.). Internal control (ß-gal activity) assays were
performed in 96-well plates. Twenty microliters of extract/well was
incubated at 37°C in a solution containing magnesium (0.1M
MgCl2, 4.5M ß-ME, 1.5 µl/well), 0.1M
NaHPO4 pH 7.5 (95.5 µl/well), and ONPG (4 mg/ml
in 0.1 M NaHPO4 pH 7.5, 33 µl/well) until a
faint yellow color appeared (10–60 min). Plates were read at 420 nm on
a spectrophotometer and units ß-gal activity/µl protein were
calculated.
Chromosome staining
Cells were resuspended in 1 ml of media, to which 10 ml of 0.8%
sodium citrate solution was added. The cells were then incubated at
room temperature for 20 min, centrifuged, and resuspended in 1 ml of
sodium citrate solution. Five milliliters of fresh 3:1 methanol:acetic
acid solution (fixative) was added dropwise while gently vortexing. The
cells were centrifuged and resuspended in 5 ml of fixative. Cells were
dropped from a height of 4 inches onto slides slanted at a 30° angle
and immediately followed with a few drops of fixative. After drying,
cells were stained with a 1:1000 dilution of DAPI mount
solution/phosphate-buffered saline for 1–2 min, rinsed, and mounted
using solution lacking DAPI (Vector Labs catalog # H-1000). Chromosomes
were imaged as described above. At least 50 metaphase spreads were
counted per cell genotype.
SCE analysis
Confluent cells were split 1:5. Twenty-four hours after passage,
3 µg/ml BrdU was added to the medium and the cells were allowed to
grow for an additional 48 h. The cells were refed with fresh
regular medium for 3 h, then treated with 10 µg/ml demecolcine
for 15 min to stop cell growth. Cells were washed, trypsinized, and
centrifuged. The cell pellets were resuspended, and 10 ml prewarmed
(37°C) 0.075M KCl solution was added dropwise while vortexing gently.
One milliliter of fixative (see above) was then added and the cells
were centrifuged. After aspiration of the media, the cells were
resuspended in 10 ml of fixative and kept at room temperature for 30
min. The fixative was changed several times, and the cells were finally
resuspended in 0.5 ml of fixative. Cells were dropped onto slides as
above and dried. Slides were then incubated in 0.1 mg/ml acridine
orange (Sigma) solution for 5 min and rinsed well. The slides were
mounted in 2.8% sodium phosphate anhydrous dibasic solution, pH 11.0,
and visualized using a green (FITC) filter. For analysis, the number of
breaks and the number of chromosomes were counted for each of 50
metaphase spreads per genotype, and the average break-per-chromosome
ratio for each genotype was calculated.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and
expression of the 172R-H p53 mutant in murine skin (13)
result in aberrantly large numbers of centrosomes and aneuploidy. As
aneuploidy is characteristic of mammary tumors arising in the WAP-p53
172R-H mice (12
, 18
, 20
, 21)
, studies were undertaken to
examine the effect of expression of this mutant p53 on centrosome
number in vitro. p53-null mouse MECs were transiently transfected with constructs encoding either wild-type p53 or the p53 172R-H mutant. Lipid-mediated transfection methods worked poorly with these cells, so an efficient adenovirus-mediated method (see Materials and Methods) was used. This resulted in a 20–40% transfection efficiency. Expression of wild-type or mutant p53 in transfected cells was initially verified by both immunocytochemistry and Western blotting (data not shown), and p53 expression could be detected in these cells for up to 5 days.
To determine the effect of transfected p53 on centrosome number in these cells with time, cells were transfected and populations grown on coverslips were fixed for analysis 2 h after transfection (day 0) and each day for the next 5 days (days 1–5). Fixed cells were then simultaneously immunostained for p53 and the centrosome component gamma-tubulin (see Materials and Methods). The double immunocytochemistry procedure permitted the identification of transfected cells for the analysis of centrosome number. The polyclonal p53 antibody used (see Materials and Methods) recognizes both wild-type and mutant forms of p53, and was therefore used for the analysis of both experimental groups.
Representative examples of centrosome and p53 staining in p53-null MECs
transfected with either wild-type (Fig. 1A
) or mutant (Fig. 1B
) p53 are shown in Fig. 1
.
Statistical analysis of centrosome numbers in cells transfected with
wild-type (Fig. 1C
) or mutant (Fig. 1D
) p53 as a
function of the day after transfection was performed as described in
Materials and Methods, and revealed that there were significantly more
cells with supernumerary centrosomes in the population transfected with
mutant p53 by day 3. (Fig. 1
, compare the day 3–5 bars for the ‘2+’
category between panels C and D). The percentage
of mutant p53-transfected cells with aberrant centrosome numbers
increased each day after transfection, reaching a maximum of over 70%
of transfected cells by day 5. A concomitant decrease in the numbers of
cells with one or two centrosomes was observed. A small percentage of
cells above background contain more than two centrosomes in the
wild-type p53 transfectants (normal background levels are defined as
10% of cells; B. R. Brinkley, personal communication). However,
the percentage of these wild-type p53-transfected cells with abnormal
centrosome numbers plateaued at 20–25% by day 2 and remained
relatively constant through day 5.
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Centrosome numbers for nontransfected (p53-null) cells on the same
coverslips at the same time points were also assessed and analyzed as
an internal control (Fig. 1E
). Nontransfected cells from
wild-type and mutant p53-transfected plates exhibited very similar
centrosome number profiles: 20% with one centrosome, 60% with
two centrosomes, and 20% with more than two centrosomes across all
time points. This did not appear to vary significantly with cell
passage number. These results essentially mirror those for wild-type
p53 expressing cells depicted in Fig. 1C
and indicate that
the introduction of the gain-of-function p53 mutant into p53 null MECs
results in centrosome abnormalities. Attempts were made to directly
examine the effect of transfected wild-type or mutant p53 on cell
ploidy by flow cytometry, but it was difficult to distinguish between
transfected and nontransfected cells by this method. However, overall
cell cycle profiles of populations transfected with mutant p53 and
wild-type p53 were similar over time, even at time points at which
centrosome number differences in the transfected populations were
significantly different (data not shown).
Functional supernumerary centrosomes in cells transfected with the
p53 172R-H mutant
Although it is possible for cells with more than two centrosomes
to form a bipolar spindle (30
; K. Murphy, unpublished
observations), one predicted consequence of the multiple centrosomes
seen with this p53 mutant is the formation of multipolar spindles
leading to aberrant genome segregation at mitosis. As illustrated in
Fig. 2
, a small percentage of cells containing multipolar spindles was
observed in these experiments. Double immunocytochemistry experiments
were performed with simultaneous staining for p53 and 
-tubulin, a
component of the mitotic spindle. These experiments demonstrated that
cells transfected with mutant p53 often contained multipolar spindles
by the fifth day after transfection (Fig. 2A/B, C/D, and
E/F
) whereas only a small percentage of untransfected
control (i.e., p53-null) cells had abnormal spindles at the same time
point (Fig. 2G/H
).
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Moderately abnormal centrosome numbers in cells stably expressing
p53 172R-H
To determine whether the abnormal centrosome numbers induced by
expression of the p53 172R-H mutant persisted for longer than 5 days,
cell lines stably expressing p53 172R-H or a control plasmid lacking
p53 were generated as described in Materials and Methods, and their p53
status was confirmed by Western blotting (data not shown). No attempt
was made to generate cells stably expressing wild-type p53, as stable
overexpression of wild-type p53 leads to growth repression and/or
apoptosis.
Mutant p53 was expressed at high levels in the stable cells (Fig. 3A
) whereas, as expected, the p53-null controls (Fig. 3B
) displayed no detectable staining. Analysis of centrosome
number in the mutant p53 stably transfected cells and controls (Fig. 3C
) indicated that there were significantly more control
cells containing one centrosome, and p53 172R-H cells with more than
two centrosomes. Multipolar spindles were also observed to coexist with
multiple centrosomes in some of the mutant p53 stable cells (Fig. 3D
). However, based on the results from the transient
transfection experiments, these differences in centrosome number were
unexpectedly small. This raised the issue of the fate of the cells
having multiple centrosomes and multipolar spindles after transient
transfection with p53 172R-H.
|
As there were only moderately elevated numbers of stably transfected
cells expressing the p53 mutant containing supernumerary centrosomes,
it was hypothesized that many of the cells with multiple centrosomes
nucleating multipolar spindles were undergoing apoptosis
(30)
. To test this hypothesis, transient transfections
were used to measure the level of activity of an integral apoptotic
caspase, caspase-3. Some apoptosis was induced by the transfection
process, but levels were equivalent between the cell types at day 1.
Apoptosis was significantly higher (P<0.001) in cells
transfected with mutant p53 than in mock-transfected cells by day 5
post-transfection (Fig. 4
), and the difference approached statistical significance
(P<0.067) by day 4. Caspase activity did not change
significantly between day 1 and day 5 in the mock-transfected cells.
These results were independently confirmed through use of a fluorescent
TUNEL assay combined with p53 immunostaining, which indicated that p53
staining frequently colocalized with TUNEL staining in day 4 and day 5
mutant p53-transfected cells (data not shown). As this coincides with
the time points in which peak numbers of multicentrosomal mutant
p53-positive cells were observed, these results suggest that the
majority of these multicentrosomal cells were undergoing apoptosis,
accounting for the observation of relatively few multicentrosomal cells
in stably transfected populations.
|
Lower basal levels of apoptosis and increased resistance to DNA
damage in p53 172R-H-expressing stable cell lines
Wild-type p53 is believed to play a role in DNA repair processes
(31
, 32)
and p53 loss or mutation is known to have
deleterious effects on these processes (33
34
35)
, although
p53-deficient cells do not exhibit an increased mutation frequency
after exposure to DNA-damaging agents (36)
. To test
whether DNA repair was influenced by the presence of the p53 172R-H
mutant in the stably transfected p53-null MECs, cells were treated with
DNA-damaging agents and cell viability after treatment was ascertained
by caspase-3 assays and/or colony assays as described in Materials and
Methods.
Cells were treated with varying doses of mitomycin C (a DNA
cross-linking agent), UV (which induces formation of cyclobutane
dimers), or ionizing radiation (which induces double-strand DNA breaks)
and cell survival was assessed at different subsequent time points. As
shown in Fig. 5
, treatment with all three of these DNA-damaging regimens yielded
similar, if unexpected, results. Control cells treated with increasing
concentrations of MMC contained significantly more caspase-3 activity
(Fig. 5A
) than correspondingly treated cells stably
expressing the p53 172R-H mutant, indicating there was more apoptosis
in control cells after MMC treatment. Treatment with UV radiation (Fig. 5B
) appeared to exert a similar effect, especially at higher
(30 or 50 J/cm2) doses. Control cells treated
with ionizing radiation (IR) also appear to die preferentially at all
doses tested (Fig. 5C
). The differences shown were
statistically significant (P=0.05) for all three agents and
at all concentrations/doses tested, including basal levels. Colony
survival assays were also performed for the latter experiment. Control
or p53 172R-H-expressing stable cells were exposed to either 5 or 8 Gy
of ionizing radiation and allowed to form colonies as described in
Materials and Methods. Colony counts indicated that the mutant-p53
expressing stables preferentially survived and formed more colonies at
both doses tested (Fig. 5D
). Figure 5A-C
also
shows that the p53 172R-H mutant can suppress basal apoptosis, as there
is substantially more apoptosis in control than in mutant
p53-expressing cells even without treatment with DNA damaging agents.
|
One potential explanation for these results is that other members of
the p53 family are inducing apoptosis in the p53-null control cells,
but that the mutant p53 protein exerts a dominant negative effect on
that alternative proapoptotic pathway when stably expressed. It has
previously been reported (37)
that coexpression of the
human p53 175R-H mutant inhibits the transcriptional activity of p73
on the bax promoter, thereby reducing the ability of
exogenous p73 to promote apoptosis in p53-null H1299 cells. To
examine the role of potential p73-mediated trans-activation
of bax in apoptosis, a bax-luciferase reporter
was transfected into cells stably expressing mutant p53 or control
cells, which were left untreated or exposed to 30
J/cm2 of UV irradiation to induce DNA damage and
apoptosis. After 24 h, cells were harvested and luciferase
activity was quantitated and normalized to an internal transfection
control. These experiments indicated that the bax reporter
is induced above basal levels by UV irradiation in both cell types and
that there is significantly more reporter activity in both untreated
and irradiated control cells than in their mutant p53-expressing
counterparts (Fig. 6B
). Slightly elevated levels of p73 protein were observed in
extracts of control cells relative to those in cells expressing mutant
p53 in two separate experiments, although p73 levels did not appear to
increase after irradiation (Fig. 6A
). Endogenous p73 levels
were extremely low in these p53-null MECs.
|
Increased chromosome number, but no change in sister chromatid
exchange in cells stably expressing p53 172R-H
Mammary tumors arising in mice carrying the p53 172R-H transgene
are frequently aneuploid (12
, 18
, 20
, 21)
. To assess the
effect of this p53 mutant on ploidy in vitro, metaphase
spreads (Fig. 7A
) were prepared from p53 172R-H stably transfected or
control cells as described in Materials and Methods, and chromosome
numbers were analyzed. Both the control and p53 172R-H transfected cell
lines averaged more than the normal 40 chromosomes per mouse cell.
However, at equivalent passage numbers, there are significantly
(P<0.05) more chromosomes (Fig. 7B
) in cells
stably transfected with the p53 172R-H mutant (mean=80, range=50–206)
than in control (i.e., p53-null) cells (mean=66, range=37–142).
|
One mechanism thought to promote abnormalities in DNA ploidy is
uncontrolled sister chromatid exchange (SCE), which is influenced by
p53 loss or mutation (38
39
40)
. Specifically, it has been
suggested that the p53 172R-H mutant protein may improperly promote
illegitimate homologous recombination (41
, 42)
.
Accordingly, the incidence of SCE (Fig. 7C
) in p53 172R-H
and control cell lines was assessed as described in Materials and
Methods. Surprisingly, no significant difference in the average number
of SCEs per chromosome was detected between the two stable cell lines
(Fig. 7D
). Average frequencies of 0.270 ± 0.015 and
0.292 ± .020 SCE/chromosome were observed for the p53 172R-H
stably transfected and control cells, respectively. Of course, this
does not preclude early changes in SCE or the occurrence of other
compensatory mutations by the time of analysis of these stably
transfected cells.
|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 18
, 20
, 21)
have shown that
expression of the p53 172R-H mutant predisposes the mouse mammary gland
to the development of aneuploid mammary tumors, but these studies did
not address the specific mechanism(s) by which this occurs. The present
studies performed in both transiently and stably transfected p53-null
MECs suggest that this p53 mutant plays a dual role in mammary
tumorigenesis, both negatively influencing apoptotic pathways and
promoting the development of aneuploid cells.
Reduction in both basal and DNA damage-induced apoptosis
It was hypothesized that p53-null MECs stably overexpressing the
p53 172R-H mutant would be defective in DNA repair and therefore more
sensitive to DNA-damaging agents. However, these cells were in fact
more resistant to DNA damage induced by MMC, UV, or IR over a range of
doses. This suggests that, at least in vitro, this p53
mutant may promote cell survival after DNA damage. This p53 mutant also
appeared to suppress basal apoptosis in the absence of DNA damage (Fig. 5A-C
). It has been argued that p53 status does not
significantly affect cellular sensitivity to DNA-damaging agents, in
contrast to what is seen in short-term DNA damage assays
(43)
, but to date no studies have directly compared the
effects of DNA-damaging agents on survival in p53-null cells vs.
p53-null cells expressing this mutant p53 isoform. The short-term and
‘gold standard’ (43)
colony survival assay results here
agree, which suggests that p53 172R-H may indeed be suppressing
apoptosis in this p53-null MEC system.
How the 172R-H p53 mutant might suppress apoptosis is not clear. This
p53 mutant may exert a dominant-negative effect on p63 or p73, other
proposed members of the p53 tumor suppressor family that can
trans-activate proapoptotic p53 target genes such as
bax (3)
. Coexpression of the human p53 175R-H
mutant is known to reduce the transcriptional activity of p73 on the
bax promoter, thereby affecting the ability of ectopically
expressed p73 to promote apoptosis in p53-null H1299 cells
(37)
. In accordance with these results, both basal and DNA
damage-induced trans-activation of a
bax-luciferase construct was significantly diminished in
MECs stably expressing the p53 172R-H mutant relative to levels seen in
control p53-null cells. This has not been directly proved to result
from p73 inactivation by the p53 mutant, but given the concordance of
these results with those of Di Como et al., it is likely that the p53
172R-H mutant in this system is reducing apoptosis, at least in part,
by blocking the activity of endogenous cellular p73. An increase in
cellular p73 levels was not detected after DNA damage induced by UV
irradiation, in keeping with the results of Fang et al., who used
mitomycin C, doxorubicin, and actinomycin D to induce DNA damage
(44)
, but in contrast to those of Gong et al.
(45)
, who induced DNA damage using cisplatin. This
suggests that there is DNA-damaging agent and possibly cell type
specificity to p73 stabilization after DNA damage.
Chromosomal consequences of p53 172R-H expression
Mammary tumors arising in mice carrying the p53 172R-H transgene
are frequently aneuploid (12
, 18
, 20
, 21)
, and exogenous
expression of this p53 mutant in p53-null fibroblasts results in
hyperdiploidy (46)
. In these experiments, both the control
and p53 172R-H stable cell lines averaged more than the normal 40
chromosomes per cell, probably because of the relatively high passage
numbers required to generate stable cell lines and the inherent
tendency of p53-null cell lines to be genomically unstable. However, at
equivalent passage numbers, there are significantly more chromosomes
(80 vs. 66) in cells stably transfected with the p53 172R-H mutant than
in control (p53-null) cells, suggesting that the p53 mutant does
exhibit a gain-of-function effect on chromosome number in this system.
It is believed that induction of SCEs represents the interchange of DNA
replication products at homologous loci, involving DNA breakage and
reunion (47
, 48)
. Functional wild-type p53 interacts
directly with RAD51 and its bacterial homologue RecA, key factors
involved with homologous chromosomal recombination, and inhibits their
function when appropriate (38)
. Defects in p53-mediated
control of homologous recombination caused by mutation
(38)
or loss (40)
of p53 may result in
inappropriate chromosomal rearrangements and genomic instability.
Previous experiments (41
, 42)
suggested that stable
expression of the human p53 175R-H mutant increases the incidence of
both spontaneous and radiation-induced homologous recombination,
although meiotic recombination is unaffected by p53 loss
(49)
. This suggested that more SCE might occur in MECs
stably expressing the p53 172R-H mutant than in the control p53-null
MECs, but SCE levels in these cells were in fact equivalent. The most
likely reason for the disparity between these results and those
reported previously is the lack of wild-type p53 in the MECs; in the
previous experiments, the mutant p53 was probably functioning as a
dominant negative. The p53 172R-H mutant does not appear to promote SCE
beyond levels resulting from the absence of wild-type p53. However,
both the control and mutant p53 stably transfected MECs appear to
exhibit a mildly elevated SCE frequency relative to what is seen in
normal mouse cells [0.185 SCE/chromosome (50)
vs. 0.270
and 0.292 SCE/chromosome in the p53 172R-H stably transfected and
control cells, respectively]. By comparison, SCE frequency is
1.94 ± 0.07 SCE/chromosome in cells derived from
Xrcc1-/-/p53-/-
mouse embryos (51)
.
Centrosome amplification in mouse MECs expressing p53 172R-H
Transient expression of the p53 172R-H mutant protein in p53-null
mouse MECs resulted in progressively increasing numbers of centrosomes
per cell, reaching a maximum of over 70% of transfected cells with
supernumerary centrosomes by day 5 post-transfection. By contrast, in
transient transfections using wild-type p53, the percentages of cells
with supernumerary centrosomes plateaued at 20–25% by day 2
post-transfection, and similar results were seen with the internal
control untransfected cells from the same coverslips at the same time
points. Any cell containing more than two centrosomes is by definition
aberrant (14)
. Loss of p53 in mouse embryo fibroblasts is
known to result in extensive centrosome amplification (31–55% of
cells with multiple centrosomes) by passage 2 (29)
. The
relatively low background percentages of control p53-null MECs with
aberrant centrosome numbers even after 8–12 passages highlight a
significant difference between fibroblasts and MECs. There are also
species-related differences within individual cell types (see ref
52
).
A variety of centrosomal kinases (e.g., STK15/BTAK; ref
53
), phosphatases and other proteins (for example, the
cdk2/cyclin E complex; ref 54
, 55
) have been implicated in
centrosome duplication (for a review, see ref 30
).
STK15/BTAK is amplified and overexpressed in 12% of
human breast cancers, and expression of this kinase leads to centrosome
amplification, chromosomal instability, and transformation in
vitro (53)
. Cyclin E is an important regulator of
S-phase entry in the mammalian cell cycle and is often overexpressed or
deregulated in tumors, leading to centrosome amplification and/or
genomic instability (54
, 55)
. Cyclin E has been localized
to the centrosome (55)
, as have wild-type p53
(56)
and STK15/BTAK (53)
. It may
be that the p53 172R-H mutant deregulates centrosome amplification by
somehow influencing the activity of either STK15/BTAK or
cyclin E-cdk2 at the centrosome.
A moderate percentage of p53-null MECs stably expressing the p53 172R-H
mutant also contained more than two centrosomes. Functional
supernumerary centrosomes might be expected to nucleate multipolar
spindles, resulting in aberrant genome segregation. As expected, this
phenomenon was observed in both the transient (Fig. 2)
and stable
transfectants (Fig. 3)
.
Brinkley and Goepfert have proposed a model to account for the
continuing presence of supernumerary centrosomes in established breast
tumors (30)
. In this model, centrosome amplification is
initiated at a very early stage in cell transformation, followed by
clonal selection of viable tumor progenitor cells. Major mitotic
spindle aberrations resulting from the presence of multiple centrosomes
would therefore be characteristic of early stages of tumorigenesis, and
would result in significant loss or gain of chromosomes. Most of the
initial progeny cells would then be removed by apoptosis, leaving an
occasional tumor progenitor cell (30)
. The results
presented in this study are consistent with this model and demonstrate
that transient expression of the p53 172R-H mutant results in dramatic
centrosome amplification and mitotic spindle aberrations followed by
apoptosis. Stable expression of this p53 mutant is characterized by a
much smaller percentage of cells with supernumerary centrosomes. Fully
transformed cells with supernumerary centrosomes may survive and
assemble bipolar mitotic spindles (30
; K. Murphy,
unpublished observations) by an unknown mechanism. It appears that in
some cases cells manage to ‘cluster’ multiple centrosomes into only
two spindle poles (57
, 58)
.
In summary, a novel p53-null mouse mammary epithelial cell model has been used in studies investigating the role of p53 in mammary tumorigenesis in vitro. The p53 172R-H mutant plays a dual role in promoting mammary tumorigenesis. Expression of this mutant leads to centrosome amplification, predominantly followed by multipolar mitotic division and apoptosis. On rare occasions, these cells that have sustained mutant p53-related genetic abnormalities survive, and then may nucleate a pretumorigenic population. The presence of this p53 mutant reduced both basal and DNA damage-induced apoptosis in stably transfected cell lines. The combination of these two pathways presents a scenario in which cells are both more likely to sustain genetic aberrations and more likely to survive the presence of these aberrations, thus providing a plausible explanation for the frequency with which this p53 mutant is found in human breast cancers.
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ACKNOWLEDGMENTS |
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Received for publication March 3, 2000.
Revision received May 17, 2000.
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REFERENCES |
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K. Kawamura, H. Izumi, Z. Ma, R. Ikeda, M. Moriyama, T. Tanaka, T. Nojima, L. S. Levin, K. Fujikawa-Yamamoto, K. Suzuki, et al. Induction of Centrosome Amplification and Chromosome Instability in Human Bladder Cancer Cells by p53 Mutation and Cyclin E Overexpression Cancer Res., July 15, 2004; 64(14): 4800 - 4809. [Abstract] [Full Text] [PDF] |
