Oncogene (2002) 21, 3043 ± 3049
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p53 mutations and tetraploids under r- and K-selection
Norio Chikatsu1, Yukari Nakamura1, Hiroyuki Sato1, Toshiro Fujita1, Shigetaka Asano2 and
Toru Motokura*,1,3
1
Department of Internal Medicine, Faculty of Medicine, the University of Tokyo, 3-28-6 Mejirodai, Bunkyo-ku, Tokyo 112-8688,
Japan; 2Advanced Clinical Research Center, Institute of Medical Science, the University of Tokyo, 4-6-1 Shirokanedai, Minato-ku,
Tokyo 108-8639, Japan
Cotransfection of rat embryo ®broblasts with c-myc and
activated H-ras oncogenes is one experimental model of the
multistep oncogenesis associated with p53 mutations and
aneuploidy. Using the model, we found that selection
processes, e.g., r- and K-selection, a€ect emergence of p53
mutants and tetraploids. Culture optimum for logarithmic
growth (r-selection) selected p53 mutants as they proliferated rapidly, while in con¯uent culture (K-selection)
tetraploids emerged regardless of the p53 status. Transfection of the mutated p53 gene with dominant negative
functions eradicated untransfected cells under both r- and
K-selection. However, these p53 mutants can be eradicated
under K-selection by cells with normal p53 function and
that had been selected under prolonged K-selection. The
presence of competitors and the type of selection should
determine whether or not p53 mutants and/or tetraploids
predominate. These observations strengthen the importance of selection processes in case of cancer.
Oncogene (2002) 21, 3043 ± 3049. DOI: 10.1038/sj/
onc/1205413
Keywords: p53; r-selection; K-selection; multistep
tumorigenesis
Introduction
The p53 tumor suppressor gene has multiple functions
and is mutated in half the number of human tumors.
The maintenance of genetic integrity is one of the
functions of p53 and dysfunctions cause genetic
instability culminating in aneuploidy, a hallmark of
cancers (Galipeau et al., 1996; Ramel et al., 1995; Yin
et al., 1999). However, genetic instability per se is not a
growth advantage unless p53 mutants gain a certain
population in which a variety of genotypes for clonal
evolution can be generated in the presence of genetic
instability. Among the consequences of p53 mutations,
there are resistance to hypoxia (Graeber et al., 1996),
ribonucleotide de®ciency (Linke et al., 1996) and an
acidi®ed microenvironment (Williams et al., 1999).
Thus a reduced apoptosis may be advantageous for
mutant cells in a corresponding microenvironment.
Therefore, p53 mutants may be selected under
unfavorable conditions.
In evolutionary biology, r- and K-selection was
initially described by MacArthur and Wilson (1967)
for species evolution in isolated islands: r is an intrinsic
growth rate and K is carrying capacity of the
environment. r-selection favors a higher population
growth rate, higher productivity and thus a small body
size. This form of selection will come to the fore during
the colonizing episode and hence must frequently build
back up to K. K-selection favors a more ecient
utilization of resources and a larger body size. This
form of selection will be more pronounced when the
species is at or near K (MacArthur and Wilson, 1967;
Pianka, 1970). We made use of r- and K-selection to
study rat embryo ®broblasts (REFs) transfected with cmyc and activated H-ras (EJ-ras) oncogenes. Cotransfection of REFs with these two oncogenes lead to in
vitro focus formation and in vivo tumor development
(Land et al., 1983). Such a tumorigenic model has
accompanying additional genetic events involving the
p53 gene and others, e.g., point mutation, allele loss,
gene ampli®cation and/or aneuploidy, recapitulating a
multistep nature of cancer development (Thompson et
al., 1989). Using this classical tumorigenic model, we
asked whether r- and K-selection would a€ect cancer
development. Initially, we speculated the emergence of
p53 mutants under unfavorable K-selection. However,
p53 mutants have growth advantages even in rselection. On the contrary, we observed emergence of
tetraploids only under conditions of K-selection, which
is reminiscent of the hypothesis described by MacArthur and Wilson (1967). We discuss the signi®cance
of r- and K-selection in cancer development.
Results
*Correspondence: T Motokura; E-mail: [email protected]
3
Current address: Department of Hematology and Oncology,
Graduate School of Medicine, the University of Tokyo, 7-3-1
Hongo, Bunkyo-ku, Tokyo 113-8655, Japan
Received 28 August 2001; revised 6 February 2002; accepted 20
February 2002
Emergence of p53 mutants and tetraploids under r- and
K-selection
We cotransfected human c-myc and EJ-ras expression
plasmids in REFs, using the calcium phosphate
p53 and tetraploids under r- and K-selection
N Chikatsu et al
3044
method. Resultant transformants expressed these
transfected genes, formed foci on the monolayer of
untransformed cells and tumors formed in nude mice,
as reported (data not shown) (Motokura et al., 1995;
Uchimaru et al., 1996). We cloned six diploid c-myc
and EJ-ras transformants, MR5, 6, 8, 9, 11 and 12,
from well-isolated foci and passaged them under
conditions of r- and K-selection. For r-selection, cells
were plated sparsely and passaged well before con¯uence in order to maintain logarithmic growth. For
K-selection, cells were passaged densely and growth
was constrained because of overcrowding. The diploid
DNA contents of these six clones were fairly constant
under r-selection (Figure 1a,b). Cells were fusiform and
overlapped and morphologies were largely unchanged.
Under K-selection, cells were initially arrested at the
G0/G1 phase, then tetraploid cells emerged and
remained in substantial proportion (Figure 1a,b).
Meanwhile, a large proportion underwent apoptosis,
as evidenced by the presence of a sub-G1 peak in ¯owcytometric analysis and the presence of DNA ladders
(data not shown). After the ploidy shift, tetraploids
were readily isolated: numbers of tetraploid subclones/
numbers of subclones established were 3/5 in MR5, 3/7
in MR8 and 2/8 in MR9 (Figure 1b). Usually these
tetraploids were ¯at cells forming a monolayer, which
di€ered from r-selected diploids (Figure 1c). Cytogenetic analysis revealed their tetraploidy (data not
shown). These tetraploids were derived from diploid
transformants because integration of the transfected
plasmid was the same as determined using Southern
blot analysis (Figure 1d).
After c-myc immortalization, p53 gene mutation or
the INK4A/ARF locus deletion occurs frequently
(Zindy et al., 1998). Therefore, we examined these
gene aberrations in clones using RT ± PCR and SSCP
analyses followed by direct sequencing in case of
aberrant signals. The REFs we used had a point
mutation of the INK4A/ARF locus, CTT to TTT,
leading to substitution of Phe for Leu 151 in p16INK4A
protein. It is located in the noncoding region of the
ARF gene and may be a polymorphism. Two clones
had the INK4A/ARF deletions and these did not
correlate with ploidy shift nor with selection process
(Figure 1e). One more clone, MR12, might have one
deleted allele or might be homozygous for the locus.
MR5, one of six clones, carried the p53 gene missense
mutation, CTG to ATG, leading to substitution of Met
for Leu 109 in the DNA binding domain. A tetraploid
subclone derived from K-selected MR5 had a deleted
wild-type allele of the p53 gene resulting in loss of
heterozygosity (LOH) (Figure 1e), a ®nding that we
initially speculated might be causally associated with
ploidy shift.
Emergence of p53LOH and not tetraploids under
r-selection
To determine if the LOH is involved in ploidy shift and
whether K-selection induces LOH, we repeated MR5
culture under r- and K-selection in quadruplicate and
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monitored the occurrence of ploidy shift and LOH.
Although ploidy shift was reproducible under Kselection (Figure 2a), it was not associated with LOH
that occurred under r-selection in two of four cultures,
despite an incomplete LOH under K-selection (Figure
2b). We also subcloned and subjected 10 diploid MR5
cells to r- and K-selection to examine the mode of
emergence of de novo LOH. Under K-selection, seven
of 10 subclones became tetraploid although p53 LOH
was newly obtained only in three of nine and preceded
ploidy shift in a single case alone (Figure 2c).
Conversely, no ploidy shift occurred under r-selection
(data not shown) and wild-type alleles were continually
deleted in parallel with shortening of doubling times:
the mean doubling times+s.d. of parent MR5 cells and
resultant cells with p53 LOH were 16.1 h+1.1 h (n=4)
and 9.8 h+0.3 h (n=7), respectively (Figure 2a,d,e).
Deletion of the wild-type allele should confer a
selective advantage under r-selection, i.e., accelerated
growth, as noted by Harvey et al. (1993) who used
®broblasts from p53-knockout mice. In addition, the
sigmoid curve of LOH emergence shown in Figure 2e
re¯ects a logistic curve of the selection process and
strongly suggests the clonal evolution of LOH (Crow
and Kimura, 1970). Delayed and incomplete emergence
of LOH shown as squares in Figure 2e can be
explained as a result of competition with rapid growers
with mutations other than p53 LOH because the
doubling times were already shortened at the beginning
of r-selection. Our ®ndings suggest that prevalent p53
mutations in human cancers can be attributed in part
to r-selection.
After 6 weeks of r-selection, we found that ploidy
shift was not reproducible under K-selection (Figure 2a).
The potential for ploidy shift in culture appeared to be
lost during r-selection although estimated cell numbers
reached under the r-selection were far more numerous
than 661013 cells present in a human body, thus an
unrealistic situation. On the contrary, when K-selection
was changed to r-selection, tetraploid cells were rapidly
replaced with diploids. The doubling times+s.d. of
diploids and tetraploids subcloned from K-selected
culture were 10.7 h (n=2) and 18.1+0.3 h (n=3),
respectively, a di€erence largely due to di€erent lengths
of G1 phases (4.7 h vs 9.0+0.4 h, calculated using ¯owcytometric analysis and the RFIT model). Therefore,
diploids grew more rapidly than tetraploids. Lower
ploidy correlates with a higher growth rate in yeast cells
(Galitski et al., 1999) and, according to our ®ndings,
even in mammalian cells. Therefore, r-selection prefers
diploids as well as p53 mutants due to a simple
competition for growth rates.
p53 dysfunction is not required for ploidy shift under
K-selection
To con®rm that p53 dysfunction is unnecessary for
ploidy shift, we examined MR8 cells. These cells had
no p53 mutation and expressed p21CIP1 protein in
response to doxorubicin (DXR) treatment, thereby
suggesting normal p53 functions in sharp contrast with
p53 and tetraploids under r- and K-selection
N Chikatsu et al
3045
Figure 1 Six c-myc and EJ-ras cotransfected clones, MR5, 6, 8, 9, 11, 12 cells, under r- and K-selection. (a) Cell numbers are
plotted on a semilogarithmic scale (upper part) and polyploid cells are indicated in per cent (lower part). Open symbols denote rselection and closed ones K-selection. (b) Cell-cycle distribution of MR5 cells was determined by ¯ow cytometry at the indicated
time under r (left side)- and K (right side)-selection. A tetraploid subclone derived from K-selection is at the top. (c) Morphologies
by phase-contrast microscopy of MR5 and MR8 cells under r- and K-selection and a K-selected tetraploid subclone, magni®cation
650. Bars denote 50 mm. (d) Southern blot analysis of MR5, MR8 and MR9 parent cells (p), r-selected cells (r) and K-selected
tetraploids (t). Conventional techniques were used for DNA extraction and for Southern blot analysis of HindIII digests (10 mg/
lane) of genomic DNAs using the ClaI/HindIII fragment of pGEMmycB (Halazonetis and Kandil, 1991) as a radioactive probe.
DNA sizes are shown on the right (kilobases). Ethidium bromide-stained gel shows loading of DNA (lower part). (e) Part of silverstained gel of RT ± PCR ± SSCP analysis shows p53 mutation and INK4A/ARF deletion before (p) and after r (r)- and K (K)selection and in tetraploid subclones (t). W, wild-type allele; M, mutant allele; *, double-strand DNAs
MR5 cells with p53 LOH that had an almost abolished
response (Figure 3a). Tetraploids emerged again in
three of four cultures under K-selection (Figure 3b). A
prolonged culture resulted in di€erent proportions of
tetraploids under K-selection but not under r-selection.
An isolated tetraploid subclone retained the p53 and
p21CIP1 response to DXR treatment, as shown in
Figure 3a. We also subjected 12 subclones derived from
MR8 cells to r- and K-selection and observed one
apparent ploidy shift and two other slight increases in
a proportion of tetraploids only under K-selection
(date not shown). We found no emergence of p53
mutation or INK4A/ARF deletion in these cultures,
except for one subclone which had one allele of the
INK4A/ARF locus deleted under r-selection (data not
shown). Therefore, ploidy shift is not dependent on
dysfunction of the p53 pathway rather it depends on
K-selection.
Predominance of transfected p53 mutants under r- and
K-selection
Because p53 mutations spontaneously developed under
r- and K-selection, we speculated that transfection with
the p53 dominant negative mutant would lead to the
predominance of transfectants under r- and K-selection
without any selection for drug resistance. Two days
after electroporation of the mutated p53-expressing
plasmid, p53KH215, to MR8 cells, cells were subjected
to r- and K-selection for 6 weeks. At the beginning of
selection, expression of a murine p53 sequence was not
detected using RT ± PCR. Weeks later, the p53KH215
sequence was readily detectable both under r- and Kselection, indicating that p53 dysfunction is advantageous for both r- and K-selection (Figure 4, lanes 4 ± 9).
All 10 subclones established after 6 weeks' r-selection
expressed the p53KH215 sequence thereby indicating
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p53 and tetraploids under r- and K-selection
N Chikatsu et al
3046
Figure 2 MR5 cells under r- and K-selection. (a) Culture conditions were renewed after 6 weeks. Open circles denote r- and then
K-selection. Closed circles denote K- and then r-selection. (b) Ethidium bromide-stained gel of PCR ± SSCP analysis for the p53
zygosity after r- and K-selection of 6 weeks. W, wild-type allele; M, mutant allele. (c ± e) Ten subclones derived from MR5 cells were
subjected to r- and K-selection and analysed for p53 zygosity, DNA ploidy and doubling times at the indicated time. (c) Ploidy shift
and p53 LOH under K-selection. Open circles denote cells in a heterozygous state and closed circles denote cells with LOH. (d)
Weekly PCR ± SSCP analysis of subclones under r-selection. Alleles are shown as in (b). Subclones after 8 weeks of K-selection are
at the very bottom. (e) Emergence of cells with p53 LOH under r-selection. Time is plotted from the point when MR5 parent cells
were plated sparsely for subcloning. The proportion of cells with p53 LOH was calculated from densitometrical analysis, using
ethidium bromide-stained gel, given that simple deletion of the wild-type allele occurred, according to the formula as follows: (m ±
w)/m, m is a signal density of the mutant allele and w is that of the wild-type. Doubling times were calculated from a fold increase
during the previous week. Circles denote subclones acquiring complete LOH and squares denote subclones with incomplete LOH
during the experiment. Closed circles correspond to more than 80% of cells with LOH
Figure 3 MR8 cells under r- and K-selection. (a) Western blot
analysis of p53 and p21CIP1 proteins in MR5 parent cells (p), a
subclone with p53 LOH (L), MR8 parent cells (p) and a
tetraploid subclone derived from K-selection (t) before (7) and
24 h after (+) 2 h treatment with doxorubicin (DXR 0.5 mM).
Ink-stained membrane shows loading of whole cell lyzates at the
bottom. (b) MR8 cells were subjected to r- and K-selection in
quadruplicate and DNA contents were measured. Open circles
denote r-selection and closed ones K-selection
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Figure 4 Emergence of p53 mutants under r- and K-selection
and competition between p53 mutants vs K-selected cells. Two
days after electroporation with the murine p53 mutant-expressing
plasmid, p53 KH215, to MR8 parent cells (p, lane 2), cells were
subjected to r- and K-selection for 6 weeks (lanes 3 ± 9). After 6
weeks' r-selection, cells were admixed in equal cell numbers with
MR8 cells which had been selected under prolonged K-selection
and were subjected to r- and K-selection (lanes 10 ± 14). RNAs
extracted at the indicated time were analysed using p53 RT ± PCR
and PvuII digests of PCR products discriminated transfected
murine from endogenous rat p53 sequences. Lane 1 denotes DNA
size markers (M) and DNA sizes (bp) are on the left; lane 15,
negative control with just water instead of cDNA
p53 and tetraploids under r- and K-selection
N Chikatsu et al
the predominance of p53KH215-transfected cells (data
not shown). Under r-selection, emergence of p53
transfectants was associated with more rapid growth
rates compared to mock transfection and under Kselection, increased cell density was readily achieved
along with expression of the transfected p53KH215
sequence (data not shown). We also used another
mutated p53, p53val135 (Michalovitz et al., 1990), and
found similar results (data not shown). Therefore, p53
mutations are selected for under both r- and Kselection.
Eradication of p53 mutants
To determine if p53 dysfunction is part of an inevitable
process and to observe e€ects of the presence of
competitors, co-culture was done. After 6 weeks' rselection, transfectants expressing the p53KH215 sequence were mixed in the equal number with cells that
had been derived from prolonged K-selection of MR8
cells and were again subjected to r- and K-selection.
Under r-selection, these transfectants readily predominated in the culture while under K-selection, these cells
were rapidly eradicated from the culture in the presence
of K-selected cells (Figure 4, lanes 13 and 14). Eradication
of p53 mutants was evidenced not only by disappearance
of murine p53 expression but also by predominance of a
¯at cell morphology seen by phase-contrast microscopy
(data not shown). We used four independently K-selected
cells as competitors and we obtained similar results.
Three of these four cells expressed p21CIP1 in response to
DXR treatment and thus appeared to have normal p53
function (data not shown). These observations suggested
that emergence of p53 mutants is not necessarily
inevitable but does depend on the type of selection and
on the absence of competitors.
Discussion
Experimental culture conditions usually correspond to
r-selection adopted in the present study. r-selected cells
tend to be homogeneous because only the fastest wins
the competition. To ensure reproducible results in any
experiment it is reasonable to use cell culture systems
that adopt r-conditions and thus maintains homogeneous populations. Conversely, under conditions of
K-selection di€erent strategies, e.g., adherence to
matrix, a decreased tendency toward apoptosis or
increased contact inhibition, may contribute to survival. Therefore, K-selection should foster a variety of
tumor cell characteristics, e.g., di€erent ploidies and
morphologies, and thus provide a variety of genotypes
for cancer development (Cavalier-Smith, 1980).
Furthermore, r-conditions should be transient in vivo
because exponential proliferation of cells would readily
exceed the tolerable cell number in a human body.
Therefore, it is reasonable that cancer development is
largely governed by K-selection in vivo.
Deregulated growth of cancer cells initially leads to
r-selection but then reaches K-selection as cells
proliferate. Such selection sequence favors the predominance of p53 mutants that have advantages in
both r- and K-selection, at least in the background of
c-myc and H-ras activation, and the prevalent p53
mutations in human cancers can be explained.
However, it is noteworthy that competitors do exist
to suppress emergence of p53 mutants, as shown in
Figure 4. The low frequency of p53 mutations in some
tumors may be associated with rapid emergence of
competitors in a tumor. Successful selection depends
on the absence of intratumor competitors as well as on
the type of selection. In addition, the growth advantage
conferred by p53 mutations is probably a€ected by the
genetic background of the tumor cells and the type of
selection depends on interactions between a tumor and
the microenvironment.
The INK4A/ARF locus deletion frequently occurred
after myc oncogene activation (Zindy et al., 1998) and
was repeatedly detected in our present study. Even one
allele deletion appeared to be advantageous under both
r- and K-selection. Because of a functional link
between ARF and p53 genes, the deletion may well
be selected in the same manner, as are p53 mutations.
Several investigators suggest a direct link between
p53 mutations and aneuploidy. In their experimental
models, emergence of tetraploids and then aneuploids
was explained by genetic instability conferred by p53
mutations (Galipeau et al., 1996; Ramel et al., 1995;
Yin et al., 1999). Even though p53 mutations are
selected for under r-selection, rapid growth readily
leads to a higher density (Harvey et al., 1993).
Therefore, the culture with p53 mutants tends to be
con¯uent and then become under K-selection, which
favors survivors rather than rapid growers and thus
facilitates ploidy shift. Genetic instability confered by
p53 mutations may increase the frequency of transition
from a diploid to tetraploid state at a single cell level
under both r- and K-selection. However, at least in our
model, p53 mutation did not lead to a ploidy shift at a
population level under r-selection. Therefore, ploidy
shift from diploids to tetraploids at a population level
was not deterministic rather it depended on the type of
selection. Tetraploid predominance required K-selection and was not necessarily associated with p53
dysfunction. In yeast cells, ploidy-dependent repression
of G1 cyclins can explain the greater cell size and the
slower growth (Galitski et al., 1999). Although underlying molecular mechanisms for slow growth of
tetraploids remain to be investigated, it seems reasonable that slow growing tetraploids never predominate
over parent diploids under r-selection.
According to our ®ndings, frequent polyploid cells in
a tumor suggest that K-selection is underway.
Clinically, a higher population density is an earlywarning change in a preinvasive state (Backman et al.,
2000). Therefore, tetraploidization can occur even in a
relatively early phase of cancer development such as in
the case of Barret's esophagus (Galipeau et al., 1996).
On the contrary, ploidy shift from diploid to polyploid
was rarely seen after a cancer had fully developed.
Consistently, in our model the frequency of the ploidy
3047
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p53 and tetraploids under r- and K-selection
N Chikatsu et al
3048
shift decreased after prolonged culture. There appeared
to be a time window when tetraploids can predominate
over parent diploids. We speculate that if a ploidy shift
occurs it is early in cancer development probably
because a growth advantage of tetraploids over
diploids is readily abrogated by genetic aberrations
accumulated in diploids during prolonged r- and Kselection.
Summary, cancer cells with a deregulated growth are
initially under r-selection. As a tumor grows, K-selection
begins. Both types of selection prefer p53 mutations in
case of c-myc and H-ras activation while ploidy shift
requires K-selection. Furthermore, a competitor with
di€erent mutations may suppress the emergence of p53
mutants. Our ®ndings strengthen the importance of
selection process that leads to cancer (Tomlinson and
Bodmer, 1999) and underscore the signi®cance of r- and
K-selection in cancer development.
Materials and methods
Expression plasmids
Expression plasmids used in this study were pucEJ for an
activated allele of human H-ras (EJ-ras) (Land et al., 1983;
Motokura et al., 1995), pSVmycB for human c-myc,
p53KH215 for a murine p53 mutant with dominant negative
functions (Finlay et al., 1988; Uchimaru et al., 1996), and
pcDneo for the neo resistance gene (Chen and Okayama,
1987; Uchimaru et al., 1996). pSVmycB was constructed by
replacing the c-jun insert of pSV2humjun (Zhang et al., 1990)
with the EcoRI/HindIII fragment of pGEMmycB (Halazonetis and Kandil, 1991).
Rat embryo fibroblasts (REFs) and transfection
REFs were prepared from 14-day pregnant Wistar rats
(Tokyo Laboratory Animals Science, Tokyo) as described
(Motokura et al., 1995; Uchimaru et al., 1996). Transfection
in REFs was done using the calcium phosphate method
described (Chen and Okayama, 1987) but with the slight
modi®cation described elsewhere (Motokura et al., 1995;
Uchimaru et al., 1996). Focus-forming cells were cloned 10 to
12 days after transfection. Transfection of p53KH215 in cmyc- and EJ-ras-transformants was done using electroporation. Ten micrograms of the plasmids were electroporated
into 26106 cells suspended in 200 ml medium at 0.5 kV with
25 mF with Gene Pulse II (Bio-Rad). Transfection eciency
was at least 261074, as determined by drug resistance.
Cell culture and r- and K-selection
Cells were initially plated at 26104 and were incubated with
Dulbecco's modi®ed Eagle's medium (Life Technologies)
supplemented with 10% fetal bovine serum (Bio Whittaker)
and kanamycin 60 mg/l (Meiji Seika Kaisha, Ltd, Tokyo) at
378C in a humidi®ed atmosphere with 5% CO2. To maintain
cells in the logarithmic phase of growth (r-selection), cells
were passaged at about 1 ± 8 : 500 (around 16104 cells) per
100-mm dish with 10 ml medium every 3 ± 4 days. For Kselection, half the number of cells was passaged and
incubated with 4 ml medium per 60-mm dish every 3 ± 4
days or weekly. Cells were counted using a hemocytometer
and Trypan blue exclusion.
Flow cytometry
After trypsinization, nuclei prepared from cells were stained
with propidium iodide (Sigma) according to Vindelùv et al.
(1983). DNA contents of at least 26104 nuclei per sample
were analysed using a FACScan and CellFIT software
(Becton Dickinson). Polyploid cells were de®ned as cells with
more than or equal to 4n DNA contents. Ploidy shift was
de®ned as an increase of polyploid cells over 50%.
Reverse transcription (RT)-polymerase chain reaction (PCR)
and single-strand conformation polymorphism (SSCP) analyses
RT was done as previously described (Uchimaru et al., 1997).
PCR was done as described (Uchimaru et al., 1997) with
primer pairs (0.5 mM each) and conditions in a volume of
20 ml were as follows: for p53 cDNA in the DNA binding
region (684 bp), 5'-CTGTCATCTTCTGTCCCTTC-3', 5'TTTCTTTTGCTGGGGAGAGG-3', 948C (1 min), 608C
(1 min), 728C (2 min), 35 cycles; INK4A/ARF cDNA
(404 bp), 5'-CAGGTGATGATGATGGG-3', 5'-TGCCAGAAGTGAAGCCAAGG-3', 948C (1 min), 588C (1 min),
728C (1 min), 35 cycles; for the exon 4 of the p53 gene
(125 bp), 5'-CTGTCATCTTCTGTCCCTTC-3', 5'-AAGCAACTCTTCAGGCCCAC-3', 948C (1 min), 608C (1 min),
728C (1 min), 35 cycles. After restriction enzyme treatment
(HpaII and KpnI for p53 cDNA and SmaI for INK4A/ARF),
SSCP was done using a non-radioactive method with silver
staining or ethidium bromide staining, as described (Sugano
et al., 1993).
Western blot analysis
Primary monoclonal antibodies were Pab240 for p53 (Santa
Cruz Biotechnology) and SX118 for p21CIP1 (PharMingen)
used at 0.2 mg/ml. A secondary antibody was alkaline
phosphatase-conjugated rabbit anti-mouse IgG1 antibody
(Zymed Laboratories) used at 1 : 1000 dilution. Western blot
analysis was done as described (Uchimaru et al., 1998).
Acknowledgments
We thank T Iiri for helpful comments and M Ohara for
language assistance. This work was supported in part by
grants (to T Motokura) from the Ministry of Education,
Science, Technology, Sports and Culture of Japan.
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