Mutagenesis vol.18 no.3 pp.287–292, 2003
Mitochondrial impairment in p53-deficient human cancer cells
Shaoyu Zhou, Sushant Kachhap and Keshav K.Singh1
Sidney Kimmel Cancer Center, Johns Hopkins School of Medicine,
Bunting-Blaustein Cancer Research Building, 1650 Orleans Street,
Room 143, Baltimore, MD 21231, USA
The mechanism linking p53 inactivation to human cell
malignancy remains unclear. Studies have indicated that
mitochondrial dysfunction is involved in carcinogenesis. In
this study we investigated the role of p53 in mitochondrial
DNA (mtDNA) mutation and maintenance of proper mitochondrial function. We measured mtDNA mutation and
found no difference in frequency of mutation between
the p53⍣/⍣ and p53–/– cell lines. However, mitochondrial
cytochrome c oxidase (COX) activity was significantly
diminished in p53–/– cells. This decrease in COX activity
was attributed to decreased protein levels of the COXII
subunit encoded by the mitochondrial genome and was not
due to mutation in the mitochondrial COXII gene. Further
investigation revealed no concomitant decrease in COXII
mRNA levels in p53–/– cells and the stability of mRNA in
p53–/– cells was unaffected. This study suggests that
decreased COX activity is likely due to post-transcriptional
regulation of the COXII subunit by p53. COX is a critical
enzyme in the mitochondrial electron transport chain and
reduced COX activity may affect mitochondrial structure.
However, examination of mitochondrial ultrastructure
revealed no obvious differences between p53⍣/⍣ and p53–/–
cell lines. Together, our study suggests that p53 is involved
in regulation of COXII at the protein level but not at the
mRNA level. p53 does not affect mtDNA mutation or
mitochondrial ultrastructure.
Introduction
Mitochondrial dysfunction is a hallmark of cancer cells. Several
notable differences between the mitochondria of normal versus
transformed cells have been reported (reviewed in Chang et al.,
1971; Pedersen, 1978; Carafoli, 1980; Singh, 1998, 1999). For
example, various tumor cells exhibit differences in the number,
size and shape of their mitochondria relative to normal cells.
The mitochondria of rapidly growing tumors tend to be fewer
in number, smaller and have fewer cristae than mitochondria
from slowly growing tumors that are larger and have characteristics more closely resembling those of normal cells. In
addition, the ultrastructural features of mitochondria in oncocytoma of thyroid, salivary gland, kidney, parathyriod and breast
cells show features in common with mitochondrial encephalomyopathies, where mitochondria are found as large aggregates
and display a variety of morphological alterations (Maximo
and Sobrinho-Simoes, 2000). Mitochondrial DNA (mtDNA)
mutations are also commonly found in many tumors (Polyak
et al., 1998; Singh, 1998; Fliss et al., 2000; Penta et al., 2001;
Modica-Napolitano and Singh, 1996, 2002).
1To
p53 is a multifunctional protein. Most of the cellular
functions of p53 have been described to be in the nucleus.
However, recent studies indicate that p53 is also localized in
mitochondria (Katsumoto et al., 1995; Merrick et al., 1996;
Marchenko et al., 2000; Donahue et al., 2001). Several lines
of evidence suggest that p53 and mitochondrial function are
interconnected. One pertains to the integral role of p53 in
regulating mitochondrial membrane potential (Li et al., 1999).
It is demonstrated that p53–/– mice are resistant to apoptosis
induced through a collapse of mitochondrial membrane potential. It is suggested that p53 mediates apoptosis through a three
step process: (i) transcriptional induction of redox-related
genes; (ii) formation of reactive oxygen species; (iii) oxidative
degradation of mitochondrial components (Polyak et al., 1997).
Several genes whose products localize to mitochondria contain
a p53 regulatory element (Miyashita and Reed, 1995; Bourdon
et al., 1997). Mitochondrial localization of p53 has also
been correlated with early changes leading to p53-mediated
apoptosis in different cell types induced by a range of apoptosis
inducers, including DNA damage and hypoxia (Marchenko
et al., 2000; Donahue et al., 2001). Mouse embryos deficient
in p53 protein show a deficiency of 16S rRNA transcript
encoded by mtDNA (Ibrahim et al., 1998).
In the nucleus, p53 plays an important role in DNA
base excision repair, both as a transcription factor and as a
component of nucleotide and base excision repair complexes
(Offer et al., 1999; Morris, 2002). p53 interacts with RAD51
and topoisomerases I and II. Each of these proteins is involved
in recombination. Consistent with this finding, an increased
rate of recombination has been reported in p53-deficient cells
(Buchop et al., 1997; Yuwen et al., 1997; Gobert et al.,
1999; Dudenhoffer et al., 1999). Loss of p53 protein and
mitochondrial function has been extensively reported in a
variety of cancers (Nigro et al., 1989; Baker et al., 1990; Auer
et al., 1994; Levine, 1997; Polyak et al., 1998; Singh, 1998).
However, it is not clear whether p53 is involved in mtDNA
mutagenesis and in maintaining proper mitochondrial function.
The purpose of the present study was to investigate the effect
of p53 inactivation on mitochondrial genetic response and
changes in mitochondrial function.
Materials and methods
Cell culture and colony formation assays
Human colon cancer cell line HCT116 (p53⫹/⫹) and its derivative HCTp53KO
(p53–/–), which has both p53 alleles disrupted (Bunz et al., 1998), were grown
in McCoy’s 5A medium supplemented with 10% fetal bovine serum and
penicillin/streptomycin, in a humidified incubator at 37°C, 5% CO2. For
colony formation assay, 100 cells were seeded in 60 mm tissue culture dishes
containing 3 ml of fresh medium supplemented with varied concentrations of
chloramphenicol (CAP) and cultured for 6 days. Surviving colonies were
fixed/stained and counted. All dose points were measured in duplicate in
each experiment and survival curves represent data from two independent
experiments.
whom correspondence should be addressed. Tel: ⫹1 410 614 5128; Fax: ⫹1 410 502 7234; Email: [email protected]
© UK Environmental Mutagen Society 2003; all rights reserved.
287
S.Zhou, S.Kachhap and K.K.Singh
Mitochondrial enzyme activity assays
Activities of mitochondrial enzymes were determined spectrophotometrically
according to previous methods with slight modifications (Singer, 1994; Trounce
et al., 1996). Cells were trypsinized and washed with phosphate-buffered
saline and then resuspended in the assay buffer. Cell membranes were
permeabilized with 0.5% Triton X-100. Samples were kept on ice for
subsequent determination of mitochondrial enzyme activities. Specifically,
Complex I (NADH dehydrogenase) activity was measured by monitoring the
reduction of ferricyanide at 420 nm (Singer, 1994). Briefly, to 1 ml of 120 mM
triethanolamine buffer, pH 7.8, containing 0.5 mM ferricyanide and 5 mM
rotenone, cells (~80 µg) were added to start the reaction. The NADHdependent reduction of ferricyanide was measured at 30°C for 2 min. The
activities are expressed as nmol reduced ferricyanide/min/mg protein, using an
extinction coefficient of 1.0 per mM/cm. Complex II (succinate dehydrogenase)
activity was measured as the rate of reduction of 2,6-dichlorophenolidophenol
(DCPIP) at 600 nm, when coupled to the complex II-catalyzed reduction of
decylubiquinone (DB) (Trounce et al., 1996). Briefly, cells (80 µg) were
added to 1 ml of 0.1 M potassium phosphate buffer, pH 7.4, containing
20 mM succinate. The mixture was incubated at 30°C for 5 min followed by
addition of antimycin (2 µg), rotenone (2 µg), KCN (2 mM) and DCPIP
(50 µM). The reaction was started by addition of 50 µM DB. The activities
are expressed as nmol DCPIP/min/mg protein, using an extinction coefficient
of 19.1 per mM/cm. Complex III (ubiquinol–cytochrome c oxidoreductase)
activity was measured by monitoring reduction of cytochrome c at 550 nm
catalyzed by complex III in the presence of reduced decylubiquinone (DBH2)
(Trounce et al., 1996). Cells were added to 1 ml of Tris–sucrose buffer
(50 mM Tris, 250 mM sucrose, 1 mM EDTA, pH 7.4), containing 50 mM
cytochrome c and 2 mM KCN. The mixture was incubated at 30°C for 5 min.
The reaction was started by adding 50 µM DBH2 and measured for 2 min.
The activities are expressed as nmol cytochrome reduced/min/mg protein with
an extinction coefficient of 19.0 per mM/cm. Complex IV (cytochrome c
oxidase) activity was measured by monitoring the oxidation of reduced
cytochrome c at 550 nm (Trounce et al., 1996). Briefly, to 1 ml of 20 mM
potassium phosphate, pH 7.4, containing 20 mM succinate, cells (~80 µg)
were added to start the reaction. Readings were recorded for 2 min. The
activities are expressed as nmol cytochrome oxidized/min/mg protein with an
extinction coefficient of 19.0 per mM/cm.
RT–PCR analysis
Levels of COXI, COXII and COXIII mRNA were evaluated by RT–PCR.
Total RNA was isolated using TRIzol reagent. For the analysis of mRNA
stability, cells were incubated with 5 µg/ml actinomycin D and RNA was
isolated at various times thereafter. RNA was annealed to oligo(dT) primers
at 70°C and reverse transcribed with SuperScript II RNase H– reverse
transcriptase (Gibco BRL) at 42°C in the presence of dithiothreitol and 200
µM each dNTP. Primer sequences were as follows: COXI forward primer, 5⬘ATGTTCGCCGACCGTTGACT-3⬘, reverse primer, 5⬘-GTATACGGGTTCTTCGAATG-3⬘; COXII forward primer, 5⬘-ATGGCACATGCAGCGCAAGG-3⬘, reverse primer, 5⬘-CTATAGGGTAAATACGGGCC-3⬘; COXIII
forward primer, 5⬘-ATGACCCACCAATCACATGC-3⬘, reverse primer, 5⬘AGACCCTCATCATAGATGG-3⬘. PCR was initially performed with different
cycle numbers to find the optimal number and sample dilution for quantitative
amplification of target and control genes. Subsequently, PCR was performed
for 20 cycles, which was optimal for a linear response. G3PDH served as a
control. PCR products were resolved on a 1.0% agarose gel and stained with
ethidium bromide. The gel was photographed with Polaroid film.
Western blot analysis
The expression of COXI and COXII was assessed by western blotting analysis
of total protein lysate. Cells at ~75% confluence were homogenized in 1 ml
of homogenization buffer (20 mM Tris–HCl, pH 7.5, 5 mM MgCl2, 0.1 mM
phenylmethylsulfonyl fluoride, 20 µM leupeptin, 20 µM aprotinin) (Takahashi
et al., 1995). The protein concentrations were determined using the Bio-Rad
protein dye with BSA as standard. Ten micrograms of each sample was loaded
onto 12% SDS–polyacrylamide gels and electrophoresed to resolve proteins.
The proteins were then transferred onto nitrocellulose membranes. Membranes
were blocked in 20 mM Tris–HCl, pH 7.5, 150 mM NaCl and 5% non-fat
dry milk. Membranes were first probed with anti-COXI and anti-COXII
antibodies (Molecular Probes, Eugene, OR) and then with peroxidase-conjugated rabbit anti-mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA).
COXI and COXII were visualized with ECL detection reagents (Amersham
Life Science, Little Chalfont, UK).
Cell proliferation assay
Cell proliferation was spectrophotometrically evaluated by the MTT reduction
assay as described previously (Mossman, 1983). Briefly, cells were seeded at
1⫻104 cells/well in 24-well plates. Surviving cells were stained with MTT at
days 0, 1, 2 and 3. The absorbance was determined at 553 nm.
288
Fig. 1. Frequency of CAP-resistant colonies in p53⫹/⫹ and p53–/– cells: One
hundred cells were seeded in 60 mm tissue culture dishes containing 3 ml
of fresh medium supplemented with various concentrations of CAP and
cultured for 6 days. Surviving colonies were fixed/stained and counted. The
data represent the means ⫾ SD for three individual experiments with
duplicate samples analyzed in each case.
Electron microscopic analysis of mitochondria
Cells in 35 mm dishes (Falcon) were fixed in 3% glutaraldehyde, 0.1 M
potassium phosphate with 3 mM MgCl2 for 1 h at room temperature on a
gentle rocker. After a 30 min buffer rinse, cells were post-fixed in 2% osmium
tetroxide in 0.1 M sodium cacodylate, 3 mM MgCl2 (on ice) for 1 h in the
dark. Samples were distilled water rinsed and en bloc stained with 2% aqueous
uranyl acetate (0.22 µm filtered) for 30 min in the dark. Plates were then
dehydrated in a graded series of ethanol then embedded in Eponate 12 (Pella)
overnight without catalyst. The next day dishes were further infiltrated with
fresh Epon plus 1.5% DMP-30 (catalyst) then cured in a 37°C oven for 3
days. Dishes were then transferred to a 60°C oven for final curing. Upon
hardening, epon discs were separated from the dishes and 3 mm buttons were
removed with a hole puncher. Samples were mounted cell side up on
Epon blanks, the Epon polymerized and trimmed for thin sectioning. Thin
compression free sections (70–80 nm) were obtained with a Diatome diamond
knife (35°, low angle). Sections were picked up on 200 mesh copper grids
and further stained with uranyl acetate and lead citrate. Grids were examined
in a Phillips CM 120 TEM under 60–80 keV.
Results
mtDNA mutational analysis
We investigated whether p53–/– cells accumulated spontaneous
point mutations in the mitochondrial genome. One of the
readily obtainable genetic markers for point mutation in the
mitochondrial genome is resistance to the antibiotic CAP. CAP
inhibits mitochondrial protein synthesis selectively by binding
to the peptidyltransferase domain of 16S rRNA in mammalian
cells (Branlant et al., 1981; Hashiguchi and Ikushima, 1998).
Resistance to CAP arises by single base substitution(s) in the
16S rRNA gene encoded by the mitochindrial genome (Blanc
et al., 1981; Kearsey and Craig, 1981; Howell and Kubacka,
1993). We treated both p53⫹/⫹ and p53–/– cell lines with varied
concentrations of CAP. Figure 1 demonstrates that there was
no difference in number of CAP-resistant colonies between
the two cell lines. This indicates that there is no difference in
frequency of spontaneous mtDNA mutations between the p53⫹/
⫹and p53–/– cell lines. In order to identify mutations in other
regions of the mitochondrial genome, we PCR amplified genes
encoding cytochrome c oxidase subunits I, II and III and found
no changes in sequence of these genes between the wild-type
cells and cells deficient in p53 function (data not shown). We
conclude that p53 does not play a role in mtDNA mutagenesis.
Mitochondrial impairment in p53-deficient cells
Fig. 3. RT–PCR quantification of COXI, COXII and COXIII transcripts.
Total RNA was isolated from cells at ~75% confluence using TRIzol
Reagent. RNA was annealed to oligo(dT) primers at 70°C and reverse
transcribed with SuperScript II RNase H– reverse transcriptase at 42°C in
the presence of dithiothreitol and 200 µM each dNTP. PCR was initially
performed with different cycle numbers to find the optimal number and
sample dilution for quantitative amplification of target and control genes.
The gene-specific primers are described in Materials and methods. G3PDH
served as a control.
Fig. 2. Comparison of mitochondrial respiratory enzymes activity. The
activity of four mitochondrial complexs was determined
spectrophotometrically in the corresponding assay as described in Materials
and methods. (A) Complex I activity. (B) Complex II activity. (C) Complex
III activity. (D) Complex IV activity. The data represent the means ⫾ SD
for three to five separate experiments. The asterisk indicates a significant
decrease compared with the wild-type, using Student’s t-test for data
analysis. The P value for (D) is 0.004, for (A)–(C) ⬎0.05 (no statistical
difference).
Decreased cytochrome c oxidase activity in p53–/– cells
p53 regulatory elements are found in the upstream promoter
region of many genes whose products play important roles in
mitochondrial metabolism (Miyashita and Reed, 1995;
Bourdon et al., 1997). In the present study we determined
whether p53 inactivation could lead to altered mitochondrial
enzyme activity. We compared activities of the mitochondrial
complexes in p53⫹/⫹ and p53–/– cells. As shown in Figure 2,
there were no differences in activities of complexes I, II and
III between the two cell lines. However, complex IV activity
in p53–/– cells was significantly lower compared with p53⫹/⫹
cells, with an activity of 1.88 ⫾ 0.29 in p53⫹/⫹ and 1.27 ⫾
0.33 in p53–/– cells (~30% lower) (Figure 2). This study
suggests that p53 inactivation leads to reduced complex IV
activity.
Decreased level of COX II protein in p53–/– cells
Subunits of complex IV are encoded by both the nuclear and
mitochondrial genomes. We investigated whether the alteration
in complex IV activity was due to lower levels of cytochrome
c oxidase protein encoded by the mitochondrial genome.
Therefore, we examined the expression of COXI, COXII and
COXIII mRNA by RT–PCR. In this experiment, G3PDH was
used as the control. The results show no differences in the
levels of COXI, COXII and COXIII mRNA between p53⫹/⫹
and p53–/– cells (Figure 3). We next examined the protein
levels of COXI and COXII by western blot analysis using
antibodies against COXI and COXII. The same blot was
probed with an antibody against actin as a control for variations
in loading. The results show that the COXII protein level was
significantly reduced in p53–/– cells (Figure 4B), while no
change was observed in COXI (Figure 4A). Since COXII
protein levels differed, we investigated whether the diminished
COXII protein level in p53–/– cells is attributable to a decreased
stability of mRNA. We treated cells with 5 µg/ml actinomycin
D, an inhibitor of mRNA synthesis. As shown in Figure 5,
there was no difference in the time-dependent decrease in
mRNA level between p53-proficient and p53-deficient cells.
The mRNA level was equally diminished even after 20 h
treatment with actinomycin D. Taken together, the discoordinate expression of COXII mRNA and COXII protein suggest
post-transcriptional regulation of COXII by p53.
Mitochondrial ultrastructure in p53⫹/⫹ and p53–/– cells
Since the complex IV activity was diminished in p53–/– cells,
we determined whether this alteration would change the
ultrastructure of mitochondria. Electron microscopy was conducted as described in Materials and methods. Figure 6A and
B shows that both p53⫹/⫹ and p53–/– cell lines contain normal
mitochondrial structure with well-organized cristae being present in both cell lines. Furthermore, no difference in cell
proliferation was observed when these two cell lines were
grown in medium containing galactose (data not shown).
Functional mitochondria are required for galactose utilization
and a difference in proliferation would reflect a defect in
mitochondrial function (Robinson et al., 1992). These studies
suggest that inactivation of p53 does not result in significant
changes in mitochondrial structure and does not affect proliferation.
Discussion
The results of the present study suggest that p53 is involved in
regulation of mitochondrially encoded cytochrome c oxidase II.
COX activity is described to be lower in colonic adenocarcinoma and hepatocellular carcinoma than in the normal
cells (Sun and Cederbaum, 1980; Sun et al., 1981; Heerdt
et al., 1990). COX deficiency is also one of the most frequent
289
S.Zhou, S.Kachhap and K.K.Singh
A
Fig. 4. Western blot analysis of COXI and COXII gene expression. The
protein lysates were prepared as described in Materials and methods. Ten
milligrams of sample was loaded in each lane onto 12% SDS–
polyacrylamide gels and electrophoresed to resolve proteins. Western blot
analyses were performed using anti-COXI and anti-COXII antibodies and
peroxidase-conjugated rabbit anti-mouse IgG. COXI and COXII were
visualized with ECL detection reagents. (A) Expression of COXI.
(B) Expression of COXII. (C) Quantitation of COXII against actin
expression. The mean intensities are 0.78 ⫾ 0.24 in p53⫹/⫹ and 0.32 ⫾
0.17 in p53–/– cells and P ⫽ 0.037, indicating a statistical difference
between the wild-type p53⫹/⫹ and p53–/–.
B
Fig. 5. Analysis of COXII mRNA stability. The stability of COXII mRNA
was analyzed by RT–PCR. Cells were incubated with 5 µg/ml actinomycin
D and total RNA was isolated at various times thereafter. RT–PCR was
performed as described in Materials and methods. 0, 2, 4, 6 and 20 h
indicates time after treatment of cells with 5 µg/ml actinomycin D.
causes of respiratory chain defects in humans. Patients with
COX deficiency present heterogeneous clinical phenotypes,
including hepatic failure and encephalomyopathy (Barrientos
et al., 2002). Although our analysis showed a decrease in
COXII subunit protein, we did not find a corresponding
decrease in COXII mRNA in p53–/– cells. It is possible that
290
Fig. 6. Mitochondrial ultrastructure in p53⫹/⫹ and p53–/– cells. (A)
Ultrastructure of p53⫹/⫹ cell. (B) Ultrastructure of p53–/– cell. The protocol
used to carry out microscopy is described in Materials and methods.
Mitochondrial impairment in p53-deficient cells
the stability of COXII mRNA was affected, which may affect
the COXII level in p53–/– cells. We therefore analyzed the
stability of mRNA in wild-type p53⫹/⫹ and p53–/– cells after
treatment with actinomycin D, an inhibitor of mRNA synthesis.
Our data suggest that the stability of mRNA was unaffected
in p53–/– cells. Interestingly, although the mtDNA sequence
shows close similarity to part of the p53 recognition site, it is
not known how p53-binding sequences in mtDNA may serve
as a recognition site for transcription of the mitochondrial
genome (Donahue et al., 2001). Together, our study suggests
that COXII is post-transcriptionally regulated by p53 and that
the lower level of COXII contributes to the reduced COX
activity in p53–/– cells. Consistent with our results in human
cells, Saccharomyces cerevisiae COXII is also regulated at the
post-transcriptional level (Pinkham et al., 1994).
Mitochondrial mutations and structural abnormalities in
mitochondria are associated with various tumors (Cavalli and
Liang, 1998; Polyak et al., 1998; Fliss et al., 2000; Yeh et al.,
2000). Studies also indicate that p53 may be intimately
involved in biogenesis and proper mitochondrial function
(Polyak et al., 1997; Giaccia and Kastan, 1998; Ibrahim et al.,
1998; Prives and Hall, 1999; Donahue et al., 2001). However,
our studies revealed no significant differences in ultrastructure
of mitochondria or deficiency in growth of p53-deficient cells
when grown in medium containing galactose (data not shown).
It is noteworthy that galactose is utilized via mitochondrial
metabolism. So if cells contain defective mitochondria they
will grow slower than cells with intact mitochondria (Robinson
et al., 1992).
p53 maintains the stability of the nuclear genome. Recent
evidence suggests that p53 is involved in base excision repair
(Zhou et al., 2001), nucleotide excision repair (Hwang et al.,
1999) and recombination (Mekeel et al., 1997) in the nucleus.
p53 expression reduces the frequency of point mutations in a
shuttle vector modified by several chemical mutagens
(Courtemanche and Anderson, 1999). Mutations in genes
encoded by mtDNA have been described in carcinoma cells
(Polyak et al., 1998). Homoplasmic mutations in mtDNA are
also reported in tumors harboring p53 mutations (Polyak et al.,
1998; Fliss et al., 2000). It is not clear whether p53 is involved
in maintaining the integrity of the mitochondrial genome.
We investigated whether genes encoding COXI, COXII and
COXIII are mutated in p53–/– cells. Our analysis revealed no
differences between the p53⫹/⫹and p53–/– coding sequences
of the COXI, COXII and COXIII genes. In addition, we
measured the frequency of mitochondrial mutation in the 16S
rRNA gene by determining chloramphenicol-resistant colonies
in wild-type and p53-null mutants cells. We did not find a
significant difference in the frequency of mutation in the 16S
rRNA gene encoded by mtDNA. These studies suggest that
p53 does not play a significant role in maintaining the integrity
of the mitochondrial genome in human cancer cells. We
conclude that mtDNA mutations and mitochondrial dysfunction
are not ascribed to defects in p53 and that another gene(s)
may be the central mediator of mitochondrial mutations and
mitochondrial dysfunction in cancer cells.
Our data suggest that p53 protein specifically affects COXII
expression at the protein level but not at the mRNA level. The
results presented in this paper point to a novel mechanism
by which p53 may regulate mitochondrial gene expression.
However, further work is needed to elucidate the exact mechanism by which p53 brings about this effect in the mitochondria.
Acknowledgements
We thank Dr Bert Vogelstein for providing the p53⫹/⫹ and p53–/– cell lines
used in this study. Research in our laboratory is supported by grants from the
National Institutes of Health RO1-097714 and American Heart Association
Scientist Development Award 9939223N.
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Received on October 11, 2002; revised on December 23, 2002;
accepted on December 24, 2002