Carcinogenesis vol.21 no.11 pp.2027–2033, 2000
Cyclosporin A inhibits chromium(VI)-induced apoptosis and
mitochondrial cytochrome c release and restores clonogenic
survival in CHO cells
Daryl E.Pritchard1,2,*, Jatinder Singh1,
Diane L.Carlisle1,3 and Steven R.Patierno1,2,3,4
1Department
of Pharmacology, 2Program in Genetics and 3Program in
Molecular and Cellular Oncology, The George Washington University
Medical Center, 2300 Eye Street NW, Washington, DC 20037, USA
4To
whom correspondence should be addressed at: Department of
Pharmacology, The George Washington University Medical Center, 2300
Eye Street NW, Washington, DC 20037, USA
Email: [email protected]
A variety of key events in the molecular apoptotic pathway
involve the mitochondria. Cyclosporin A (csA) affects the
mitochondria by inhibiting the mitochondrial permeability
transition (MPT), thereby preventing disruption of the
transmembrane potential. The role of the MPT in apoptosis
is not fully understood, but inhibition of the MPT may
prevent the release of mitochondrial caspase activators,
such as cytochrome c (cyt c), into the cytosol. Certain
hexavalent chromium [Cr(VI)] compounds are known occupational respiratory tract toxins and carcinogens. We have
previously shown that these compounds induce apoptosis
as a predominant mode of cell death and that this effect
can be observed in cell culture using soluble Cr(VI).
We show here that Cr(VI)-induced apoptosis in Chinese
hamster ovary (CHO) cells involves disruption of mitochondrial stability. Using a cyt c-specific monoclonal antibody,
we observed a dose-dependent release of mitochondrial cyt
c in cytosolic extracts of CHO cells exposed to apoptogenic
doses of sodium chromate. Co-treatment of these cells with
csA inhibited the release of cyt c and abrogated Cr(VI)induced apoptosis as determined by a reduction in internucleosomal DNA fragmentation. Co-treatment with csA
also markedly increased clonogenic survival of Cr(VI)treated CHO cells. In contrast, the general caspase inhibitor
Z-VAD-FMK markedly inhibited most of the morphological
and biochemical parameters of apoptosis but did not
prevent cyt c release and did not increase clonogenic
survival. These results suggest that the MPT plays an
important role in the regulation of mitochondrial cyt c
release and that this may be a critical point in the apoptotic
pathway in which cells are irreversibly committed to death.
Introduction
The apoptotic signaling pathway consists of a cascade of
molecular events which lead from the initiation of cell death
to eventual dismantling of the cell. The point at which the cell
is irrevocably committed to death is unclear. Inhibitors of the
Abbreviations: csA, cyclosporin A; cyt c, cytochrome c; DMSO, dimethyl
sulfoxide; IDF, internucleosomal DNA fragmentation; MPT, mitochondrial
permeability transition; PBS, phosphate-buffered saline; Z-VAD-FMK, Z-ValAla-Asp(OMe)-FMK.
*This work was conducted in partial fulfilment of the requirements for the
PhD degree in Genetics, Columbian Graduate School of Arts and Sciences,
The George Washington University.
© Oxford University Press
proteolytic caspase cascade are able to block some or all of
the morphological consequences associated with apoptosis, but
they appear to be unable to maintain clonogenic or long-term
survival (1–3). Cells exposed to pro-apoptotic stimuli in the
presence of caspase inhibitors will either undergo terminal
growth arrest or will die by some slower non-apoptotic
mechanism of cell death (4). Upstream of the caspases, a
variety of key events in apoptosis occur which involve the
mitochondria, including loss of mitochondrial transmembrane
potential, activation of the mitochondrial permeability transition (MPT), the participation of bcl-2 family proteins and the
release of caspase activators. If a mechanism for commitment
to cell death exists, it may involve these mitochondrial events.
Cytochrome c (cyt c) is released from the mitochondrial
intermembrane space to the cytosol where it forms a complex
with Apaf-1 and caspase-9, thereby triggering caspase-9 activation and initiating the caspase cascade (5–7). It has been
suggested that cyt c release may occur in response to a collapse
of the mitochondrial inner transmembrane potential (3). The
loss of membrane potential indicates a MPT caused by opening
of a large conductance MPT pore (8). Studies with isolated
mitochondria show that the MPT pore favors a closed state,
but certain physiological and pathological signals trigger pore
opening (9–11). The fully opened state creates a channel for
艋1.5 kDa molecules, resulting in dissipation of the H⫹ gradient
across the membrane and uncoupling of the respiratory chain
(12). Two models have been established which describe how
MPT pore opening may effect cyt c release. In the first model,
opening of the MPT pore may result in mitochondrial volume
expansion, eventually causing outer membrane rupture and
thereby releasing intermembrane caspase-activating proteins,
such as cyt c. The alternative model suggests that the MPT
may trigger a signal for the opening of an outer membrane
channel which allows cyt c release without concomitant
organellar swelling. Cyclosporin A (CsA), an immunosuppressant, can inhibit the MPT. CsA binds to Cyp-M, a
cyclophilin-family protein associated with the MPT pore,
causing it to dissociate from the pore complex (13,14). This
reaction is believed to increase the probability of pore closure.
In this study we have examined whether csA can block
apoptosis and restore clonogenic survival to cells exposed to
apoptogenic doses of hexavalent chromium [Cr(VI)].
Exposure to particulate Cr(VI) is strongly associated with
lung toxicity and increased incidence of lung cancer (15,16).
Soluble sodium chromate (Na2CrO4) can be used to study the
effects of Cr(VI) in culture. Cr(VI) is transported into cells
where it undergoes metabolic reduction to reactive genotoxic
species (17–19). This process leads to diverse genotoxic and
cytotoxic effects, including oxidative DNA damage (20),
DNA–DNA interstrand crosslinks (21–23), Cr–DNA adducts
(24,25), single-strand breaks (23), DNA–protein crosslinks
(25), chromosomal aberrations (26,27), DNA polymerase
arrest, RNA polymerase arrest (28) and inhibition of transcription and translation (29). Apoptosis is the predominant cellular
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D.E.Pritchard et al.
response to Cr(VI) in Chinese hamster ovary (CHO) cells in
culture (30,31). Therefore, we used CHO cells exposed to
Na2CrO4 as a model for apoptotic induction caused by diverse
genotoxic exposure.
In this study we show that Cr(VI) exposure causes a dosedependent release of cyt c and we examine the effect of csA
on cyt c release, apoptosis and clonogenic survival in Cr(VI)exposed CHO cells. csA affects the apoptotic signaling pathway
upstream of the caspase cascade, therefore, we investigated
the difference in its effects compared with the broad spectrum
caspase inhibitor Z-VAD-FMK. The ability of csA to inhibit
cyt c release and increase clonogenic survival, whereas ZVAD-FMK does not, suggests that apoptotic events that occur
at the mitochondria may denote a ‘point of no return’ in the
cell death pathway. Furthermore, cells that are rescued from
Cr-induced apoptosis by treatment with csA would survive
despite the genotoxic effects of Cr. This would potentially
promote the survival of genetically damaged cells which may
contribute to Cr(VI)-induced carcinogenesis.
Materials and methods
Cell culture
CHO AA8 cells were maintained in Eagle’s basal medium containing 10%
fetal bovine serum (Hyclone Laboratories, Logan, UT), 100 U/ml penicillin,
100 µg/ml streptomycin (Life Technologies, Gaithersburg, MD) and 2 mM
glutamine (Life Technologies) in a 95% air/5% CO2 humidified atmosphere
at 37°C.
Treatment of cells with chromium, Z-VAD-FMK and csA
Sodium chromate (Na2CrO4·4H2O) (J.T. Baker Chemical Co., Phillipsburg,
NJ) was dissolved in double distilled H2O and sterilized through a 0.2 µm
filter before use. Cells were treated with a final concentration of 0, 3, 6, 9 or
12 µM Na2CrO4 for 24 h in complete medium. After 24 h the medium was
replaced and the cells were incubated for an additional 24 h period. csA
(Sigma, St Louis. MO) was dissolved in dimethyl sulfoxide (DMSO). Cells
were pre-treated for 30 min prior to the Na2CrO4 exposure and co-treated for
the 24 h Na2CrO4 exposure period with a final concentration of 5 µM
csA. Z-Val-Ala-Asp(OMe)-FMK (Z-VAD-FMK) (Enzyme Systems Products,
Livermore, CA) was dissolved in DMSO and added to cells immediately prior
to the 24 h Na2CrO4 exposure period at a final concentration of 50 µM. The
final concentration of DMSO in the medium never exceeded 0.1%. Control
samples were exposed to an equivalent concentration of DMSO.
Internucleosomal DNA fragmentation (IDF) analysis
IDF was analyzed after the 24 h post-Na2CrO4 exposure period. Cells were
collected by trypsinization and combined with non-adherent cells from the
culture medium. The cells were centrifuged at 600 ⫻ g for 5 min. Cell pellets
were washed once in phosphate-buffered saline (PBS) and lysed in 500 µl of
digestion buffer (100 mM Tris–HCl, pH 8.0, 200 mM NaCl, 10 mM EDTA,
4% SDS and 0.2 mg/ml proteinase K), followed by incubation at 55°C for 18
h. DNA was extracted with phenol/chloroform and then chloroform alone,
incubated with 0.1 mg/ml RNase A for 30 min at 37°C and finally precipitated
with 100% ethanol. Sample volumes equivalent to 7.5 µg were electrophoresed
on a 1% agarose gel with 0.5 µg/ml ethidium bromide and visualized with an
Eagle Eye II still video imaging system (Stratagene, La Jolla, CA).
Preparation of cytosolic fractions
Cr-treated cells were harvested by gentle cell scraping and combined with
non-adherent cells from the culture medium. The cells were centrifuged at
300 ⫻ g for 5 min at 4°C and washed once with ice-cold PBS. Cell pellets
were resuspended at 1.0–2.0⫻107 cells/ml in ice-cold isotonic cytosol buffer
[210 mM mannitol, 70 mM sucrose, 5 mM HEPES, pH 7.5, 0.2 mM EGTA, 1
mM MgCl2, 5 mM glutamic acid, 5 mM malate, 0.1 mM phenylmethylsulfonyl
fluoride, supplemented with protease inhibitors (5 µg/ml pepstatin A, 10 µg/
ml leupeptin, 2 µg/ml aprotonin) (Sigma), and containing.004% digitonin to
permeabilize the cells]. Cell suspensions were stirred for 10 min and then
centrifuged at 12 000 ⫻ g for 15 min at 4°C to remove cell membrane
debris, mitochondria and other organelles. The resulting supernatants were
concentrated through Millipore Ultrafree-CL filters and stored at –20°C until
used for PAGE and western blot analysis.
For measurement of the full cyt c content of a cellular sample, cytoplasmic
extracts were isolated in a hypotonic solution. This cell fraction contains lysed
2028
mitochondria and pieces of plasma membrane and other organelles, but not
whole cells or nuclei. Cell pellets were resuspended at 1.0–2.0⫻107 cells/ml
in ice-cold hypotonic cytosol buffer [20 mM KCl, 20 mM HEPES, pH 7.5,
1.5 mM MgCl2, 1 mM EDTA, 1 mM DTT, 0.1 mM phenylmethylsulfonyl
fluoride, 0.2 mM CaCl2, supplemented with protease inhibitors (5 µg/ml
pepstatin A, 10 µg/ml leupeptin, 2 µg/ml aprotonin) (Sigma)]. Cell suspensions
were vortexed for 10 min and then centrifuged at 5000 ⫻ g for 15 min at
4°C. The resulting supernatants were concentrated through Millipore UltrafreeCL filters and stored at –20°C until used for analysis.
Protein electrophoresis and western blotting
Protein concentrations of the cytosolic fractions were determined with a DC
Protein Assay Kit II (Bio-Rad, Hercules, CA). Samples containing an equal
amount of concentrated proteins were separated on a 4–20% gradient SDS–
polyacrylamide gel and transferred to a polyvinylidene difluoride membrane
by electroblotting for 1 h at 30 V at 4°C. Non-specific membrane binding
sites were blocked overnight at 4°C with blocking solution [TBS-T (2.7 mM
KCl, 138 mM NaCl, 20 mM Tris base, pH 7.4, 0.1% Tween-20) containing
5% non-fat dry milk]. The membrane was incubated with primary mouse anticyt c monoclonal antibody (Pharmingen, San Diego, CA) diluted 1:2000 in
blocking solution (0.5 µg/ml) for 1 h at room temperature. The membrane
was washed thoroughly with TBS-T and then incubated for 1 h with
a horseradish peroxidase-conjugated anti-mouse IgG secondary antibody
(Amersham, Arlington Heights, IL) diluted 1:2000 in blocking solution.
The secondary probe was detected by using the Renaissance enhanced
chemiluminescence detection system (NEN, Boston, MA).
Integrated density analysis
Relative densities of protein or DNA bands were determined using an Eagle
Eye II still video imaging system with Eaglesight software (Stratagene).
Clonogenicity analysis
CHO cells were seeded at 105 cells/60 mm2 dish and incubated for 24 h prior
to csA or Z-VAD-FMK treatment and Na2CrO4 exposure. After the 24 h
exposure, cells were collected by trypsinization, counted, and reseeded at
2⫻102 cells/60 mm2 dish in triplicate. The plates were incubated for 9–10
days, then rinsed with PBS and incubated with crystal violet stain (80%
methanol, 2% formaldehyde, 2.5 g/l crystal violet) for 15–30 min at room
temperature. The plates were thoroughly rinsed with dH2O and allowed to
dry. Colonies were counted, the triplicates were averaged and long-term
survival was determined as a percent of control.
Cr uptake analysis
Conical tubes (15 ml) containing 5⫻105 CHO cells/sample were pre-treated
with either 5 µM csA or an equivalent volume of DMSO (final concentration
0.1%) for 30 min at 37°C. The cells were then exposed to appropriate
concentrations of Na2CrO4 spiked with Na251CrO4 for 3 h at 37°C. The cells
were centrifuged at 300 ⫻ g for 5 min at 4°C. Cell pellets were washed twice
in PBS and lysed in 500 µl lysis buffer (100 mM Tris–HCl, pH 8.0, 200 mM
NaCl, 10 mM EDTA, 4% SDS). An aliquot of 100 µl of each sample was
transferred to a 5 ml scintillation vial and combined with 1 ml of Ecolite
scintillation cocktail (ICN, Irvine, CA). Values for d.p.m./105 cells were
determined in a Beckman LS6500 scintillation counter (Beckman Instruments,
Fullerton, CA).
Results
To determine if Cr(VI)-induced apoptosis involves mitochondrial disruption, we isolated the cytosolic fractions of apoptotic
CHO cells and analyzed them for the presence of cyt c. We
based our Cr(VI) concentrations on previously reported dosedependant increases in CHO cell apoptosis and decreases in
clonogenicity following Cr(VI) exposure (32). We isolated the
cytosolic fractions using a method based on selective plasma
membrane permeabilization in isotonic buffer containing low
concentrations of digitonin (33,34). This method allows isolation of cytosol without contamination with mitochondriaassociated cyt c. We carefully titrated the level of digitonin
used in the isotonic cytosol buffer to determine the optimal
conditions for plasma membrane permeability without mitochondrial membrane permeability in CHO cells. We observed
that a 24 h exposure to different concentrations of Na2CrO4
caused a dose-dependent increase in cytosolic levels of cyt c
(Figure 1A). Densitometric analysis of independent western
Cyclosporin A inhibits Cr(VI)-induced apoptosis
Fig. 1. Cr(VI) causes dose-dependent release of cyt c. (A) Cytosolic
fractions of CHO cells exposed to different concentrations of Na2CrO4 were
collected and analyzed for cyt c by western blotting. The Na2CrO4 doses
ranged from 0 to 12 µM as indicated. Purified bovine heart cyt c was used
as a standard (St). (B) Densitometric analysis of cyt c western blots shows
that the relative density of cytosolic cyt c increases significantly at 6, 9 and
12 µM Na2CrO4. Results are the means ⫾ SE of three independent
experiments. The * denotes the sample mean of two independent
experiments.
blots showed that at the 3 µM dose only a minor increase in
cytosolic cyt c was observed (Figure 1B). However, the
cytosolic cyt c levels increased as the Na2CrO4 dose increased
from 3 to 12 µM. The increases in cytosolic cyt c levels were
statistically significant by Student’s t-test at the 6, 9 and 12
µM Na2CrO4 doses (P ⫽ 0.024, 2.3⫻10–7 and 7.4⫻10–4,
respectively). cyt c release was maximal at the highest dose
tested (12 µM), which corresponds to nearly 100% apoptotic
cell death in CHO cells (data not shown). Cytosolic samples
with the full mitochondrial cyt c content isolated using a
hypotonic buffer were also analyzed. The level of cyt c release
at the 12 µM dose was ~75% of the total cyt c content of the
hypotonic control sample (data not shown).
Cytosolic fractions isolated from cells exposed to 9 µM
Na2CrO4 and pre-treated for 30 min and co-treated for 24 h
with 5 µM csA were analyzed for the presence of cyt c by
western blotting. Treatment with csA markedly inhibited the
cyt c release induced by Na2CrO4 (Figure 2A). In contrast,
the general caspase inhibitor Z-VAD-FMK had no effect on
the release of cyt c (Figure 2B). Densitometric analysis of
independent western blots showed that only 24% of the cyt c
release induced by 9 µM Na2CrO4 exposure was observed
when cells were treated with csA as compared with cells
treated with Z-VAD-FMK or solvent alone (Figure 2C).
Degradation of DNA into 180–200 bp oligonucleosomal
Fig. 2. Inhibition of Cr(VI)-induced mitochondrial cyt c release by csA but
not by Z-VAD-FMK. (A) A 30 min pre-treatment and 24 h co-treatment
with 5 µM csA was given to CHO cells exposed to 0 or 9 µM Na2CrO4.
Cytosolic fractions of these cells were compared with cytosols of cells
without csA treatment for the presence of cyt c by western blot analysis. An
aliquot of 10 ng of purified bovine heart cyt c was used as a standard (St)
in the first lane. (B) CHO cells exposed to 0 or 9 µM Na2CrO4 were either
treated with 50 µM Z-VAD-FMK (Z-VAD) or a solvent control and the
cytosols were compared for the presence of cyt c by western blot analysis.
An aliquot of 10 ng of purified bovine heart cyt c was used as a standard
(St) in the first lane. (C) Densitometric analysis of western blots shows the
percentage of cytosolic cyt c in samples treated with 9 µM Na2CrO4 and
co-treated with either csA or Z-VAD-FMK compared with the samples
which received solvent only. Results are the means ⫾ SE of three
independent experiments.
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D.E.Pritchard et al.
Fig. 4. Increased clonogenicity of Cr-exposed CHO cells by csA but not by
Z-VAD-FMK. CHO cells exposed to increasing concentrations of Na2CrO4
for 24 h, and either treated with 5 µM csA, 50 µM Z-VAD-FMK or a
solvent control were analyzed for cloning efficiency. The number of
colonies for the indicated concentrations of Na2CrO4 are expressed as a
percentage of the 0 µM control for each group. Results are the means ⫾ SE
of three independent experiments.
Fig. 3. Reduction of Cr-induced apoptosis by csA and Z-VAD-FMK. (A)
Genomic DNA was isolated from CHO cells exposed to 0 or 9 µM
Na2CrO4 for 24 h, and either treated with 5 µM csA, 50 µM Z-VAD-FMK
or a solvent control. The DNA was isolated 24 h after exposure and
analyzed for IDF. A 1 kb ladder was used as a DNA marker in lane 1. (B)
Densitometric analysis of IDF shows the percentage of cytosolic cyt c from
samples treated with 9 µM Na2CrO4 and co-treated with either csA or ZVAD-FMK compared with the samples which received solvent only. Results
are the means ⫾ SE of three independent experiments.
fragments is a hallmark indicator of apoptosis (35). IDF is
considered a late stage apoptotic event and is induced by
active caspases (36). CHO cells exposed to 9 µM Na2CrO4
undergo extensive IDF (Figure 3A). Inhibition of the caspases
with 50 µM Z-VAD-FMK was able to markedly reduce Cr2030
induced IDF, verifying the dependence for IDF on caspase
activity. Treatment with 5 µM csA was also able to reduce
IDF induced by 9 µM Na2CrO4, indicating that cyt c release
may also be a necessary component of the IDF pathway.
Densitometric analysis of three independent IDF gels showed
that Z-VAD-FMK is able to reduce the amount of IDF to 36%
of that caused by 9 µM Na2CrO4 alone, while csA can reduce
Cr-induced IDF to a comparable 41% (Figure 3B).
Since csA was shown to block mitochondrial cyt c release
and reduce apoptosis, we examined whether it could restore
clonogenic survival to Cr-treated CHO cells. Clonogenicity is
an indicator of a cell’s long-term survival and ability to grow
and form colonies in culture after exposure to a toxic agent.
Clonogenicity reflects both apoptosis and long-term growth
arrest caused by the toxic exposure. The clonogenicity of CHO
cells exposed to different concentrations of Na2CrO4 alone
and with pre- and co-treatments with csA or Z-VAD-FMK
was determined (Figure 4). At the 3 and 6 µM doses csA was
able to increase the number of colonies that formed. The
increase in clonogenicity was statistically significant by Student’s t-test at the 3 and 6 µM Na2CrO4 doses (P ⫽ 0.021
and 0.003, respectively). Exposure to 3 µM Na2CrO4 decreased
clonogenic survival to 57% compared with the 0 µM control.
Pre- and co-treatment with 5 µM csA at 3 µM Na2CrO4
increased the clonogenicity to nearly 100% of control. The 6
µM dose reduced clonogenicity to 9% of control, but csA
increased this clonogenic potential to 30%. We observed the
same trend at the highly toxic 9 µM dose, although it was not
statistically significant. In contrast, Z-VAD-FMK had no effect
on clonogenicity of Cr-exposed CHO cells.
Because csA, but not Z-VAD-FMK, increased clonogenic
survival after chromium treatment, we sought to determine
whether csA affected uptake of Cr(VI) from the medium.
Cyclosporin A inhibits Cr(VI)-induced apoptosis
Fig. 5. CsA has no effect on Cr uptake. CHO cells exposed to increasing
concentrations of Na2CrO4 spiked with Na251CrO4 for 3 h were either
pre-treated for 30 min and co-treated with 5 µM csA or treated with a
solvent control. The cells were then lysed and analyzed for intracellular
51Cr on a scintillation counter. Results are the means ⫾ SE of three
independent experiments.
Importantly, csA did not alter Cr uptake by CHO cells (Figure
5). CHO cells treated with csA and exposed to different levels
of Na251CrO4 for 3 h had similar levels of intracellular 51Cr
to the solvent controls.
Discussion
Dysregulation of the homeostasis between cell proliferation
and cell death is involved in a vast array of human health
consequences, including tumorigenesis. If apoptosis fails to
occur normally when cells have been damaged or injured, then
it may contribute to the progression of cancer. In studying
the relationship between apoptosis and cancer, genotoxic
carcinogens are particularly interesting because in order to
induce neoplastic transformation, doses that induce a certain
level of apoptosis are often required (16). In the case of
Cr(VI), exposure to levels of particulate Cr compounds that
are associated with lung cancer usually also cause respiratory
toxicity that manifests as perforation of the nasal septum and/
or respiratory tract ulcerations (16). These conditions are
associated with high levels of cell death, thus a key question
is how and why do certain cells exposed to a carcinogen
escape death when others die? In tissue exposed to a Cr(VI)
compound, individual cells may experience different local
concentrations that are possibly genotoxic but not apoptotic.
The populations of cells which either escape or are resistant
to apoptosis may become the precursor pools out of which
neoplastic variants will emerge. Therefore, it is important to
determine the point in the apoptotic pathway when a cell is
irrevocably committed to death. Our results here suggest that
the ‘point of no return’ for chromium-induced apoptosis occurs
at the mitochondria and that cells that survive an apoptotic
challenge may have a selective advantage in their ability to
maintain mitochondrial stability.
Exposure to apoptotic doses of Cr(VI) causes mitochondrial
instability, as indicated by the release of cyt c into the cytosol
(Figure 1). It is believed that cyt c release is necessary for
activation of the caspase cascade or, in some cases, at least
for the amplification of caspase activity required for a normal
apoptotic response (6,7,12). Here we show that treatment with
the caspase inhibitor Z-VAD-FMK is able to inhibit DNA
fragmentation associated with apoptosis (Figure 3) but does
not affect long-term clonogenic survival (Figure 4). Despite
inhibition of the morphological apoptotic characteristics caused
by active caspases, the cells never regain replicative competency and eventually die. The point at which a cell is committed
to death should, therefore, occur upstream of the caspase
cascade.
csA functions at the mitochondria by promoting mitochondrial stability and preventing the release of caspase activators.
It is believed to bind at the MPT pore, thereby increasing the
probability of pore closure and preventing disruption of the
mitochondrial inner membrane potential (13,14). Our results
indicate that treatment with csA is able to inhibit apoptosis
(Figure 3) and block mitochondrial cyt c release (Figure 2).
Most importantly, csA treatment is able to restore long-term
clonogenic survival to cells that have been exposed to apoptotic
doses of Cr(VI) (Figure 4). These results suggest that maintaining mitochondrial stability may allow cells to escape
apoptotic death and that the point along the apoptotic pathway
in which cells are committed to death involves mitochondria.
It is unclear whether the ‘point of no return’ in cell death is
disruption of the mitochondrial membrane potential or release
of mitochondrial caspase activators. These two events may be
intricately linked, however, some studies have shown that the
release of cyt c can occur before or in the absence of disruption
of the transmembrane potential (37,38). It is possible that
different regulatory events which control mitochondrial membrane permeability are dependent on cell type. Nevertheless,
it seems likely that uncoupling of the respiratory chain and
subsequent release of cyt c from the mitochondria would
prevent long-term cell survival. Release of cyt c from a
mitochondrion should cause an irreversible disruption of oxidative phosphorylation, rendering the mitochondrion non-functional for energy metabolism and cell growth. The extent of
cyt c release may vary for each cell depending on the individual
molecular and cellular factors that govern apoptosis. Thus it
is possible that when a critical amount of cyt c is released
from mitochondria the cell is committed to death, either
apoptotic or otherwise.
At lower doses (3 µM) of Cr(VI) only a small amount of
cyt c release (reproducible but not statistically significant)
could be observed at one particular time point (Figure 1).
Interestingly, these low doses caused a significant decrease in
clonogenic survival (57% of control) over a longer course of
time. Cyt c release at lower doses of Cr(VI) may occur
gradually followed by a protracted increase in apoptosis.
Therefore, this response accounts for the significant decrease
in clonogenic survival, but a marked release of cyt c at any
one moment in time would not be expected. Nevertheless,
treatment with csA was able to significantly increase the longterm survival of the cells, raising clonogenicity from 57 to
nearly 100% (Figure 4). csA was not able to significantly
increase the clonogenic survival of cells treated with the
highest dose of Cr(VI) (9 µM). This is undoubtedly due to the
presence of an insurmountable amount of damage to critical
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D.E.Pritchard et al.
macromolecules making it impossible for the cells to regain
replicative competence even if apoptosis is prevented.
Here we have shown that the anti-apoptotic and pro-survival
effects of csA are not the result of altered cellular uptake of
Cr(VI) (Figure 5). We have previously shown that treatment
of cells with Cr(VI) causes structural DNA damage in the
form of adducts, single-strand breaks, DNA crosslinks and
chromosomal abnormalities (39). Some of this structural damage leads to functional damage in the form of base-specific
DNA and RNA polymerase arrest and inhibition of DNA
replication and transcription. The cellular response to this
damage includes altered gene expression, G1/S and S phase
cell cycle arrest, terminal growth arrest and apoptosis
(21,23,28–31). Survivors exhibit DNA deletion mutations and
may progress to neoplastic transformation (39). We have
observed that p53 levels are increased in Cr(VI)-treated cells
(40) and, presumably, this increase would be part of the cellular
response to genotoxicity, contributing to G1/S checkpoint arrest
and apoptosis in the presence of excessive unrepairable damage.
CHO cells express a mutant p53 which alters the G1/S
checkpoint arrest arm of the p53 molecular response pathway
(41,42), but they still undergo apoptosis after Cr(VI) treatment.
Nevertheless, the p53 status of the cell line does not negate
the observation that csA inhibited apoptosis and increased
clonogenic survival, whereas Z-VAD-FMK inhibited apoptosis
but had no effect on clonogenic survival. Experiments on p53
wild-type diploid human lung cells are in progress but are
difficult and slow due to the nature of the primary cell cultures.
The ability of cells to undergo apoptosis in response to
genotoxic insult may represent a protective mechanism against
neoplastic transformation in the organism by eliminating cells
that contain unrepaired genetic lesions. The cells that survive
genotoxic damage by escaping apoptosis and gradually
regaining replicative competence may harbor the deleterious
effects of the apoptotic insult that induced death in the rest of
the cell population. Thus, these cells may be predisposed to
further progression towards a neoplastic phenotype. CsA,
widely used as an immunosuppressive drug to treat autoimmune
diseases and prevent rejection in organ transplantation, has
many common side-effects, including an increased risk of
cancer (43). The ability of csA to reduce apoptosis may be a
consideration as a mechanism for the increased risk of cancer
in patients who take csA long term. In this study, cells acquired
resistance to apoptosis by interference with the mitochondrial
instability conveyed by the pharmacological treatment. However, this raises the interesting question of whether the cells
that survive apoptotic doses of a chemical carcinogen such as
Cr(VI), in the absence of any pharmacological intervention,
have a stochastic resistance to mitochondrial instability. By
way of this resistance, such a sub-population of cells may be
selected for by the apoptotic inducer, thereby increasing the
population of cells resistant to apoptosis. If early tumor growth
requires a net accumulation of cells due to alterations in the
growth/death ratio, then selection of cells with resistance to
apoptosis may facilitate the early steps of carcinogenesis.
Acknowledgements
The authors thank Bronwyn Owens and Kristy Markos for their expert
technical assistance and Dr. Anne Murphy for her valuable assistance in
method development.
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Received February 4, 2000; revised July 6, 2000; accepted July 11, 2000
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