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Oncogene (1999) 18, 6641 ± 6646
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Mitochondrial DNA determines the cellular response to cancer therapeutic
agents
Keshav K Singh*,1, James Russell1, Barbara Sigala1, Yonggong Zhang1, Jerry Williams1 and
Kylie F Keshav2
1
Johns Hopkins Oncology Center, 600 North Wolfe Street, Room 2-121, Baltimore, Maryland, MD 21287, USA; 2Department of
Bioscience and Biotechnology, Drexel University, 32nd and Chestnut Streets, Philadelphia, Pennsylvania, PA 19104, USA
Mutations in the mitochondrial genome leading to
mitochondrial dysfunction have been reported in a
variety of cancers. However, the potential implication
of these ®ndings in the cellular response to cancer
therapeutic agents is unclear. To examine the importance
of mitochondrial DNA (mitDNA) encoded functions in
cancer therapeutic response, we determined the clonogenic survival of HSL2 (Rho+, HeLa subline), and its
derivative cell line lacking mitDNA (Rho0) after
exposure to di€erent anticancer agents. We found that
isogenic Rho0 cells lacking mitDNA were extremely
resistant to adriamycin and photodynamic therapy
(PDT) induced cell death, whereas the Rho+ cell line
was sensitive. However, there was no measurable
di€erence in the responses of these cell lines to either
alkylating agent or g-radiation. We show that the
development of resistance to adriamycin was not due to
changes in apoptotic cell death, cell cycle response or to
the uptake of adriamycin in isogenic Rho0 cells. We also
demonstrate that exposure of HeLa cells to adriamycin
leads to mutations in mitDNA. These studies provide
direct evidence that mitDNA plays an important role in
cellular sensitivity to cancer therapeutic agents.
Keywords: mitochondria; mitochondrial DNA; cell
death; adriamycin
Introduction
With the exception of erythrocytes, all human cells
contain mitochondria (Schatz, 1995; Singh, 1998). Cells
derive up to 95% of their energy through oxidative
phosphorylation carried out in mitochondria. Mitochondria, therefore, are regarded as the powerhouse of
the cell (Schatz, 1995; Singh, 1998). In addition to
producing energy, mitochondria also support many
essential cellular functions. These include intermediatory metabolism, ion homeostasis, synthesis of lipids,
amino acids, and nucleotides, active transport processes, cell motility, cell proliferation and initiation of
programmed cell death (apoptosis) (Schatz, 1995;
Singh, 1998; Wallace et al., 1997; Petit and Kroemer,
1998). Thus mitochondria participate in numerous
functions in the cell.
*Correspondence: KK Singh
Received 18 February 1999; revised 28 June 1999; accepted 6 July
1999
Interestingly, one of the most common and
profound phenotypes of many cancer cells is their
defective mitochondrial function (Pedersen, 1978, 1997;
Rempel, 1996). Mitochondria occupy up to 50% of the
cytoplasmic volume of most animal cells. However,
most cancer cells have few mitochondria, less
mitochondrial content and are highly glycolytic
(Pedersen, 1978, 1997; Rempel et al., 1996). Cancer
cell mitochondria have a characteristic shape and size
(Pedersen, 1978, 1997; Rempel et al., 1996). Studies
indicate that cancer cell mitochondria are small, have
few cristae, and have altered membrane composition
and altered membrane potential (Pedersen, 1978, 1997;
Rempel, 1996). Studies also indicate that the commitment to apoptosis is coupled with an early drop in
mitochondrial membrane potential, a drop in the rate
of mitochondrial translation and defects in maturation
of precursors of mitochondrial proteins (Petit and
Kroemer, 1998).
Mitochondria house approximately 1000 proteins
that are encoded by nuclear DNA (Schatz, 1995; Singh,
1998). These include anti-apoptotic Bcl2 and apoptosis
inducing factor (Petit and Kroemer, 1998). Mitochondria contain their own DNA (Schatz, 1995; Keshav
and Yoshida, 1998). Mitochondrial DNA (mitDNA)
encodes 13 proteins consisting of four enzyme
complexes of the respiratory chain (Complex I, III,
IV and ATP synthase) and genes specifying 2 rRNA
and 22 tRNA that are components of mitochondrial
protein synthesizing system (Schatz, 1995; Singh, 1998).
Changes in the mitochondrial genome have been
reported in cancer cells (Welter et al., 1989; Boultwood et al., 1996; Liang, 1996), and are implicated in
carcinogenesis and other diseases (Singh, 1998).
Furthermore, microscopic study of oncocytic tumors
revealed mitochondrial hyperplasia (Tallini, 1997), and
di€erential expression of mitochondrial cytochrome
oxidase II in benign and malignant breast tissues
(Sharp et al., 1992) and mitochondria dependent
increased sensitivity to cis-platinum is reported (Liang
and Ullyatt, 1998). Taken together, these studies
underscore the importance of the mitochondrion and
its genome in cancer and cancer therapy.
In order to de®ne the role of mitochondria in cancer
therapy, we have examined the response of a tumor cell
line completely lacking mitDNA to di€erent anticancer
agents. Our results demonstrate that mitDNA is
important for cell death induced by adriamycin and
PDT but not by other anticancer agents. This study
indicates that the mitochondrial genome may play an
important role in the response of cancer cells to
therapeutic agents.
Mitochondrial DNA and cell death
KK Singh et al
6642
Results
Mitochondrial DNA is important for cellular sensitivity
to cancer therapeutic agents
Ethidium bromide is a known inhibitor of mitDNA
replication and transcription because it selectively
intercalates with mitDNA (Nass, 1972). As a result
human cell lines devoid of mitDNA can be isolated
(Morais et al., 1994). To analyse the importance of
the mitDNA encoded functions in cellular response to
di€erent anticancer agents, HeLa and its mitDNAless derivative were exposed to various agents. Figure
1a,b demonstrates that both cell lines were equally
sensitive to ionizing radiation and to a model
alkylating agent, MNNG. However, the response of
mitDNA-less cells to adriamycin was di€erent (Figure
1c). HeLa cells lacking mitDNA were approximately
twice as resistant to adriamycin compared to the
parental cells. The doses required for 90% cell kill
were 1.6 mM (Rho0) and 0.75 mM (HeLa parental
cells).
Mitochondria have been implicated as a primary
target of photodynamic therapy (PDT) (Sharkey et al.,
1993; Hilf et al., 1987; Woodburn et al., 1992).
However, the importance of mitDNA in PDT is not
clear. We assayed the cytotoxic response of HeLa cells
to PDT after exposure to Mesoporphyrin IX (MPIX),
Suramin and light (Figure 1d). Interestingly, Rho0 cells
showed complete insensitivity to photosensitization by
MPIX and Suramin. We conclude that adriamycin and
PDT-induced cytotoxicity requires mitDNA-encoded
functions. However, these functions are not required
for cell death induced by ionizing radiation or by
model alkylating agent MNNG.
Resistance to anticancer agents is not due to inhibition of
apoptosis in Rho0 cells
Mitochondria play a central role in programmed cell
death or apoptosis (Petit and Kroemer, 1998).
Mitochondria can trigger cell death in a number of
ways including a disruption in energy metabolism
(Kiberstis, 1999). Because Rho0 cells have altered
(a)
(b)
(c)
(d)
Figure 1 E€ect of anticancer agents on cell survival. Exponentially growing parental HSL2 (Rho+) and its derivative cell line
devoid of entire mitDNA (Rho0) were exposed to (a) g-radiation; (b) MNNG; (c) adriamycin; (d) PDT. For PDT treatment cells
were exposed to indicated concentration of suramin, mesoporphyrin IX (MPIX), or suramin plus MPIX for 4 h. Cells were then
irradiated for 3 min, washed immediately with PBS and plated. Intensity of light irradiation ranged from 2.4 ± 2.6 mW/cm26100
measured by Blak-Ray ultraviolet meter (Ultraviolet Products, Inc San Gabriel, CA, USA). The cell survival curve was plotted as
described in Materials and methods. All dose points were measured in triplicate. Each point indicates the mean+s.d. of three
independent experiments
Mitochondrial DNA and cell death
KK Singh et al
mitochondrial functions and are resistant to adriamycin, we investigated whether adriamycin resistance was
due to inhibition of apoptosis. HeLa cells with
functional mitochondria underwent apoptosis after
treatment with adriamycin (1.5 mM, 45 min). When
Rho0 cells were treated with the same drug apoptosis
was also observed. The number of apoptotic cells was
counted microscopically as described in Materials and
methods. Table 1 indicates that there was no signi®cant
reduction in number of cells undergoing apoptosis
either after 24 or 48 h adriamycin treatment of Rho0
cells. We conclude that removal of mitochondrial
function does not prevent apoptotic death and it does
not contribute to resistance of Rho0 HeLa cells to
adriamycin.
Loss of mitDNA does not alter cell cycle response
Adriamycin inhibits DNA replication because it
directly intercalates between the bases and blocks
DNA topoisomerase II function (Singhal et al., 1997)
resulting in S phase block. This suggests that resistance
to cytotoxicity could arise due to an altered cell cycle
in Rho0 cells as opposed to Rho+ cells. As cells in S
phase are more sensitive to adriamycin toxicity (To€oli
et al., 1996; Nass, 1972; Singhal et al., 1997), we
reasoned that di€erences in cell cycle distribution
between the two cell lines may account for their
di€erent drug sensitivities. To test this possibility we
measured the cell cycle response of HeLa and the
Table 1 Per cent apoptosis in Rho+ and Rho0 HeLa cells after
adriamycin treatment. Cells were exposed to 1.5 mM adriamycin for
45 min and apoptosis was measured as described in Materials and
methods
Cell line
Rho+
Untreated control
24 h after treatment
48 h after treatment
Rho0
Untreated control
24 h after treatment
48 h after treatment
Apoptotic
total
Apoptotic
fraction (%)
5/209
6/214
14/223
2.4
2.8
6.3
1/210
2/217
4/215
0.5
0.9
1.8
isogenic Rho0 cells. The S phase fraction for the HeLa
parental cells was approximately 0.3, and for the Rho0
cells 0.28 (Figure 2). This slight di€erence does not
seem to explain the observed twofold di€erence in drug
sensitivity between the cell lines. Adriamycin treatment
causes cells to accumulate in S phase. After a short
exposure (45 min) to 1.25 mM adriamycin, the S phase
fraction of Rho+ cells increased by a factor of 2.5; for
Rho0 cells, the S phase fraction doubled (data not
shown). Because the cell cycle distributions are so
similar, we conclude that mitDNA does not govern the
cell cycle response to adriamycin.
Adriamycin uptake is not altered in cells lacking
mitDNA
The high level of adriamycin resistance observed in
Rho0 cells suggests that adriamycin cytotoxicity is
dependent on mitDNA-encoded functions. However, it
is possible that resistance may arise because of
decreased uptake of adriamycin. To test this possibility, HeLa cells and its Rho0 derivative were grown in
DMEM and exposed to 1.2 mM adriamycin for 45 min.
The level of adriamycin uptake was quantitated by
FACS analysis as described previously. Figure 3
demonstrates that level of adriamycin uptake was
independent of presence or absence of mitDNA
(mean peak value 173.2 for Rho+ versus the 178.5
for Rho0). We conclude that loss of mitDNA-encoded
functions do not a€ect the level of uptake of
adriamycin.
Adriamycin induces damage to mitDNA
Several studies have suggested that adriamycin-induced
cardiomyopathy may be due to increased oxidative
damage (Singhal et al., 1997; Palmeira et al., 1997).
Adriamycin undergoes redox cycling between quinone
and semiquinone which, when reoxidized in the
presence of O2, generates highly reactive oxygen
species, ROS (Singh, 1998; Kule et al., 1994). Human
mitDNA has no protective histones and is continuously exposed to ROS generated endogenously during
normal oxidative phosphorylation in mitochondria
(Singh, 1998). We therefore envisioned that mitDNA
may be susceptible to damage by adriamycin. To test
Figure 2 Flow cytometry analysis of parental HSL2 (Rho+) and its derivative cell line devoid of entire mitDNA (Rho0). Cell lines
were grown to exponential phase and analysed for cell cycle distribution as described in Materials and methods
6643
Mitochondrial DNA and cell death
KK Singh et al
6644
Figure 3 Uptake of adriamycin: parental HSL2 (Rho+) and its
derivative cell line devoid of entire mitDNA (Rho0) was grown to
exponential phase and exposed to adriamycin (1.25 mM) for
45 min. Cells were washed and analysed as described in
Materials and methods
this possibility, we isolated total DNA from Rho0 cells
treated with adriamycin (0.75 mM) for 45 min and from
untreated control cells. Total DNA was digested with
PvuII, which gives rise to a single 16.6 Kb fragment of
mitDNA. Southern blot analysis using a mitDNA
speci®c probe revealed a 16.6 Kb fragment in the
untreated control (Figure 4). Figure 4 shows that
exposure to adriamycin causes damage to mitDNA
(lane 3) resulting in deletion of mitDNA (smaller
mitDNA fragments generated after treatment). Figure
4 demonstrates that the level of wild-type mitDNA
(16.6 Kb fragment) was noticeably less after 24 h and a
new smaller fragment was generated 48 h after short
exposure to adriamycin. We did not ®nd any
quantitative di€erences in DNA loading between the
treated and untreated control as judged by ethidium
bromide staining. We conclude that adriamycin causes
damage to mitDNA.
Discussion
Mutations in the mitochondrial genome occur in a
variety of cancer cells (Polyak et al., 1998; Liang, 1996;
Habano et al., 1998). However, the potential implication of mitDNA mutations in cancer therapeutic
response has until now not been evident. Our study
demonstrates that functions encoded by the mitochondrial genome are important in cancer therapeutic
response. Cell death induced by adriamycin in Rho0
cells was signi®cantly less when compared to cells
Figure 4 Adriamycin-induced damage to mitochondrial DNA:
parental HSL2 cells were grown to exponential phase and exposed
to adriamycin (1.25 mM) for 45 min. Cells were then washed and
grown to indicated time. Total DNA was extracted (as described
in Materials and methods), digested with PvuII and fractionated
by 1% agarose gel electrophoresis. Southern blot analysis was
performed using the mitochondrial DNA probe as described in
Materials and methods. Marker; Lambda phage DNA digested
with HindIII and EcoRI
containing mitDNA. Results of this study also
demonstrate the importance of mitDNA in PDT
response because Rho0 cells show resistance to PDT
(Figure 1d). We have demonstrated that the di€erence
in cell death between Rho+ and Rho0 cells was not due
to a di€erence in the cell cycle response or di€erences
in drug uptake. Rather the Rho0 derivative, like the
parental HeLa cells, arrests in the S phase of cell cycle
in response to adriamycin (To€oli et al., 1996; Singhal
et al., 1997) and both of these cell lines also show equal
uptake of adriamycin into the cell. Notably, the
mitochondrial genome does not play an important
role in cell death induced by g-radiation or model
alkylating agent MNNG because the presence or
absence of mitDNA did not a€ect the level of cell
survival (see Figure 1). These results suggest that
responses to these agents are independent of functions
encoded by the mitochondrial genome.
Rho0 cells possess all the essential structures
involved in apoptosis, i.e. they contain (a) a normal
mitochondrial transmembrane potential, (b) are capable of pre-apoptotic disruption of mitochondrial
transmembrane potential, (c) contain mitochondrial
apoptosis inducing factor(s) and (d) notably are
competent in apoptosis (Marchetti et al., 1996; Petit
and Kroemer, 1998). Consistent with the observations
of Marchetti et al. (1996) and Petit and Kroemer
(1998), we found no signi®cant di€erences between the
Mitochondrial DNA and cell death
KK Singh et al
number of Rho+ or Rho0 cells undergoing apoptosis
after adriamycin treatment (Table 1). Our data
therefore con®rms that removal of mitDNA-encoded
functions does not a€ect apoptosis and indicates that
apoptosis does not play a role in resistance to
adriamycin. Furthermore, Eguchi et al. (1997) have
demonstrated that necrotic death in HeLa cells is
independent of mitochondrial function. Together, these
studies indicate neither apoptosis nor necrosis is likely
to play a role in the observed resistance to adriamycin.
p53 was recently shown to control the expression of
several genes involved in protecting cells from
mitochondria mediated apoptosis (Polyak et al.,
1997). Since HeLa cells are de®cient in p53 function
(Isaacs et al., 1997), our results also suggest that
resistance to adriamycin and PDT is independent of
p53 function. These results are further supported by
Fisher et al. (1999) who recently provided direct
evidence that tumor cell sensitivity to PDT is
independent of p53.
Adriamycin activation in mitochondria requires oneelectron reduction of the quinone to semiquinone free
radicals by Complex I. The semiquinone is subsequently reoxidized in mitochondria to reactive oxygen
species (ROS) that at suciently high levels results in
cell death (Singh, 1998). It is conceivable that the
removal of mitochondrial function leads to the inability
of Rho0 cells to both activate adriamycin and to
produce signi®cant amounts of ROS necessary for cell
death. Reactive oxygen species produced by PDT is
also required for cytotoxic e€ects of PDT (Munday et
al., 1996; Fisher et al., 1999). Consistent with these
observations, our results (Figure 1d) and those of
Munday et al. (1996) show that cells lacking
mitochondrial function are resistant to cytotoxic
e€ects of PDT. These studies indicate that ROS
produced by mitochondria must play a critical role in
cell death induced by both adriamycin and PDT.
Development of drug resistance to chemotherapeutic
agents is a major clinical problem. Several underlying
mechanisms have been suggested to contribute to drug
resistance. These include: (1) ampli®cation or overexpression of the P-glycoprotein family of membrane
transporters, (2) changes in cellular proteins involved
in detoxi®cation or activation, (3) changes in proteins
involved in DNA repair and (4) activation of
oncogenes such as Her-2/neu, bcl-2 and p53 (ElDeiry, 1997). In this study, we provide evidence that
mitDNA also plays an important role in development
of drug resistance in cancer cells.
We report that damage to mitDNA by adriamycin
leads to deletion mutations in mitDNA (Figure 4). The
mitochondrial genome appears to be a vulnerable target
of adriamycin because it lacks protective histones and is
continuously exposed to ROS produced within the
mitochondria (Richter, 1988; Singh, 1998). ROS
produced by adriamycin may make mitDNA even
more vulnerable to damage. ROS produced by
adriamycin modi®es bases in DNA and lead to
preferential accumulation of 8-hydrooxyguanosine (8Oxo-G) lesions in mitDNA of adult rats (Palmeira et
al., 1997). Repeated repair of 8-Oxo-G base by 8-OxoG DNA glycosylase (hOGG1) may cause strand breaks
in mitDNA resulting in deletions in mitDNA (Figure 4,
and Takao et al., 1998). Increased damage to mitDNA
by other anticancer agents has also been reported
(Segal-Bendirjan et al., 1988; Modica-Napolitano et al.,
1996). Our system utilizing Rho0 cancer cells should
serve as a tool for elucidating the mechanism of
cytotoxicity of potential anticancer agents.
Adriamycin, a member of the anthracycline class of
drugs is one of the most e€ective anticancer agents and
is widely used in therapy of childhood leukemias,
lymphomas and solid tumors (Ito et al., 1990; Von
Ho€ et al., 1979). However, as cumulative dose
increases, the incidence of congestive heart failure
increases rapidly (Von Ho€ et al., 1979; Shan et al.,
1996). Recent evidence suggests that anthracycline
therapy also leads to arrythmias, pericarditis-myocarditis syndrome, ventricular dysfunction and cardiomyopathies, sometimes 2 ± 15 years after treatment has
ceased (Ito et al., 1990; Von Ho€ et al., 1979; Shan,
1996). Studies reported in this paper suggest that
adriamycin-induced mutation in mitDNA may accumulate and contribute to cardiac pathology observed in
cancer patients. Current studies are aimed at testing
this possibility.
Materials and methods
Cell culture and clonogenic assays
HSL2, (Rho+, a HeLa subline) and its derivative cell line
devoid of mitDNA (Rho0) were maintained as described
previously (Morais et al., 1994). Cells were grown in
humidi®ed incubators at 378C, 5% CO2. Culture medium
consisted of high-glucose DMEM supplemented with FBS
(10%), L-glutamine (2 mM), sodium pyruvate (110 mg/ml)
and uridine (4 mg/ml) (Morais et al., 1994). Cells were
subcultured weekly. Rho0 cells were refed twice weekly.
Clonogenic assays were performed using exponentially
growing cells. Three days prior to assay, 56105 cells were
seeded into 75 cm2 ¯asks. Cells were trypsinized and
resuspended in medium containing drug, at a ®nal cell
concentration of 26104/ml. Exposure lasted for 45 min, after
which cells were spun down, rinsed twice with PBS and
plated out. For colony formation, cells were seeded at
densities ranging from 100 ± 10 000/plate (Rho+) and 200 ±
20 000/plate (Rho0). Radiation experiments were performed
on cells that had been plated out 18 h before exposure to
radiation. Irradiation was performed using a 137 Cs source
(Gammacell, Atomic Energy of Canada) at a dose rate of
0.8 Gy/min. Suramin was obtained from Dr Steve Howard
(Mobay Chemical Corporation, Germany). Mesophyrin IX
was from Sigma Chemicals (USA). After 10 ± 14 days, plates
were stained, and colonies counted. Colonies were de®ned as
containing at least 50 cells. For each experiment, all dose
points were measured in triplicate, and survival curves
represent data from three independent experiments. Survival
was ®tted using SPSS software.
Measurement of apoptosis
For determination of apoptosis in response to adriamycin
(1.5 mM) exponentially growing cells were treated for 45 min.
Medium was removed and saved. Cells were trypsinized and
added back to the medium they had grown in. Cells were
spun down and resuspended in PBS. An equal volume of icecold methanol/acetic acid was added. Cells were spun down
and resuspended in methanol/acetic acid. Cells were held on
ice 10 min, spun down, resuspended in 100 ml methanol/acetic
acid and dropped onto slide. After the ®xative evaporated,
slides were immersed in Acridine Orange (6 mg/ml in PBS) for
5 min. Slides were washed in PBS, mounted with ddH2O, and
apoptotic cells were counted on UV microscope.
6645
Mitochondrial DNA and cell death
KK Singh et al
6646
Flow cytometry analysis
Exponentially growing cells were trypsinized and ®xed in
70% alcohol for 10 min. For cell cycle analysis, cells were
resuspended in propium iodide (40 mg/ml) and RNase
(Sigma, St. Louis, MO, USA), (100 mg/ml) for 30 min at
room temperature. Cells were spun down, resuspended in
PBS and analysed on Coulter Epics ¯ow cytometer, (Coulter
Electronics, Hialeah, FL, USA) with excitation at 488 nm.
Cell cycle distribution was analysed using Multicycle software
(Phoenix Flowsystems, San Diego, CA, USA). FACS
analysis was also used to quantitate adriamycin uptake
(Chevillard et al., 1990; To€oli et al., 1996). Exponentially
growing cells were exposed to 1.25 mM drug for 45 min. The
drug was rinsed o€, cells were trypsinized, ®xed and analysed
as above.
Southern blot analysis
Total DNA from untreated and adriamycin treated HeLa
cells was isolated as described by Modica-Napolitano et al.
(1996) and Tanaka et al. (1996). The DNA was digested with
PvuII restriction enzyme which linearizes the mitDNA
resulting in a single 16.6 Kb fragment (Modica-Napolitano
et al., 1996). PvuII-digested DNA was size fractionated by gel
electrophoresis on 1% agarose gel in Tris-borate EDTA (pH
8.0). Equal loading was visualized by ethidium bromide
staining. The DNA was transferred overnight to a nylon
membrane and probed with 32P-labeled mitDNA fragment
spanning the region 7441 ± 8287 of mitDNA (King and
Attardi, 1988). Hybridization, washing and labeling of probe
was carried out as described (Modica-Napolitano et al.,
1996).
Acknowledgments
This work was supported by grants from Alexander and
Margaret Stewart Trust, CA/ES 66204, ES 09714-01 and
American Heart Association 9930223N to KK Singh and
the Plotkin ± Katz Foundation to KF Keshav. We thank
Dr Rejean Morais for the kind gift of HSL2 and its
mitDNA-less derivative cell lines. We also thank Dr
Michael King for the mitDNA probe used in this study.
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