Download Selective Loss of Mitochondrial DNA after Treatment of Cells with

[CANCER RESEARCH 48, 4982-4992, September 1, 1988]
Selective Loss of Mitochondrial DNA after Treatment of Cells with Ditercalinium
(NSC 335153), an Antitumor Bis-intercalating Agent
Evelyne Segal-Bendirdjian,1 Dominique Coulaud, Bernard P. Roques, and Jean-Bernard Le Pecq
Unité
de Physicochimie Macromoléculaire(CNRS UÀ147, INSERM U140) ¡E.S-B., J-B. L.] and Laboratoire de Microscopie Cellulaire et Moléculaire[D. C.J, Institut
Gustave Roussy, 94805 Villejuif Cedex; and Départementde Chimie Organique (CNRS VA 498, INSERM U 266), UER des Sciences Pharmaceutiques et Biologiques,
4, Avenue de l'Observatoire, 75006 Paris; France
ABSTRACT
Ditercalinium (NSC 335153), a bifunctional intercalating molecule
with antitumor activity, is found to express its toxicity through a mech
anism of action completely different from that of other monointercalating
agents.
Electron microscopic observation of ditercalinium-treated cells shows
a drastic alteration of mitochondria! structure. Cells deficient in mito
chondria! respiration (GSK3 cells) isolated by A. Franchi et al. (Int. J.
Cancer, 27: 819-827, 1981) are about 25-fold more resistant than cells
deficient in glycolysis (DS7 cells) isolated by J. Pouysséguret al. (Proc.
Nati. Acad. Sci. USA, 77: 2698-2701, 1980). Revenants have been
isolated from GSK3 cells. In these cells, the sensitivity to ditercalinium
has been recovered with mitochondria! respiration. Ditercalinium treat
ment of 1.1210 leukemic mouse cells leads to a specific elimination of
mitochondria! DNA detected by DNA-DNA hybridization. No measur
able alteration of nuclear DNA is observed. In contrast, the monomeric
analogue of ditercalinium only alters nuclear DNA and does not change
the mitochondria! DNA content. The activity of cytochrome c oxidase,
an enzyme which contains a subunit coded by the mitochondria! DNA,
decreases exponentially in treated cells with a half-life of 24 h, corre
sponding to the turnover of the enzyme.
These results suggest that ditercalinium exerts a specific cytotoxic
effect at the level of mitochondria! DNA. This action could account for
the delayed cytotoxicity induced by this compound.
INTRODUCTION
In an effort to obtain new antitumoral agents with high
binding affinity for DNA, various bifunctional intercalators
have been synthesized (1-3). In the series of 7H-pyridocarbazole, dimerization yielded molecules which bis-intercalate into
DNA, depending on the nature, the flexibility, and the inni/.ation state of the linking chain. Among these molecules, diter
calinium, which bis-intercalates into DNA through the large
groove (4), displays anticancer activity on a variety of animal
tumor models (2, 5).
Several observations suggested that the mechanism of action
of mono- and bis-intercalators are different (6, 7).
The dimers are about 10- to 40-fold more toxic than their
respective monointercalating counterparts; they are character
ized by a delayed cytotoxicity: treated cells continue growing
for seven to eight generations before arrest; they do not provoke
a block of cell cycle progression in the G2 + M phase of the cell
cycle, as other intercalating agents.
In a bacterial study, ditercalinium has also been shown to be
cytotoxic on Escherichia coli polA mutants and not on polA
uvrA double mutants, suggesting that this dimer which binds
reversibly to DNA, is able to induce in vivo DNA conformational changes recognized by the uvr ABC repair system in E.
coli. This recognition could lead to a futile and abortive DNA
repair process (8).
Received 1/11/88; revised 5/24/88; accepted 6/2/88.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1To whom requests for reprints should be addressed, at the Institut Gustave
Roussy, 39 rue Camille Desmoulins, 94805 Villejuif Cedex, France
In addition, preliminary electron microscopy analyses re
vealed that the cytotoxic effects of ditercalinium is accompanied
with alterations of the mitochondria! morphology (9).
Because recent reports underlined frequent alterations of
mitochondria in carcinoma cells and had suggested that mito
chondria could represent a specific target for antitumor drugs
(10, 11), we have investigated the effect of ditercalinium on
mitochondria.
In this study, we present evidence that ditercalinium causes
a selective degradation of mitochondrial DNA.
MATERIALS
AND METHODS
Conditions of Cell Culture. The Chinese hamster lung fibroblast clone
023 and their derivatives DS7 and GSK3 were isolated by Pouysségur
et al. and their properties are described in previous works (12, 13).
Briefly, DS7 and GSK3 are derivatives of 023 cells after ethylmethanesulfonate mutagenesis and tritium suicide selection. The DS7 cell
line has a total block in the glycolytic pathway. The GSK3 cell line
maintains a permanent high rate of glycolysis.
Those cells were grown in Dulbecco's modified Eagle medium
(GIBCO) supplemented with 10% fetal calf serum and antibiotics (50
lU/ml penicillin and 50 Mg/ml streptomycin). L1210 cells were main
tained in culture in RPMI 1640 Medium (Flow Laboratories), supple
mented with fetal calf serum (9%), penicillin (100 lU/ml), streptomycin
(50 Mg/ml), and ,8-mercaptoethanol (60 MM).All cells were maintained
at 37°Cin a 5% CO2 atmosphere.
Drugs. Ditercalinium (2,2'-[4,4'-bipiperidine-l,l'-bis(ethane-l,2diyl)]bis( 10-methoxy-7H-pyrido[4,3-c]carbazolium
d¡chloridedihydrochloride), a 7H-pyridocarbazole dimer (NSC 335153) was from the
Roger Mellon Laboratory. The monomeric derivative, 2-[2-(Ar-piperidyl)ethyl]-10-methoxy-7H-pyrido[4,3-c]carbazole
chloride hydrochloride, and the dina-rii- non-antitumor analogue with a longer linking
chain between the two intercalating rings, l,3-bis|Ar-[2-(10-methoxy7H-pyrido[4,3-c]carbazole-2-yl)ethyl]-4-piperidyl)propane
tetramesylate, were synthesized as previously described (1, 2).
All drugs were dissolved in water, and sterilized by filtration through
a 0.2-fim polycarbonate membrane (Nucleopore Corp., Peasanton, CA).
Drug concentrations, in the sterile solutions, were determined by ab
sorption spectroscopy. These solutions were stored at 4"C, in the dark.
Immediately before use the solutions were serially diluted to the appro
priate concentration in culture medium.
Drug Exposure and Cell Survival. Throughout this study, the effects
of the drugs were determined on exponentially growing cells.
For drug treatment of the Chinese hamster lung fibroblasts, cells
were seeded the day before in 35-mm diameter Petri dishes at a density
of 5 x 10" cells/dish for the 023 and DS7 cell lines and 1 x 10s cells/
dish for the GSK cell line and left overnight for the attachment. The
medium was then replaced either with 1 ml of fresh medium (controls),
or with 1 ml of medium containing the drug at the indicated concentra
tions. After 3 h of incubation at 37°C,the drug was washed off by
rinsing the dishes twice with 3 ml of medium and trypsinized for the
determination of the colony forming ability. About 500-1000 cells were
plated in triplicate in 60-mm diameter Petri dishes containing 5 ml of
medium. The colonies were counted about 1 week later. In these
conditions, the cloning efficiency of the controls was about 40% for
023 and DS7 lines and 30% for GSK.
For drug treatment of LI 210 cells, exponentially growing cells (1 x
10s cell/ml) were incubated in medium containing the drug for 24 h at
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LOSS OF MITOCHONDRIA!.
37°C.The cells were then centrifugea and washed twice with phosphate
buffered saline (2.7 mM KC1, 1.5 mM KH2PO4, 137 mM NaCl, 8.1 mM
Na2HPO.i) and once with RPMI before dilution in fresh medium at the
initial density.
At different times, after washing, aliquots were removed for DNA
analysis or cytochrome oxidase activity determination.
The L1210 cells were treated either with 1.4 fiM of the monomeric
analogue, or with 0.14 ^M of ditercalinium. These doses were known
to be toxic doses giving less than 1% of surviving LI 210 cells (7).
Total cell numbers in the different cultures were determined by means
of a Coultronic Coulter Counter.
Lactic Acid Determination in 023, DS7, GSK3 Cells. 72 h after
treatment the cells were washed twice with DMEM2 without serum and
incubated at zero time with DMEM2 supplemented with 10% dialyzed
fetal calf-serum. At different times, the medium of four dishes (two
control and two treated ones) was removed, filtered, and assayed for
lactate content using the Boehringer lactic acid assay kit following the
supplier's instructions.
The cells in each dish were counted. The content of lactic acid was
expressed in mg/106 cells.
Electron Microscopy. For the ultrastructural studies, the cells were
grown in 90-mm dishes. After 3 h of treatment, the drug was removed,
the cells were washed twice and refed with fresh medium for 3 days.
After this postincubation time, the cell monolayers were fixed at room
temperature by adding 1.5% glutaraldehyde (6% in volume of 25%
solution, from Merck) to the culture medium. In this solution, the cells
were scraped off the plastic and centrifuged. The pellet was resuspended
in new fixative containing Sifirensen phosphate buffer (0.66 mM, pH
7.4) with 1.5% glutaraldehyde for l h at 4°C.After 2-h washing with
buffer, cells were postfixed in 2% OsO4 in the same buffer. Dehydration
in graded ethanol and propylene oxide, Epon embedding, and uranyllead staining were classical. Observations were made in the Zeiss
EM902; optimal contrast was obtained by selecting the elastic electrons
with the slit of the spectrometer.
Extraction of Total Cellular DNA from L1210 Cells. Cells (5 x IO7
to 1 x IO8cells) were washed twice with phosphate buffered saline and
kept at —70°C
until used. Cell pellets were then suspended in 5 volumes
of 0.15 M NaCl-10 mM EDTA (pH 7.0) were lysed in 1% sodium
dodecyl sulfate at 60°Cfor 15 min. The lysate was incubated for 2 h at
37°Cwith proteinase K (100 Mg/ml), deproteinized once with phenol,
once with phenol-chloroform-isoamylalcohol (25:24:1), and once with
chloroform-isoamylalcohol (24:1). The DNA extract was then dialyzed
overnight against Tris-HCl IO"2 M, EDTA IO"3 M (pH 7.5). Then the
DNA extract was incubated with 200 Me of heat treated RNase A/ml
(15 min at 100°C)for 2 h at 37'C.
After deproteination, as indicated above, NaCl was added (100 mM)
and the preparation was precipitated overnight at —20°C
with 2 volumes
of 95% ethanol. The precipitate was then solubilized in Tris-HCl 10~3
M, EDTA 10"3M (pH7.5).
DNA
formamide, with nick-translated 32P-labeled heat-denatured mouse mt
DNA probe (0.2 Mg;5-9 x IO7cpm/Mg).
The filters were washed several times at room temperature in 2x
SSC - 0.1% sodium dodecyl sulfate and several times at 62°Cin the
same buffer. Filters were then autoradiographed at —70°C
with Kodak
X-Omat RP film and a Dupont Cronex Lightning-Plus intensifying
screen.
Probes. The mitochondria! DNA probe (a gift from Dr. D. A.
Clayton, Stanford University, CA), consisted of the entire mouse mi
tochondria! DNA genome with plasmid pACYC 177 as the vector
cloned into Escherìchiacoli HB101.
The nuclear DNA probe (pMRB 1.1), generously given by Dr. M.
Meunier-Rotival, UnitéINSERM 56, Hôpital de Kremlin Bicêtre,
France, contained interspersed repeats of mouse DNA (16).
Cytochrome Oxidase and Protein Determination. For cytochrome
oxidase activity, cultures were centrifuged, washed twice with 0.25 M
sucrose, and resuspended in water. The cell suspension was transferred
to a Dounce homogenizer and a few up-and-down strokes of the piston
were sufficient to break all the cells. The homogenates obtained were
used as source of enzyme.
Cytochrome oxidase was measured in the following incubation me
dium: 0.1 ml of 0.1 M potassium phosphate buffer (pH 7.0); 0.07 ml of
reduced ferrocytochrome c 1% (type III, Sigma). The blank cuvet was
oxidized with 0.01 ml potassium ferricyanide 0.1 M.
Immediately before the assay, 0.2 ml of a suitable amount of homogenate was added to the sample cuvet to initiate the reaction. The volume
of the incubation medium was adjusted to 1.0 ml with distilled water.
Temperature of the reaction was 38°C.The decrease in absorbancy was
measured at 550 nm during 30 min.
The concentration of protein was determined by the method of
Bradford using bovine serum albumin as reference standard (17).
RESULTS
Comparison of Ditercalinium Cytotoxicity on Cells Deficient
in Glycolytic or Respiration Pathway. Pouysséguret al. (12, 13)
recently characterized cell lines of Chinese hamster lung fibroblasts deficient in either the glycolytic or the respiration path
way. The DS7 cell line is deficient in the phosphoglucoisomerase enzyme and derives all its energy from mitochondria! res
piration. The GSK3 cell line is deficient in respiration and
derives all its energy from glycolysis. These cells were used to
estimate the role of mitochondria in controlling drug cytotoxicity. Fig. 1 shows the cytotoxicity of ditercalinium of both
these cell lines compared to the parental line 023.
100
Southern Blot Hybridization. Hindlll restriction enzyme was pur
chased from Amersham. DNAs were digested for 3 h with Hindlll
according to the supplier's recommendations. Digests were analyzed by
gel electrophoresis on 0.8% agarose gel in Tris-borate EDTA (pH 8.0)
containing 0.5 Mfi/mlof ethidium bromide. Electrophoresis was carried
out in a horizontal slab gel apparatus at 30 V for 18 h. After electro
phoresis, the DNA fragments were acid depurinated in 0.25 M HC1
twice for 15 min, then denaturateli by soaking the gel in several volumes
of 0.4 M NaOH for 30 min. After neutralization of the gel in several
volumes of a 0.5 M Tris-hydrochloride (pH 7.4)-3 M NaCl solution for
30 min, the gel was blotted onto nitrocellulose filter by the method of
Southern (14). The nitrocellulose filters were moistened with 5x SSC
(Ix SSC is 0.15 M NaCl plus 0.015 M sodium citrate), and prehybridized for 2 to 4 h at 42°Cin solution containing 5x SSC, 5x Denhardt
io
GSK3
O)
C
C
o
solution (15), 0.1% sodium dodecyl sulfate, salmon sperm DNA (0.35
mg/ml), and 50% formamide. Hybridization was carried out overnight
at 42°Cin solution containing 5x SSC; Ix Denhardt solution, 0.1%
30
sodium dodecyl sulfate, salmon sperm DNA (0.1 mg/ml), and 50%
2The abbreviations used are: DMEM, Dulbecco's modified Eagle's medium;
mt DNA, mitochondrial DNA; <T;,-, thèdose required to inhibit the cloning
efficiency to a factor of 0.37.
Fig. 1. Effects of ditercalinium on cell survival as a function of drug concen
tration. After 3 h of drug treatment, the cell cloning efficiency were measured as
described in "Materials and Methods." Insert, effect of ditercalinium on the DS7
cell survival. Curve, average of three independent experiments.
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LOSS OF MITOCHONDRIAL
The DS7 cell line appears extremely sensitive to ditercalinium. The CE37, after only 3 h of treatment, was found to be
0.26 (¿M.
In the same conditions, the GSK3 cells were 25-fold
less sensitive than DS7 (CE37 = 6.24 UM). The response of the
023 parental cell line is close to that of the DS7 cells (CE37 =
0.52 /IM). As a control of the specificity of this observation, the
cytotoxicities of the monomeric analog and the dimeric nonanalog antitumor were measured. Values of CE37 are presented
in Table 1. Interestingly, the three cell lines elicit similar
sensitivity either for the monomeric derivative or for the dimeric
analog of ditercalinium. In addition, the ditercalinium analog
and the monomeric derivative are much less toxic than diter
calinium on the two lines 023 and DS7.
In addition the GSK3 cell line does not appear to be more
resistant than the 023 and DS7 lines to these two analogs.
A delayed arrest of cellular growth in observed after ditercal
inium treatment with all these cell lines. This is well indicated
by the appearance of small size colonies as previously observed
(6, 7). Even the most sensitive cell line which is unable to make
glycolysis displays such a delayed cytotoxicity effect.
Measurement of Glycolysis after Treatment. Mitochondria!
inactivation was expected to induce a cellular adaptive response
leading to a stimulation of glycolysis. Therefore, the kinetics of
lactate formation, a product of anaerobic glycolysis, were meas
ured on the three cell lines after ditercalinium treatment, at a
toxic (1.4 A<M)and a nontoxic dose (0.14 ¿tM).
Cells were treated for 3 h with ditercalinium. The ditercali
nium was then washed out and the accumulation of lactate
measured in the medium as a function of time. Fig. 2 shows
that after ditercalinium treatment, an accumulation of lactic
acid is observed in the parental 023 cell culture medium. This
indicates that there is a stimulation of glycolysis in the parental
cells. However, this stimulation does not occur in DS7 cells
which are deficient in the glycolytic pathway, nor in GSK3 cells
which maintain a permanently high glycolysis rate because of a
deficiency in the respiration pathway. The kinetics of lactate
formation were also measured 3 days after treatment of the 023
cell line as shown in Fig. 3/4. Glycolysis is clearly stimulated
but interestingly, the glycolysis stimulation is also observed
after treatment with ditercalinium at the nontoxic dose. Con
trastingly, the monomeric derivative has no effect on glycolysis,
even at toxic doses (Fig. 3B).
Ultrastructural Studies. Mitochondria! ultrastructure of ditercalinium-treated cells substantiates further the biochemical
data.
Fig. 4, A and B, and Fig. 5, A and B, show the ultrastructure
of 023 and DS7 control cells, respectively. Mitochondria gen
erally appeared oblong with cristae that traversed the entire
width of the organelle. The density of the mitochondria! matrix
was greater than that of the surrounding cytoplasm. Treatment
with ditercalinium induced important structural changes of 023
cell mitochondria (Fig. 4, C and D). Most of them appeared
enlarged and swollen and cristae were lacking or segregated at
the periphery of a low density mitochondrial matrix. A few
mitochondria contained small electron dense particles.
The effect of ditercalinium on the very sensitive DS7 cells
was more puzzling (Fig. 5, C-E). Mitochondria looked to be
less modified than those of 023-treated cells in their shape, size,
cristae content, and matrix density, although annular-shaped
electron-dense bodies were frequently observed (Fig. 5Z>).Rare
swollen mitochondria with a clear matrix were found beside
normal-looking ones (Fig. 5/:"). In addition, treated cells pos
sessed a quantity of lysozome-like
Table 1 Cloning efficiency (CE31) of the parental O23 and the two variant DSF
and GSK3 sublines after treatment with ditercalinium and its monomeric and
dimeric analogs
Ditercalinium
Monomeric derivative
Dimeric analog
1Means ±SE.
6
023 (MM)
0.52 ±0.05°
21.5 ±0.02
5.5 ±0.5
DS7 (MM)
0.26 ±0.03
18.3 ±2.0
2.34 ±0.2
DNA
Õ OS
e
m 04
£
o
GSK3 (MM)
6.24 ±0.06
11.7 ±0.1
6.5 ±0.6
<
<X2
TIME
-
l
"s
\
large cavities containing
(h)
B
4
-
GSK
0.4
023
<
0.2
01
24
48
TIME
72
96
23456
TIME
(h)
ih)
Fig. 3. Rates of glycolysis of Chinese hamster fibroblasts 023. 3 h after
Fig. 2. Lactate accumulation in Chinese hamster fibroblasts medium with (•, treatment, the cells were washed and grown in DMEM supplemented with 10%
•,A) and without (O, D, A) ditercalinium treatment. 023 (•,O); GSK3 (•.D);
fetal calf serum. 3 days later, cells were washed and changed to fresh medium
DS7 (A, A). 3 h after treatment, the cells were washed and grown in DMEM
containing 10% of dialysed fetal calf serum at time 0. Lactic acid secreted into
medium was measured as described in "Materials and Methods." A, ditercalinium:
supplemented with 10% fetal calf serum. Lactic acid secreted into medium was
measured as described in "Materials and Methods." Each point was carried out
•,0.14MM;O, 1.4 MM.fi, monomeric derivative: A, 2.7 MM;A 27 MM.Untreated
in duplicate.
cells: x. Each point was carried out in duplicate.
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LOSS OF MITOCHONDRIA!.
DNA
Fig. 4. Electron micrographs of 023 cell line: control cells (. I and B); ditercalinium treated cells (C and D); monomeric derivative treated cells (/•.').
Cells were
treated 3 h either with ditercalinium (1.4 /IM) or with the monomeric derivative (27 //M), then they were washed and refed with fresh medium for 72 h. After
ditercalinium
treatment,
markedly
can /:')
be and
identified
by theand
persisting
peripheral membranes; treatment with the monomeric analogue
does not induce
alterationstheofmitochondria
mitochondria are(/;').
Bars, 1swollen
imi (.I. and
C, and
O.S ;<n>(B
/').
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LOSS OF MITOCHONDRIA!,
>
•
. • ^.
t
DNA
.-, '"
i
3'Ä *»
•\
Jt
•
m
•:•&'
.Ã->'¿*ág3Õ
W %*»
Fig. 5. Electron micrographs of DS7 cell line: control cells (A and fi); ditercalinium treated cells (C, D, and E); monomeric derivative treated cells (F). The
conditions of treatment were the same as in Fig. 4. After ditercalinium treatment, some mitochondria with normal structure remain (E). Dense annular bodies are
frequent in the matrix. The large vesicles, filled with irregular dense material, are much more larger and numerous in treated cells (C, D, and E). No alterations are
induced by the monomeric analogue (F). Bars, I pm (A, C, and F) and 0.5 urn (B, D, and E).
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LOSS OF MITOCHONDRIAL
filamentous material very similar to that found in a swollen
mitochondria, as well as very dense irregular bodies (Fig. 5C),
and membraneous residues either isolated, or connected to the
membrane limiting the vesicle. Such single-membrane-limited
vesicles were also present in the control cells, although rare and
small. Their identification as strongly altered mitochondria is
possible but uncertain.
Those alterations were not seen after treatment of cells with
equitoxic doses of the corresponding monomer (Fig. 4E and
Fig. 5F).
In control GSK3 cells, inactivation of the respiration pathway
was associated with an alteration of the structure of mitochon
dria. Many mitochondria appeared devoid of cristae (Fig. 6/1).
So, no additional modification of the ultrastructure of mito
chondria could be seen after treatment of those cells with
ditercalinium (Fig. 6B).
To further establish the specific relation between the diter
calinium resistance of GSK3 cell lines and its mitochondria!
deficiency, revenant cells were isolated. Cells were regularly
reseeded. After 95 passages a beginning of reversion was ob
served as glycolytic activity decreased. After 117 passages
(about 14 months), a complete reversion of the GSK3 phenotype was observed. The growth rate and glycolytic activity could
not be distinguished from that of control cells. In addition, the
normal appearance of mitochondria under electron microscope
was recovered (Fig. 6C). This reversion was accompanied by an
increase in ditercalinium sensitivity. Revenant cells became as
ditercalinium sensitive as the reference cell line 023 (Fig. 7/4).
In addition glycolysis could be stimulated in these revenant
cells by ditercalinium treatment (Fig. IB).
Mitochondria! DNA Content of 1.1210 Cells Treated with
Ditercalinium. The fate of mt DNA after ditercalinium treat
ment was investigated on the murine leukemic LI210 cell line
using a DNA probe containing the entire mouse mt DNA. Total
cellular DNA was extracted during and at different times fol
lowing ditercalinium treatment. DNA was treated with Hindlll
restriction enzyme and analyzed using agarose gel electrophoresis. After transfer to nitrocellulose filter according to the
Southern technique (14), DNA was hybridized with either a mt
DNA probe or with a nuclear DNA probe. Hindlll digestion
gave a single major band for mt DNA.
We selected as a nuclear probe a cloned interspersed repeti
tive sequence. This allowed us to analyze a global effect on
nuclear DNA rather than a specific effect on a given gene. In
that case a smear was observed on the autoradiogram, as
previously shown (16).
Results of these experiments are shown in Fig. 8, A and B.
During the first 24 h of treatment with ditercalinium (Fig. 8Ä),
no significant variation of mt DNA was observed. However 24
h after washing out ditercalinium, the mt DNA band almost
completely disappeared. No reappearance of mt DNA was
observed even in cells maintained in culture for 6 days. Con
trastingly, the autoradiogram obtained with the nuclear probe
did not reveal any alteration of nuclear DNA after ditercalinium
treatment. Treatment with the ditercalinium monomeric analog
showed a very different effect on mt DNA as well as on nuclear
DNA (Fig. 8/1). No variation was observed in the content of mt
DNA after treatment. But a clear modification of nuclear DNA
was observed after treatment with the monomer analog. The
nuclear DNA probe revealed the appearance of low molecular
DNA fragments. A reduction of the bulk DNA molecular
weight was also observed on the gel after ethidium staining,
with appearance of a pattern characteristic of subnucleosomal
material (18, 19).
DNA
Effects on Cytochrome c Oxidase Activity of LI210 Cells.
Because of the observed disappearance of mt DNA after diter
calinium treatment, we measured the activity of cytochrome c
oxidase which contains a subunit coded by mt DNA as shown
in Fig. 9.
Cytochrome c oxidase activity decreased exponentially im
mediately after treatment with an half-life of 24 h. A small and
significant decrease was also observed after monomer treatment
but, after washing out the compound, the activity of the enzyme
returned to that of control cells 24 h after the wash.
DISCUSSION
Ultrastructural studies of 023 cells treated with ditercalinium
reveal a striking alteration of mitochondria with complete loss
of cristae. The effect is less clearly interpretable in DS7 cells.
There were less altered mitochondria in those cells than in 023
ones. Probably DS7 cells, in which metabolism depends only
on mitochondria! respiration, are more sensitive than 023 ones
to fewer mitochondria! alterations. Similar structural changes
have already been observed after treatment with ditercalinium
of another cell line in culture (9) and they looked similar to
those which have been observed after long term treatment with
ethidium bromide or chloramphenicol (20-22). However, contrarily to what was observed after ethidium bromide treatment,
the mitochondria! structural changes that resulted from diter
calinium treatment were irreversible even after removal of the
drug. In the case of ethidium bromide, the morphological
alterations showed a gradual return to almost normal mito
chondria! ultrastructure after the removal of the drug. Contrast
ingly, the monomeric analog of ditercalinium does not induce
such an effect. These mitochondria! alterations are accom
panied with a significant stimulation of glycolysis of ditercali
nium treated cells, which is not observed with the monomeric
analog.
The mitochondrial alterations are associated with an almost
complete loss of the mitochondrial DNA. As expected the loss
of mitochondrial DNA is accompanied with the disappearance
of the activity of the cytochrome c oxidase, an enzyme which
contains a subunit coded by mt DNA. Interestingly, the rate at
which cytochrome c oxidase activity disappears (ii/2 = 24 h) is
close to that reported for the turnover of this enzyme, measured
after chloramphenical treatment (21). One must also underline
that the loss of cytochrome c oxidase activity is irreversible. In
contrast, when cells are treated with the monomeric analog of
ditercalinium (our results), with chloramphenicol or ethidium
bromide (21, 23), the cytochrome c oxidase activity is fully
recovered after removal of the drugs.
After ditercalinium treatment, the loss of mt DNA is observed
in the absence of detectable nuclear DNA alterations. However,
treatment of cells with the monomeric analog results in altera
tion of nuclear DNA. After treatment with the monomeric
derivative, subnucleosomal bands of DNA corresponding to
mono- di- and oligo-nucleosomes appear on agarose gel, sug
gesting that selective degradation at the level of internucleosomal DNA occurs (18, 19). Such results are consistent with the
observations of Markovits et al. (24) which showed that diter
calinium did not cause any DNA protein-associated breaks in
the nuclear DNA. Such breaks were clearly observed with
antitumor monointercalating agents (25) and the monomeric
analog of ditercalinium.3
It is therefore very likely that the primary effect of ditercali3J. Markovits, personal communication.
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LOSS OF MITOCHONDRIA!.
DNA
iT
9'T
^
'
f-
cf
\ m^i
*^ A
=- A* _
-'
K.
fc »•
'
«
•".-•-.
•
::
' •
" ^*"
•%»:.:
i?
..X*
i?*"02
'_v '
A»
•
":
:.#ÃŒP
T:V,;;--
^^
••
' -:-
'"' *1
:<r4A
Fig. 6. Electron micrographs of GSK3 cell lines:. I. GSK3 in the 27th passage; B, GSK3 in the 27th passage treated with 1.4 ¿IM
ditcrcalinium (the conditions of
treatment were the same as in Fig. 4); C, GSK3 in the 117th passage, after the reversion of the phenotype. Mitochondria of GSK3 cells at early passages are swollen
and have short, round cristae. Treatment with ditercalinium does not modify their morphology. Mitochondria of GSK3 cells revertants appear similar to those in
control 023 parental cells. Bars, 1 //in.
4988
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LOSS OF MITOCHONDRIAL
leads to a futile and abortive repair cycle (8). It is possible,
therefore, to speculate that the DNA structural alteration may
also be recognized in mitochondria.
The existence of a DNA repair process at the level of mt
DNA has been investigated (27, 28). This question is of impor
tance because several compounds and carcinogens react pref
erentially with mt DNA, probably because mt DNA is not
associated with proteins (29-32). For instance, it was reported
that the reaction of dihydrodiol-epoxide
derivative of
benzo(a)pyrene with mt DNA is 40 to 90 times greater than
with nuclear DNA (29).
Several authors have reported that there is no DNA repair at
the level of mt DNA (27). It was suggested that damaged mt
DNA is degraded and leads to a preferential replication of
nondamaged DNA. Replacement of mt DNA instead of its
repair would represent an alternative defense mechanism.5 If
100
GSK3
O
C
.5
u
10
O)
O
"3
ditercalinium mt DNA complexes were recognized as a defect,
as in bacteria, one could understand how such a compound
leads to rapid mt DNA elimination. Such a process is observed
with many compounds leading to the "petite" mutation induc
B
95 th
co
0.8u"b
3
06O)LU
\
/•
^/_..,„
0.4
.<§0,m-
123456
123456
TIME
DNA
1 23456
(h)
Fig. 7. A, effects of ditercalinium on GSK3 and GSK3 revenant cell lines
survival. (Il) GSK3 cells were treated during their 27th passage (about 3 months
after thawing) and (O) during their 117th passage (about 14 months after). (+)
cell survival of treated 023 cells. Each curve represents an average of three
independent experiments. H. rates of glycolysis of GSK3 cells. (D) GSK3 control
cells, (•)3 h ditercalinium treated GSK3 cells ( 1.4 MM),(x) 023 control cells, (+)
ditercalinium treated 023 cells (1.4 MM).Each point was carried out in duplicate.
nium is at the level of mt DNA and that the irreversible
mitochondria! alterations result from the irreversible loss of mt
DNA. A compound like chloramphenicol which specifically
prevents the synthesis of mitochondrial proteins causes mor
phological, as well as metabolic, alterations very similar to that
of ditercalinium, but these alterations are readily reversible (21,
22). Interestingly, under continuous exposure to chloramphen
icol, a delayed toxicity is observed which resembles the one
observed after ditercalinium treatment. Induction of a delayed
cytotoxicity might represent a common feature of drugs acting
on mitochondria. This delayed cytotoxicity was also observed
in vivo. In tumor-bearing mice an unusual delayed cytotoxicity
appeared. In Phase I clinical trials, ditercalinium caused lethaldelayed hepatotoxicity at therapeutic antineoplastic doses (26).
The causes of this hepatotoxicity are under investigation. Some
preliminary results4 suggest that this toxicity is related to the
pharmacokinetic characteristics of the molecule and its concen
tration in liver.
Presently the mechanisms leading to mt DNA loss after
ditercalinium treatment are unknown. However, it has been
shown that ditercalinium induces a structural deformation on
DNA which is recognized in bacteria by the repair system and
4 R. Fellous, D. Coulaud, I. El Abed, B. P. Roques, J. B. Le Pecq, E. Detain,
and A. Gouyette, manuscript submitted for publication.
tion in yeast (33).
Comparison of the activity of ditercalinium and other drugs
on cells deficient either in mitochondrial respiration or in
glycolysis is of importance in studying the role of mitochondria
in determining cytotoxicity. Inactivation of mitochondrial me
tabolism in GSK3 cells results in increased resistance to diter
calinium. This resistance appears correlated with mitochondrial
deficiency since reversion of the mitochondrial defect in the
GSK3 cells is associated with a parallel increase of ditercali
nium cytotoxicity. In contrast, the lack of glycolysis in respi
ration proficient cells (DS7) does not cause a significant in
crease of ditercalinium toxicity. Furthermore, ditercalinium
toxicity on DS7 cells still appears delayed although these cells
are unable to produce energy through glycolysis. This observa
tion suggests that the primary effect of ditercalinium must be
mainly at the mt DNA level and not directly on the enzymes of
the respiratory chain. If ditercalinium was able to directly
inactivate the respiration chain, DS7 cells would be expected
to stop growth immediately. Indeed, when DS7 cells have been
treated with oligomycin, a specific inhibitor of mitochondrial
ATPase, the DS7 cells die within l h (13). However, if mt DNA
is the major toxic event after ditercalinium treatment, mito
chondrial respiration could be expected to decline at a rate
corresponding to the turnover of the respiratory enzyme units
coded by mt DNA. These variant cell lines would therefore be
extremely useful in demonstrating the role of mitochondrial
functions in drug action.
Chen et al. (34) have proposed that the membrane potential
of mitochondria in some tumor cells might be higher than in
normal cells. The mitochondria of tumor cells could therefore
concentrate lipophilic cations such as rhodamine 123 and dequalinium (10, 11). Like dequalinium, ditercalinium, which
bears at least two positive charges on its quaternary ammonium
groups, because of the membrane potential, could also be con
centrated by mitochondria. However, it cannot be excluded that
ditercalinium is also able to induce some defects at another
level, for instance on nuclear DNA. Tráganos et al. (35) have
found an increase of denaturated DNA in nucleus after diter
calinium treatment and Markovits et al. (24) have found a
condensation of chromatine which was not observed in resistant
cells.
Clearly, the cellular action of ditercalinium is different from
* E. T. Snow, S. Mita, and L. A. Loeb, Congress Abstract Mittelwhir, 1985.
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LOSS OF MITOCHONDRIA!. DNA
TI
after
wash
C 244872
TIME
(h)
after
wash
TIME
(h)
after
wash
ME
Ch!
C
24 48 72
C 24 48 72
kb
— 23.1
_
94
_
65
.
43
23
2.0
— 1.0
— 0.5
electrophoresis
hybridization
nuclear
B
ME(h)withdrug3
TI
6 9 24afterwash62448
probe
ME(h)withdrug3
TI
6 924afterwash62448
mt
probe
TIME(h)withdrug3
2448
6 9 24afterwash6
_
1 .0
_
0.5
hybridization
electrophoresis
nuclear
probe
mt
probe
Fig. 8. Electrophoresis and hybridizations with either a mt DNA probe, or a nuclear DNA probe to the Hindlll treated cellular DNA prepared from I.I 2 Id cells.
A, cells were treated 24 h with 1.4 >iMof the monomeric derivative. DNA extractions were made 24, 48, and 72 h after removing of the drug. Lane C, DNA from
nontreated cells. The DNAs were treated for 3 h with Hindlll. Digests were analyzed by gel electrophoresis. The gel was then blotted onto nitrocellulose niter and
hybridized with the 32P-labeled mt DNA probe and autoradiographed during 6 days. The niter was then dehybridized and rehybridized with a "P-labeled DNA nuclear
probe containing repetitive sequences. The filter was then autoradiographed. B, cells were treated 24 h with 0.14 pM of ditercalinium. DNA extractions were made
either during (3, 6, 9, 24 h) the treatment or 6, 24, and 48 h after the removing of the drug. The DNAs were then treated as above. Molecular weight markers were A
phage DNA digested by Hindlll and 1-kilobase ladder fragments (Bethesda Research Laboratories). Only the first major band has been shown because it corresponds
to approximately 90% of the mt DNA.
4990
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LOSS OF MITOCHONDRIA!.
100
0
24
48
72
96
TIMEIh)
Fig. 9. Effect of treatments on cytochrome c oxidase activity of L1210 cells.
Cells were treated 24 h either with 0.14 »Mditercalinium (•),or with 1.4 ^M of
the monomeric derivative (+). Arrow, time at which the cells were treated. The
activity is expressed as the percentage of control. Each point was carried in
triplicate.
that of its monomeric derivative and other monointercalating
agents. Ditercalinium's effect is mainly at the level of mt DNA
whereas monointercalating agents act at the level of nuclear
DNA and DNA topoisomerase II (25). Furthermore, Lim and
Neims (36) reported that Adriamycin had no effect on mt DNA
and that bleomycin induced short breaks in mt DNA but at a
dose much larger than that inducing nuclear DNA breaks.
However, it is not clearly stated that the effect of ditercalinium
is related to mt DNA elimination alone. The viability of a cell
like GSK3, as well as other respiration deficient cells (37), with
no measurable respiration and clear morphological alteration
of the mitochondria, suggests that a mammalian cell can derive
all its energy from glycolysis without need of a functional
respiratory chain. Recently, Desjardins et al. (23, 38) have
shown that chick embryo cells completely devoid of mt DNA
are still viable. If this result could be extended to cells of other
species, it would suggest that elimination of mt DNA alone is
not sufficient to lead to cell death.
These results extend previous observations (6-9) suggesting
that ditercalinium, a bifunctional DNA intercalator, elicits antitumor activity by a mechanism of action completely different
from that of monointeracalating antitumor drugs. Ditercali
nium appears to have a selective action on mt DNA which may
be responsible for the delayed cytotoxicity which has also been
observed with other ant ¡mitochondria! agents. But it is not clear
that such an effect is sufficient to account for cytotoxicity and
antitumor action of ditercalinium. Additional effects, for in
stance at the level of nuclear DNA, may also contribute to cell
toxicity.
Acknowledgments
The authors are very grateful to Dr. A. Jacquemin-Sablon
E. Delain for fruitful discussions and suggestions. We wish
Dr. Pouyssegur for providing 023, DS7, and GSK3 cell lines
Clayton and Meunier-Rotival for providing DNA probes. D.
acknowledged for his help in editing the manuscript.
and Dr.
to thank
and Drs.
Fortin is
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Selective Loss of Mitochondrial DNA after Treatment of Cells
with Ditercalinium (NSC 335153), an Antitumor Bis-intercalating
Agent
Evelyne Segal-Bendirdjian, Dominique Coulaud, Bernard P. Roques, et al.
Cancer Res 1988;48:4982-4992.
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