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INTRODUCTION |
Retinoids are a group of natural and synthetic analogs of vitamin
A that have been shown to play an important role in cell differentiation and proliferation (1, 2). Because retinoid treatment
inhibits the growth of a variety of epithelial cancers, retinoids have
great promise in the area of cancer chemotherapy and chemoprevention
(3-5). However, many tumors have been found to be resistant to the
growth inhibitory activity of natural retinoids. More recently, several
synthetic retinoids such as
6-((1-admantyl)-4-hydroxyphenyl)-2-naphthalenecarboxylic acid
(CD437)1 and fenretinide
N-(4-hydroxyphenyl)retinamide (4-HPR) have been shown to
inhibit the growth and induce apoptosis in both
all-trans-retinoic acid (ATRA)-sensitive and -resistant
tumor cell lines. Thus determination of the mechanism by which these
synthetic retinoids inhibit growth and induce apoptosis of ovarian
tumors could lead to the development of more effective cancer treatments.
CD437 is a conformationally restricted synthetic retinoid that has been
studied in a number of cancer models including non-small cell lung
carcinoma, cervical, breast, and ovarian carcinomas (6-15). This
retinoid is currently being evaluated in a number of clinical trials.
Our laboratory has previously reported that CD437 inhibits growth and
induces apoptosis in ATRA-resistant (SK-OV-3) as well as ATRA-sensitive
(CA-OV-3) ovarian carcinoma cells lines (6, 16). CD437 has been
reported to be an RAR-
-/RAR-
-selective ligand when used at
apoptotic inducing concentrations (10
6 M) (6,
7, 15, 17, 18). Our previous studies (16) demonstrated that the effects
of CD437 on ovarian carcinoma cell growth and induction of
apoptosis is at least partially RAR-dependent.
4-HPR, another synthetic retinoid, also induces growth arrest and
apoptosis in both the ATRA-sensitive, CA-OV-3, and ATRA-resistant, SK-OV-3, ovarian tumor cell lines. Although, the mechanism of action of
4-HPR is not fully understood, it has been reported that this compound
could induce apoptosis and growth inhibition by both
RAR-dependent and -independent pathways (19-25). We also have found that in CA-OV-3 and SK-OV-3 ovarian tumor cells, 4-HPR acts
independent of RAR function or
activation.2
In this investigation we describe the isolation and characterization of
an ovarian carcinoma cell line, CA-CD437R, which has been made
resistant to the apoptotic effects of CD437. This cell line, isolated
from the CA-OV-3 ovarian tumor cell line, is resistant to the effects
of CD437 treatment, but is not resistant to apoptosis induced by 4-HPR.
Interestingly this cell line is also resistant to the apoptotic effects
of TNF-
. Our results suggest that these two retinoids use separate
apoptotic inducing pathways. This cell line is a valuable tool, which
can be used to compare molecular mechanisms leading to apoptosis
induced by CD437 and 4-HPR in ovarian carcinoma cells.
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MATERIALS AND METHODS |
Cell Lines and Culture Conditions--
CA-OV-3 cells were
obtained from the American Type Culture Collection (Rockville, MD).
Stock cultures of CA-OV-3 and CA-CD437R were maintained in Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine serum
(FBS), 2 mM L-glutamine, 100 units/ml penicillin and streptomycin, and 100 units/ml nystatin. All cell lines
were routinely split at a ratio of 1:10 weekly. The plates were
incubated in an atmosphere of 98% humidified 5% CO2.
Retinoids and TNF---
ATRA was kindly supplied by
Hoffmann-La Roche (Nutley, New Jersey). CD437 was a generous
gift of Galderma Research and Development (Sophia Antipolis, France).
4-HPR was generously provided by R. W. Johnson Pharmaceutical
Research Institute (Raritan, NJ). Retinoid stock solutions
(103 M) prepared in Me2SO were
stored at 
70 °C. Recombinant human TNF- was purchased from
Promega (Madison, WI). All procedures involving retinoids were carried
out under subdued light. In each experiment, control cultures were
treated with an equivalent amount of Me2SO.
Isolation of the CA-CD437R Cell Line--
The CA-CD437R cell
line was isolated from the CA-OV-3 ovarian tumor cell line. CA-OV-3
cells were cultured for 6 months in complete medium with increasing
concentrations of CD437. The concentration of CD437 synthetic retinoid
was progressively increased from 1010 M to a
concentration of 106 M over the 6-month
period of time. The CA-CD437R cell line has been cultured for over 18 months, during this time every 14 days the medium was supplemented with
106 M CD437 for a period of 5 days to ensure
continued resistance. The CA-CD437R cell line did not revert to a
CD437-sensitive phenotype similar to the parental CA-OV-3 cell line
upon removal of selective pressure. Before CA-CD437R cells were used in
experiments, CD437 selective pressure was applied to stock CA-CD437R
cultures, for a period of 48 h. The stock cultures were then
trypsinized and plated in complete medium without CD437 as needed for
each specific experiment.
Cell Proliferation--
Ovarian carcinoma cells were seeded onto
100-mm tissue culture plates at a density of 2 × 104
cells/plate. The cells were incubated for 48 h prior to treatment. The medium, with or without varying concentrations of retinoids, was
changed every 2 days. On the days indicated, cells were washed with
phosphate-buffered saline, trypsinized, and counted using a
hemocytometer. Cells were assessed for viability using trypan blue. All
cell counts were repeated in triplicate.
Apoptosis ELISA--
Detection of apoptosis in ovarian cancer
cells was performed via an ELISA as described by Salgame et
al. (26). The ELISA uses a capture monoclonal antibody (LG11-2),
which is specific for residues 1-25 of the amino-terminal domain of
histone H2B, and a biotinylated detection monoclonal antibody (PL2-3),
which is specific for the nucleosome subparticle composed of histones H2A, H2B, and DNA. The presence of histones in the cytoplasm is indicative of apoptosis. The ELISA was quantitated by measuring absorbance at 405 nm using a Vmax Kinetic Reader
(Molecular Devices, Sunnyvale, CA).
RAR Transcriptional Activity--
RAR transcriptional activity
was assayed using a pRARE-CAT reporter plasmid (a generous gift from
Dr. R. Evans of the Salk Institute, La Jolla, CA) and a commercially
available pCMV--galactosidase plasmid
(Clontech). Transient transfection was performed
using the Effectene transfection kit from Qiagen (Valencia, CA).
Briefly, 5 × 105 cells were seeded into 60-mm tissue
culture plates in complete DMEM and incubated at 37 °C in a
humidified CO2 incubator 1 day prior to transfection. 1 µg of pRARE-CAT plasmid DNA and 1 µg of pCMV--galactosidase
plasmid DNA was diluted in the DNA condensation buffer to a final
volume of 150 µl. The mixture was centrifuged and mixed with 25 µl
of Effectene transfection reagent. The medium was then removed from the
culture plates, the cells were washed once with phosphate-buffered
saline, and 4 ml of fresh complete DMEM was added. 1 ml of complete
DMEM was added to the DNA mixture and the mixture was added dropwise
onto the cells. The cells were incubated at 37 °C for 24 h at
which time the cells were treated with either 106
M ATRA or Me2SO carrier. After an additional
24 h the cells were harvested and assayed for chloramphenicol
acetyltransferase activity (CAT) (27) and -galactosidase activity
(28). CAT was normalized with respect to -galactosidase activity to
control for efficiency of transfection. All experiments were repeated
in triplicate.
Caspase Activity Assays--
The activity of caspase proteases
was measured using an ApoAlert CPP32 colorimetric assay kit
(Clontech). Briefly, whole cell lysate from
~4 × 106 ovarian cells was incubated for 1 h
at 37 °C with the caspase-specific substrate in the provided
reaction buffer. Cleavage of the caspase-specific substrate by active
caspase resulted in the liberation of pNA (p-nitroanilide)
into solution. Release of pNA was quantitated spectrophotometrically by
measuring absorbance at 405 nm using a Vmax
kinetic reader (Molecular Devices, Sunnyvale, CA). All experiments were
repeated in triplicate. The following specific caspase substrates were
utilized in these studies: Ac-DEVD-pNA (caspase-3/7)
(Clontech), Ac-VEID-pNA (caspase-6), Ac-IETD-pNA (caspase-8) and Ac-LEHD-pNA (caspase-9) (Biomol, Plymouth Meeting, PA).
Caspase Inhibitor Studies--
Ovarian carcinoma cells were
seeded onto 100-mm tissue culture plates at a density of 2 × 104 cells/plate. The cells were incubated for 48 h
prior to treatment. 1 h prior to retinoid treatment, 20 µg/ml of
the caspase-3-like inhibitor DEVD-cmk (Bachem, King of Prussia, PA) was
added. The effect of inhibiting caspase-3-like activity on
retinoid-induced growth arrest and apoptosis was determined by direct
cell counting and/or apoptotic ELISA. All experiments were repeated in triplicate.
Oxygen Consumption Measurements of Cultured Cells--
The rate
of oxygen consumption was measured using a YSI model 5300 biological
oxygen monitor that employs a Clark-type oxygen electrode (YSI Inc.,
Yellow Springs, OH). 6 × 106 cells were trypsinized
and suspended in 3 ml of complete DMEM culture medium in a 3-ml
respiration chamber with continuous stirring. The respiration chamber
was placed in a circulating water bath at 37 °C and the oxygen
electrode was inserted into the respiration chamber eliminating any air
space between the oxygen electrode and the surface of the culture
medium. The oxygen electrode was calibrated using complete DMEM
without cells. The rate of oxygen consumption was measured over 10-min
time periods and the respiration rates were normalized (nanomole of
O2/min/106 cells) assuming 220 µM
O2 concentration in air-saturated medium at 37 °C (29).
Retinoid and control treatments were added directly to the culture
medium through a channel in the electrode holder, using a Hamilton
syringe, without separating the electrode from the surface of the
culture medium. Measurements were carried out under low light
conditions. CD437 treatments were done at a concentration of
106 M. Treatments greater than 1 h were
performed as reported in Hail et al. (29) with the
modification that the number of cells tested was 6 × 106 cells. Cell numbers were adjusted using numbers
determined by direct cell counts after respiration determinations.
35S-PCR Analysis of Mitochondrial DNA--
A PCR
method reported by Wei et al. (32) was used to detect
mitochondrial DNA sequences. This method was employed to determine the
effects on mitochondrial number associated with CD437 exposure for long
periods of time and to determine the relative proportion of
mitochondrial DNA in the CA-CD437R cell line as compared with the
CA-OV-3 cell line. To determine the linear range of the PCR reactions,
the reactions were carried out for 37, 40, and 43 cycles, as
recommended in Wei et al. (32), in a thermal cycler using primers specific for mitochondrial DNA (L1-5'-AACATACCCATGGCCAACCT-3' and H1-5'-GGCAGGAGTAATCAGAGGTG-3'). The L1-H1 primers were used for
the amplification of a 533-bp fragment from the total mitochondrial DNA. The first cycle involved a 3-min denaturation at 94 °C, 3-min annealing at 55 °C, and a 1-min extension at 72 °C. The other cycles were as follows: denaturation of 40 s at 94 °C,
annealing for 40 s at 55 °C, and a 50-s extension at 72 °C.
Two µl of mitochondrial DNA was amplified in a 50-µl reaction
mixture containing 200 µM of each dNTP, 0.4 µM of each primer, 1 unit of TaqDNA polymerase (Promega, Madison, WI), 50 mM KCl, 1.5 mM
MgCl2, and 10 mM Tris-HCl, pH 8.3. We modified
the original method by adding 1 µl of 35S-labeled dATP
(10 mCi/ml) to the reaction mixture. The proper size and relative
amounts of PCR product was determined by phosphorimaging (Cyclone Phosphor System and Optiquant Image Analysis software, Packard
Instrument Co., Meridian, CT).
CMXRos Cell Staining--
A method described by Poot et
al. (30) was used to determine the permeability of mitochondria
after treatment with 4-HPR and CD437. After a 12-h treatment with
retinoids, cells were stained with MitoTracker Red CMXRos
(8-(4'-chloromethyl)phenyl-2,3,5,6,11,12,14,15-octahydro-1H,4H,10H,13H-diquinolizino-8H-xanthylium chloride) from Molecular Probes (Eugene, OR). Culture medium was added to the cells and the fluorescent mitochondrial probe CMXRos was
added at a final concentration of 0.25 µg/ml for 15 min at room
temperature. After two rinses for 2 min each with culture medium the
cells were wet mounted with culture media and sealed with silicone.
Images were visualized using a fluorescent microscope (Nikon Eclipse
TE300, Japan) and camera (Nikon Digital Camera DXM1200, Japan).
35S-RT-PCR Analysis of TR3 Expression--
RT-PCR
was used to detect TR3 expression using the Advantage RT-for-PCR kit
(Clontech). In brief, the RT reaction was carried out with 1 µg of total RNA, from cells treated for 8 h with
CD437, in H2O to a total volume of 12.5 µl into which was
added 1 µl of oligo(dT)18 primer, 4 µl of 5× reaction
buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl, and
3 mM MgCl2), 1 µl of dNTP mixture (10 mM each), 0.5 µl of RNase inhibitor, and 1 µl of
Moloney murine leukemia virus-reverse transcriptase. The mixture was
incubated for 1 h at 42 °C, then heated for 5 min at 94 °C
to stop the reaction. The cDNA mixture was diluted to 100 µl. The
PCR reaction was carried out in a thermal cycler using primers specific
for human TR3 (5'-TCATGGACGGCTACACAG-3' and 5'-GTAGGCATGGAATAGCTC-3')
(31). To determine the linear range of the reaction, samples are
removed from the PCR reaction at 20, 25, and 30 cycles. These primers
were used for the amplification of a 517-bp fragment of the TR3
cDNA. The cycles were as follows: denaturation of 45 s at
94 °C, annealing for 45 s at 60 °C, and a 2-min extension at
72 °C. Five µl of the cDNA mixture was amplified in a 50 µl
of reaction mixture containing 200 µM of each dNTP, 0.4 µM of each primer, 1 unit of TaqDNA polymerase
(Promega), 50 mM KCl, 1.5 mM MgCl2,
10 mM Tris-HCl, pH 8.3, and 1 µl of
35S-labeled dATP (10 mCi/ml). Quantitation of the PCR
product was determined by phosphorimaging (Cyclone Phosphor System and
Optiquant Image Analysis software, Packard Instrument Co.).
Antibodies Used for Western Blotting--
Differences in the
level of a variety of mitochondrial proteins associated with apoptosis
including Bid, Bad, Bax, Bcl-2, and Bcl-x (Transduction Laboratories),
caspase-3, caspase-12, and cytochrome c (Santa Cruz
Biotechnologies) were determined by Western blotting as we have
described previously (33).
Antisense Caspase-12 Treatment--
An antisense approach was
used to determine the importance of caspase-12 function after treatment
with CD437 using a modification of a method described by Nakagawa (34).
The sequence of the antisense caspase-12 oligo was: 5'-thiol
modification-TGTCCTCCTGGCCGCCATGGCTGT-3'. A scrambled caspase-12
oligo was used as a control: 5'-thiol
modification-GTCGCTCTGTACGCCTGTGCCTG-3'. In brief, 5 × 105 cells were plated into 100-mm culture dishes in
complete DMEM. Twenty-four hours later the cells were treated with
antisense caspase-12 oligonucleotides or the control oligonucleotide
representing a scrambled caspase-12 sequence. 1 h after the
addition of oligonucleotides, retinoids were added to the culture. The
cells were assessed for reduction of caspase-12 protein expression by
Western blot, viability by direct cell count, and induction of
apoptosis by ELISA.
MAP Kinase Inhibitors and in Vivo Kinase Assay--
To determine
the role of p38 MAP kinase activation in induction of apoptosis the
cell-permeable p38 MAP kinase inhibitors SB 203580 and PD 169316 (Calbiochem, San Diego, CA) were added to culture medium 1 h prior to CD437 or 4-HPR treatments. A commercially available kit was
used to measure MAP kinase activity (MAPK In Vivo Kinase
Assay kit Clontech). Briefly, 4 × 105 cells/well were cultured in 6-well plates. These cells
were transfected with the p38 target protein ATF, a negative control or
a positive control using the Effectene (Qiagen, Valencia, CA)
transfection protocol. The cells were co-transfected with luciferase
reporter and pCMV--galactosidase (Clontech).
Cells were treated with CD437, 4-HPR, or the p38 inhibitor PD 169316 (Calbiochem) 24 h after transfection. After 6 h cell lysates
were collected and luciferase activity was measured using a luminometer
(Femtomaster FB 12, Zylux Corp., Oak Ridge, TN). The results of
the luminometer measurements were normalized by -galactosidase levels.
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RESULTS |
CA-CD437R Cells do Not Exhibit Growth Arrest or Apoptosis after
CD437 Treatment--
We have previously demonstrated that CD437
treatment induced apoptosis in the CA-OV-3 ovarian cancer cell line (6,
16). The CA-CD437R cell line was generated by culturing CA-OV-3 cells in the presence of increasing concentrations of CD437 over a period of
6 months. CA-CD437R cells were treated with a concentration of CD437 of
106 M because this concentration was
previously found to be the most effective at inducing apoptosis in
ovarian tumor cell lines (6, 16). Fig.
1A shows that over a treatment
period of 120 h, the CA-CD437R cell line did not undergo apoptosis
as determined by apoptosis ELISA. For comparison, the parental cell
line CA-OV-3 did undergo apoptosis after treatment with CD437. It
should be noted that we have treated CA-CD437R cells for as long as 7 days with CD437 and have failed to observe apoptosis. Thus, these
results clearly indicate that the cell line selected over the 6-month treatment period is resistant to CD437 and not merely less sensitive, requiring longer treatments.
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Fig. 1.
CA-CD437R ovarian tumor cell line is
resistant to CD437-induced apoptosis and growth suppression.
A, CA-CD437R ovarian tumor cells (2 × 105) were plated in 100-mm culture dishes in 10 ml of DMEM
supplemented with 10% FBS, and treated with CD437 at a concentration
of 106 M for 120 h. A 120-h
Me2SO treatment was used as a negative control. CA-OV-3
ovarian tumor cells treated with 106 M CD437
for 120 h were used as a positive control. The apoptosis ELISA was
performed as described under "Materials and Methods." Data are
expressed as an index of the absorbance of the Me2SO
control treatment, which was set to a value of 1 apoptotic unit. The
data represent the mean ± S.D. of three separate experiments
performed in triplicate.  , CA-OV-3 cells treated with
Me2SO.  , CA-OV-3 cells line treated with CD437.  ,
CA-CD437R cells treated with Me2SO (slightly hidden behind
the filled circles).  , CA-CD437R cells treated with
CD437. B, ovarian tumor cells (2 × 105) were plated in 100-mm culture dishes in 10 ml of DMEM
with 10% FBS, and treated with CD437 at a concentration of
106 M for 7 days. Cell counting was performed
each day for 5 days using a hemocytometer. Data are expressed as cell
numbers × 104. The data represent the mean ± S.D. of three separate experiments performed in triplicate. ,
CA-OV-3 cells treated with Me2SO. , CA-OV-3 cells line
treated with CD437. , CA-CD437R cells treated with
Me2SO. , CA-CD437R cells treated with CD437.
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Whereas CA-CD437R cells were resistant to CD437-induced apoptosis it is
possible that these cells might still be sensitive to growth arrest by
CD437. It has been reported by our laboratory and others that CD437
treatment is able to induce growth arrest in a number of tumor cells
(6-15). Parental CA-OV-3 and CA-CD437R cell lines were treated with
106 M CD437 and viable cell counts were
determined daily following treatment. Fig. 1B shows that
CA-CD437R cells were not growth arrested by CD437 treatment. As
expected, treatment with CD437-induced growth inhibition and reduction
in cell number of parental CA-OV-3 cells. Moreover, resistance to
growth inhibition did not change with increased time of exposure to
CD437. These results are consistent with our previous findings that
CA-CD437R cells have been selected to be resistant to the effects of
CD437 treatment. Taken together, our results using the apoptotic ELISA
and direct cell counts indicate that the CA-CD437R cell line is
resistant to both the growth inhibitory and apoptotic effects of
CD437.
Stability of the CA-CD437R Cell Line--
The results described
above indicate that the CA-CD437R cell line is resistant to
CD437-mediated growth inhibition and apoptosis. We next wished to
determine the stability of this cell line when the selective pressure
of CD437 treatment was removed for extended periods of time. If this
cell line remained resistant after removal of the selective pressure it
is likely that resistance to CD437 results from a permanent change to
the genome of the CA-OV-3 cells. CA-CD437R cells are normally cultured
with CD437 selective pressure 5 of every 14 days. CD437 was removed
from the CA-CD437R cell culture for extended periods of time, and then
these cells were exposed to CD437 for 5 days and assayed for growth
inhibition and induction of apoptosis. We found that CD437 treatment of
the CA-CD437R cell line does not induce growth inhibition or apoptosis even when selective pressure is removed for as long as 84 days (data
not shown). Thus the CA-CD437R cell line does not revert back to a
CD437-sensitive phenotype when grown for extended periods of time in
the absence of CD437.
Sensitivity of the CA-CD437R Cell Line to ATRA, 4-HPR, and
TNF---
To determine whether the CA-CD437R cell line was also
resistant to ATRA we determined the effect of ATRA treatment on cell growth and their ability to undergo RAR transcriptional activation. Fig. 2A shows that CA-CD437R
cells remain sensitive to growth inhibition by 106
M ATRA. To further determine whether RAR function was
altered in these cells, the ovarian tumor cell lines were transfected with a RARE--CAT reporter plasmid. Twenty-four h after transfection the cells were treated with 106 M ATRA. No
distinguishable difference in RAR-mediated transcriptional activation
could be discerned between the parental CA-OV-3 and the CA-CD437R cell
lines (Fig. 2B).
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Fig. 2.
CA-CD437R retains RAR-mediated
transcriptional activity and ATRA-induced growth inhibition similar to
the parental CA-OV-3 cell line. A, ovarian tumor cells
(2 × 105) were plated in 100-mm culture dishes in 10 ml of DMEM with 10% FBS, and treated with either Me2SO or
106 M ATRA for 5 days. Cell counting was
performed on the indicated days using a hemocytometer. Data are
expressed as percent of Me2SO-treated control. The data
represent the mean ± S.D. of three separate experiments performed
in triplicate. B, 5 × 105 ovarian
tumor cells were transfected with a pRARE-CAT as described under
"Materials and Methods." After 24 h, fresh medium and
either 10 µl of Me2SO or 106 M
ATRA treatments were added. Cell extracts were collected after 24 h and measured for ATRA-induced CAT activity. The data represent the
mean ± S.D. of three separate experiments performed in
triplicate.
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To further characterize the CA-CD437R cells we next determined the
sensitivity of this cell line to other apoptotic inducing agents. The
agents chosen for study, fenretinide (4-HPR) and TNF-, both induce
apoptosis in the CA-OV-3 parental cell line. 4-HPR was used at a
concentration of 105 M and TNF- treatments
were carried out at a concentration of 50 ng/ml. The CA-CD437R cell
line, which is resistant to CD437, was also resistant to the induction
of apoptosis by TNF- but was not resistant to the induction of
apoptosis by 4-HPR as measured by an apoptotic ELISA (Fig.
3, A and B).
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Fig. 3.
CA-CD437R ovarian tumor cell line is
resistant to TNF--induced apoptosis but not
4-HPR-induced apoptosis. Ovarian tumor cells (2 × 105) were plated in 100-mm culture dishes in 10 ml of DMEM
with 10% FBS, and treated with either 50 ng/ml TNF- or
105 M 4-HPR for 120 h. A 120-h
Me2SO treatment was used as a negative control. The
apoptosis ELISA was performed as described under "Materials and
Methods." Data are expressed as an index of the absorbance of the
Me2SO control treatment, which was set to a value of 1 apoptotic unit. A: , CA-OV-3 cells treated with
Me2SO; , CA-OV-3 cells line treated with TNF-; ,
CA-OV-3 cells treated with 4-HPR. B: , CA-CD437R cells
treated with Me2SO; , CA-CD437R cells line treated with
TNF-; , CA-CD437R cells treated with 4-HPR. The data represent
the mean ± S.D. of three separate experiments performed in
triplicate.
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Caspase Activity in CA-CD437R Cells after Retinoid
Treatment--
To begin to understand the mechanism by which retinoids
such as CD437 and 4-HPR induce apoptosis, we next examined the
activation of a number of effector caspases including
caspase-3/caspase-7 and caspase-9 activity. Fig.
4, A and B, shows
that no caspase-3-like activity could be detected in the CA-CD437R cell
line, in response to CD437. However, the level of caspase-3 activity
assayed in CA-CD437R cells following 4-HPR treatment was similar to
that induced by both CD437 and 4-HPR in the CD437-sensitive parental cell line CA-OV-3. Furthermore, treatment with DEVD-cmk (a
caspase-3-like inhibitor) 1 h prior to retinoid treatment
significantly inhibits 4-HPR-induced caspase-3-like activities in the
CA-CD437R cell line. These results demonstrate that the caspase-3-like
protease is not altered in CA-CD437R cells. We also investigated the
activation of other caspases in these cell lines after treatment with
CD437 and 4-HPR. In the 24 h after treatment no caspase-6 or
caspase-8 activity could be detected in either CA-OV-3 or the CA-CD437R cells (data not shown). Similar to our results with caspase-3-like activity, caspase-9 activity was present in both cell lines after treatment with 4-HPR and could be detected in the CA-OV-3 cell line
after CD437 treatment (Fig. 4, C and D). However,
no caspase-9 activity could be detected following CD437 treatment of
the CA-CD437R cell line. These results suggest that the alteration in
CA-OV-3 cells, which is responsible for CD437 resistance, must map
upstream of caspase-3-like and caspase-9 activation.
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Fig. 4.
Retinoid and TNF-
induced caspase activity in ovarian carcinoma cell line
CA-CD437R. A and B, caspase-3-like activity.
Ovarian tumor cells (8 × 106) were plated in 100-mm
culture dishes in 10 ml of DMEM with 10% FBS, and treated as indicated
for 48 h. Whole cell lysate from ~4 × 106
ovarian cells was collected at the indicated times and the activity of
caspase-3-like proteases was measured by the cleavage of the caspase
substrate Ac-DEVD-pNA. Data are expressed as absorbance at 405 nm. The
data represent the mean ± S.D. of three separate experiments
performed in triplicate. A, CA-OV-3, and
B, CA-CD437R cells were treated with Me2SO
(), 4-HPR (), TNF- (), CD437 (), and a combination of
4-HPR and the caspase-3 inhibitor DEVD-cmk ( ). C and
D, caspase-9 activity. Cell lysates were prepared by the
same method as caspase-3-like activity. The activity of caspase-9
proteases was measured by the cleavage of the caspase substrate
Ac-LEHD-pNA. Data are expressed as absorbance at 405 nm. The data
represent the mean ± S.D. of three separate experiments performed
in triplicate. C, CA-OV-3, and D,
CA-CD437R cells were treated with Me2SO (), 4-HPR (),
TNF- ([itrif), CD437 (), and a combination of 4-HPR and the
caspase-3 inhibitor LEHD-cmk ().
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Caspase-12 has been reported to be important for induction of apoptosis
following endoplasmic reticulum stress (34-37). Because retinoids are
lipophilic and might act via an endoplasmic reticulum-mediated process,
we wished to determine whether there was a difference in levels or
activation of caspase-12 in CA-CD437R cells versus CA-OV-3
cells following CD437 treatment. Interestingly, Western blot analysis
showed that the CA-CD437R cell line expressed only 10% of the
caspase-12 observed in the parental CA-OV-3 cells (Fig. 5A). However, the levels of
caspase-12 did not change following CD437 treatment (Fig.
5A). To determine whether this reduction in caspase-12
levels was responsible for the resistance of CA-CD437R cells to CD437
we determined the effect of reducing caspase-12 levels on the ability
of CD437 to induce apoptosis in parental CA-OV-3 cells. Fig.
5B shows that treatment of CA-OV-3 cells with antisense to
caspase-12 (50 µg/ml) substantially reduced the level of caspase-12
within 6 h after treatment. Fig. 5C shows that
pretreatment with concentrations of antisense caspase-12, which
significantly reduced caspase-12 protein levels, did not prevent the
induction of apoptosis as measured by an apoptotic ELISA (Fig.
5C), or the reduction of cell number (Data not shown) even
when assayed 120 h after CD437 treatment (Fig. 5D).
These results indicate that reduction of caspase-12 levels in CA-OV-3
cells does not in and of itself result in resistance to induction of
apoptosis by CD437.
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Fig. 5.
Modulation of caspase-12 expression does not
affect the ability of CD437 or 4-HPR to induce apoptosis in ovarian
carcinoma cells. A, Western blotting was used to detect
endogenous caspase-12 levels in both the CA-OV-3 and CA-CD437R cell
lines after 6 h treatment with Me2SO, CD437, or 4-HPR.
B, Western blots were used to detect the levels of
caspase-12 after CA-OV-3 cells were treated with antisense caspase-12
oligonucleotide (AS) for 6 and 24 h. CA-OV-3 cells
treated with CD437 and 4-HPR for 6 h were used for comparison of
caspase-12 levels. C, CA-OV-3 ovarian tumor cells (5 × 105) were plated in 100-mm culture dishes in 10 ml of DMEM
with 10% FBS, and treated with either 106 M
CD437 or 105 M 4-HPR for 5 days and antisense
caspase-12. Cell counting (C) and apoptosis ELISA
(D) were performed on day 5 after treatment with the
retinoids and various doses of antisense caspase-12. Me2SO
was used as a control for comparison. In C, CA-OV-3
cells were treated with antisense caspase-12 (50 µg/ml) and
Me2SO () or CD437 (), or 4-HPR () for 5 days and
cell counts were used to determine the effect of treatment in a
time-dependent manner.
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CMXRos Cell Staining--
Apoptosis characterized by induction of
caspase-9 activity followed by activation of caspase-3 has been
reported to be associated with permeabilization of the mitochondrial
membrane (38-40). Because CD437 and 4-HPR both induced caspase-9 and
caspase-3 in the CA-OV-3 cell line, we next examined the mitochondrial
permeability of CA-OV-3 and the CA-CD437R cells using the probe CMXRos.
Mitochondria accumulate the CMXRos probe unless the membranes of the
mitochondria are depolarized. The micrographs in Fig.
6 clearly show that compared with
vehicle-treated control cells (Fig. 6, A and B)
the mitochondria of CA-OV-3 did not accumulate the CMXRos stain
following treatment with either CD437 (Fig. 6, C and
D) or 4-HPR (Fig. 6, E and F). This
indicated that the induction of apoptosis by these retinoids involves
depolarization of the mitochondrial membranes. In contrast, CD437
treatment of the CA-CD437R cell line did not induce the depolarization
of the mitochondrial membranes (Fig. 7,
C and D). As expected, 4-HPR was found to induce
depolarization in both CA-CD437R cells and parental CA-OV-3 cells as
indicated by the fact that the mitochondria were no longer able to
accumulate CMXRos (Figs. 6, E and F, and 7,
E and F). It should be noted that the use of a
second similar stain, DIOC(6) gave the same results (data not shown). Thus, CD437 fails to induce mitochondrial membrane depolarization in the CA-CD437R cells indicating that the alteration in
the CA-CD437R cells maps upstream of mitochondrial
permeabilization.
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Fig. 6.
Loss of mitochondrial membrane potential
after treatment of CA-OV-3 cells with apoptosis-inducing retinoids
CD437 and 4-HPR. CMXRos staining was performed as described
under "Materials and Methods." CA-OV-3 cells were treated for
12 h with Me2SO (A and B), CD437
(C and D), or 4-HPR (E and
F) and then stained with CMXRos to determine mitochondrial
membrane potential. The lack of retention of CMXRos (red)
occurs in mitochondria, which loses membrane potential and is
indicative of apoptosis induction. For comparison purposes cells are
shown in bright field in panels A, C, and
E. Images were visualized at ×1000 using a fluorescent
microscope and digital photo camera.
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Fig. 7.
Loss of mitochondrial membrane potential
after treatment of CA-CD437R cells with apoptosis-inducing retinoids
CD437 and 4-HPR. CMXRos staining was performed as described under
"Materials and Methods" to determine the extent of retinoid-induced
loss of mitochondrial membrane potential in the CA-CD437R cell line
treated for 12 h with Me2SO (A and
B), CD437 (C and D), or 4-HPR
(E and F). Bright field panels (A,
C, and E) are for comparison purposes.
Images were visualized at ×1000 using a fluorescent microscope and
digital photo camera.
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35S-RT-PCR Analysis of TR3 Expression--
Increased
expression of TR3 has been reported to occur in cells induced to
undergoing apoptosis by a number of agents including CD437 (8, 31,
41-45). Because the CMXRos staining indicated a depolarization of the
mitochondrial membrane in conjunction with reports that CD437-induced
apoptosis can be associated with the increased expression of TR3 and
the eventual translocation of TR3 to the mitochondria, we next examined
TR3 expression in our cells (8, 31, 43-45). We used a semiquantitative
RT-PCR to measure expression of TR3 after 8 h of CD437 treatment.
This method was employed to determine whether CD437 treatment induced expression of TR3 in CA-OV-3 cells and to determine whether
differential expression of TR3 was responsible for the resistance of
the CA-CD437R cell line to the effects of CD437. As indicated in Fig.
8, both the parental CA-OV-3 cell line
and the CA-CD437R cell line express TR3 and exhibited an increase in
the expression of TR3 following CD437 treatment as measured by
35S-RT-PCR. This increase was similar for both cell lines.
The data were normalized to glyceraldehyde-3-phosphate dehydrogenase
levels as determined by 35S-RT-PCR. Quantitation of the
25-cycle bands indicated a 5-fold increase in the expression of TR3 in
the parental CA-OV-3 cell line and an 8-fold increase in the expression
of TR3 in the CD437-resistant CA-CD437R cell line. These data indicate
that expression of TR3 is not impaired in the CA-CD437R cell line
following CD437 treatment.
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Fig. 8.
TR3 expression increases in both parental
CA-OV-3 cells and CA-CD437R cells after CD437 treatment. Total RNA
was collected after 12 h of treatment with either
Me2SO or CD437. The total RNA was used in an RT-PCR assay
to determine the expression of TR3 in the parental CA-OV-3 cell line
(A) as compared with the CA-CD437R cell line (B).
The 35S-RT-PCR reactions were carried out as indicated
under "Materials and Methods" and the image of the PCR products
and the relative band density was determined at three different cycle
numbers by phosphorimaging. GAPDH,
glyceraldehyde-3-phosphate dehydrogenase.
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Oxygen Consumption Measurements of Ovarian Carcinoma Cells--
It
has recently been reported that CD437 could decrease cellular oxygen
consumption in human cutaneous squamous cells and that reduction of
mitochondria numbers by ethidium bromide treatment resulted in
resistance to CD437 (29). We compared the oxygen consumption rate in
the parental ovarian carcinoma cell line, CA-OV-3, and the CA-CD437R
cell line. We found that the resting rate of oxygen consumption in the
CA-CD437R cells was 65-70% lower than the parental CA-OV-3 cells
(Fig. 9A). Treatment of the
parental CA-OV-3 cell line with CD437 resulted in a decrease in the
rate of cellular oxygen consumption by ~75% (Fig. 9A).
This reduction in the rate of oxygen consumption continued to decrease
for 6 h. In contrast, CD437 treatment of CA-CD437R cells does not
further reduce oxygen consumption. To determine whether the lower
resting rate of oxygen consumption of the CA-CD437R cell line compared with the parental CA-OV-3 cells was because of a reduction in the
number of mitochondria in CA-CD437R cells, a 35S-PCR method
was used to detect mitochondrial DNA sequences. Fig. 9B
shows that the relative amount of mitochondrial DNA and thus the
relative number of mitochondria was similar in both cell lines indicating that the reduced oxygen consumption by the CA-CD437R cell
line was not because of a reduction in the number of mitochondria. This
suggests that the alteration in CA-CD437R cells resulting in resistance
to CD437-induced apoptosis maps at the level of the mitochondria
and most likely involves events that regulate oxygen
consumption.
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Fig. 9.
Oxygen consumption measurements of cultured
cells after treatment with CD437 and 4-HPR. A, the rate
of oxygen consumption was measured using a Clark-type oxygen electrode.
6 × 106 cells were trypsinized and suspended in 3 ml
of DMEM culture medium in a 3-ml chamber with continuous
stirring at 37 °C. The oxygen electrode was calibrated using
complete DMEM without cells. The rate of oxygen consumption was
measured over 10-min time periods at 1, 3, 6, and 12 h and the
respiration rates were normalized (nanomole of
O2/min/106 cells) assuming 220 µM
O2 concentration in air-saturated. CD437 (106
M) and control treatments were added directly to the
culture medium. Cell numbers were adjusted using numbers determined by
direct cell counts after respiration determinations. CA-OV-3 was
treated with Me2SO () and 106
M CD437 (), and the CA-CD437R cell line was treated with
Me2SO () and 106 M CD437
(). The data represent the mean ± S.D. of three separate
experiments performed in triplicate. B, a
35S-PCR method was employed to determine the relative
proportion of mitochondrial DNA in the CA-CD437R cell line as compared
with the CA-OV-3 cell line. The PCR reaction was carried out as
indicated under "Materials and Methods" and the image of the PCR
products at three different cycles and the relative band density was
determined by phosphorimaging.
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Western Blotting for Proteins Associated with the Induction of
Apoptosis--
Because the resistance of the CA-CD437R cell line to
CD437 appeared to involve mitochondria function but was not because of a decrease in the number of mitochondria, we examined the expression of
a number of mitochondrial proteins associated with apoptotic induction.
Western blot analysis indicated that no differences in the levels of
expression of Bad, Bid, Bax, and Bcl-x mitochondrial proteins occur
between the CA-OV-3 and CA-CD437R cell lines (data not shown). Whereas
levels of the mitochondrial protein Bcl-2 were slightly greater in the
parental cell line and were observed to increase only after 4-HPR
treatment, Bcl-2 levels did not change after CD437 or 4-HPR treatment
of the CA-CD437R cell line (Fig. 10A). Likewise cytochrome
c was released from the mitochondria to the cytoplasm after
treatment of the CA-OV-3 cell line with CD437 (Fig. 10B) but
not after treatment of the CA-CD437R cell line (data not shown). 4-HPR
treatment of both the parental CA-OV-3 and the CA-CD437R cell lines
induced cytochrome c release at similar levels (data not
shown).
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Fig. 10.
Expression of mitochondrial proteins Bcl-2
and cytochrome c in the CA-OV-3 and CA-CD437R cell
lines. CA-OV-3 and CA-CD437R cells were examined for differences
in the level of the mitochondrial protein Bcl-2 (A) after
24 h treatment with CD437 106 M or 4-HPR
105 M. The positive control is from a Jurkat
cell line known to have high expression of Bcl-2. Cytochrome
c levels (B) were examined in the mitochondrial
fraction of cell lysates after 6 and 24 h treatment with
Me2SO or CD437 (106 M) or 4-HPR
(105 M). The Western blots were visualized by
chemiluminescence.
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MAP Kinase (p38)--
It has been reported that activation of p38
MAP kinase, following treatments that induce apoptosis, can lead to the
activation of caspase-3 and the depolarization of the mitochondrial
membrane (39, 46-48). Fig.
11A shows that 6 h of
CD437 treatment can induce the activity of p38 in both the CA-OV-3 and
CA-CD437R cell lines. In fact p38 activity was induced to a greater
extent in CA-CD437R cells following retinoid treatment. We used p38 MAP
kinase inhibitors in conjunction with retinoid treatment to determine
whether the inhibition of p38 activity would prevent retinoid-induced
apoptosis (Fig. 11B). The apoptosis ELISA was used to
measure the extent of apoptosis after 5 days treatment with the
synthetic retinoids and MAP kinase inhibitors. We found that both p38
inhibitors, PD169316 and SB202190, could inhibit the induction of
apoptosis by CD437 in the CA-OV-3 cell line. In contrast, the MAP
kinase inhibitors did not inhibit 4-HPR-induced apoptosis. These
results confirm that CD437 and 4-HPR induce apoptosis by completely
different pathways. Moreover because CD437 can induce p38 activity in
CA-CD437R cells, the alteration in CA-CD437R cells responsible for
resistance to apoptosis must map downstream of the activation of p38 by
CD437.
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Fig. 11.
CD437-induced apoptosis requires MAP kinase
(p38) activity. A, MAP kinase (p38) activity was
detected after CD437 treatment in the CA-OV-3 cell line but not the
CA-CD437R cell line. CA-OV-3 and CA-CD437R cells (4 × 105 cells/well) were cultured in 6-well plates. The next
day cells were transfected with a luciferase reporter to measure p38
activity and a pCMV--galactosidase to determine transfection
efficiency. Twenty-four h after transfection cells were treated with
CD437 or 4-HPR and/or the p38 inhibitor PD169316. After 12 h of
treatment cell lysates were collected and luciferase activity was
measured using a lumenometer. UVB-treated cells were used as a positive
control. -Galactosidase levels normalized the luminometer results.
The data represent the mean ± S.D. of three separate experiments
performed in triplicate. B, MAP kinase (p38) inhibitors
modulate CD437- but not 4-HPR-induced apoptosis in CA-OV-3 and
CA-CD437R cells. CA-OV-3 and CA-CD437R ovarian tumor cells (5 × 105) were plated in 100-mm culture dishes with 10 ml of
DMEM with 10% FBS, and treated with either 106
M CD437 or 105 M 4-HPR and MAP
kinase inhibitor, 20 µg/ml PD169316 or 10 µg/ml SB202190. The
apoptosis ELISA, described under "Materials and Methods," was
performed on 5-day treated cells to determine the extent of apoptosis
induction. Me2SO and the retinoids alone were used as
controls. The data represent the mean ± S.D. of three separate
experiments performed in triplicate.
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DISCUSSION |
Using a cell line selectively resistant to the growth suppressive
and apoptotic effects of CD437 treatment, we have determined the
following new information about the molecular events responsible for
induction of apoptosis by CD437: 1) the pathway utilized by CD437 to
induce apoptosis appears to be similar to that induced by TNF- but
not that induced by another apoptotic inducing retinoid, 4-HPR. 2)
CD437-induced apoptosis in ovarian carcinoma cells is dependant upon
caspase-3 and caspase-9 but not caspases-6, caspase-8, or caspase-12.
3) CD437-induced apoptosis in ovarian carcinoma cells is dependent and
involves a change in mitochondrial oxygen consumption, depolarization
of the mitochondria, and subsequent release of cytochrome c,
resistance of CA-CD437R cells to CD437 does not involve a reduction in
ATRA responsiveness or RAR activity, mitochondrial number, or ability
to activate p38 kinase.
To characterize the mechanism involved in induction of apoptosis
by CD437 we explored the response of a CD437-resistant cell line
(CA-CD437R) to other agents that induce apoptosis. The parental CA-OV-3 ovarian tumor cell line is sensitive to apoptosis induced by
treatment with 4-HPR and TNF-. Surprisingly, the CA-CD437R cell line
was not responsive to the effects of TNF- treatment but was just as
sensitive as the CA-OV-3 cell line to 4-HPR treatments. This suggests a
common mechanism for the induction of apoptosis by TNF- and CD437
treatments. Furthermore, the ability of 4-HPR to induce apoptosis in
CA-CD437R cells indicates a clear divergence in the pathway of
apoptosis induction by these two synthetic retinoids.
Because retinoids are known to function by activation of RARs and
induction of apoptosis by CD437 is at least partially RAR dependent in
CA-OV-3 cells (16), it was important to determine whether RAR function
had been altered in the CA-CD437R cell line. Two approaches were used
to determine whether the RAR response was altered: sensitivity to ATRA
growth inhibition and transcriptional transactivation of a reporter
plasmid construct that contained the ATRA responsive RAR--RARE. We
found that CA-CD437R cells responded to ATRA-induced growth inhibition
and RARE-dependent transcriptional activation in a manner
identical to that of the parental CA-OV-3 cells. These experiments show
that RAR function and the sensitivity to ATRA-induced growth inhibition
has not been altered in the CA-CD437R cell line. This suggests that the event responsible for CA-CD437R resistance to CD437 maps downstream of
RAR activation and does not involve elements common to both the ATRA
and CD437 pathways. This is consistent with the work of Ponzanelli
et al. (49) who showed that a promyelocytic leukemia cell
line resistant to CD437 retained sensitivity to ATRA and the RAR-
selective synthetic retinoid AM580.
Recent studies in other cell models to determine the molecular events
that constitute the apoptotic pathway induced by CD437 have suggested
that the mechanism of CD437 action is extremely complex and at times
contradictory. For example, CD437 was shown to induce growth arrest and
apoptosis in tumor cell lines via a caspase-dependent
pathway in some tumor cells and via caspase-independent pathways in
others (50, 51). CD437-induced apoptosis is associated with the
activation and degradation of many caspase isoforms including caspase-3, -6, -7, and -9 (38, 49, 52). In addition, treatment with
CD437 has been shown to modulate the expression of apoptotic-related proteins such as Bax, Bcl-2, DR5, p53, and p21 (WAF1/CIP1) in lung, breast, and small cell carcinoma cells (8, 13, 38, 51, 53).
We have found that in ovarian carcinoma cells the induction of
apoptosis by both CD437 and 4-HPR is caspase-dependent.
Addition of the caspase-3-like inhibitor, DEVD-cmk, 1 h prior to
the addition of CD437 or 4-HPR blocked apoptosis induced by these
retinoids. We could not detect caspase-3-like activity in the CA-CD437R
cells after treatment with CD437 or TNF-. To determine whether lack of caspase-3 or -7 was responsible for CD437 resistance in CA-CD437R cells, we used 4-HPR to determine whether the caspase-3-like proteases were present and functional in this cell line. Measurements of the
activity of the caspase proteases using the caspase-3-like substrate
DEVD-pNA mixed with 4-HPR-treated cell lysates indicates that 4-HPR
induces a caspase-3-like protease activity. Moreover, treatment of
CA-CD437R cells with DEVD-cmk protected these cells from 4-HPR-induced
apoptosis. Together these two findings show that caspase-3-like
proteases are functional and unaltered in the CA-CD437R cell line and
are thus not responsible for the resistance of these cells to
CD437-induced apoptosis.
We also looked at other caspases that are known to function upstream of
caspase-3 activation. Assays for caspase-8 and -6 after treatment
showed that neither CD437 nor 4-HPR activated these caspases.
Caspase-12 has been reported to be activated upstream of caspase-3-like
activity during endoplasmic reticulum stress (34-37). Moreover
CA-CD437R cells express less caspase-12 protein than parental CA-OV-3
cells. However, reduction of caspase-12 levels in parental CA-OV-3
cells did not protect them from CD437 or 4-HPR-induced apoptosis.
Reports in other cell models systems have indicated that CD437 and
4-HPR treatments cause an apoptosis-related depolarization of the
mitochondrial membrane (38, 49, 54-56). Consistent with these reports,
we show that depolarization also occurs in both CA-OV-3 and CA-CD437R
cells after treatment with 4-HPR but that CD437 induced depolarization
of the mitochondria only in the parental CA-OV-3 cells and not in the
CA-CD437R cells. Depolarization of the mitochondrial membrane results
in the release of caspase-9, cytochrome c, and APAF-1,
which form a complex capable of activating caspase-3, -6, and -7 (57).
It has been suggested that cytochrome c may be central to
CD437-induced caspase activation (38, 49, 52, 53, 55). Consistent with
these reports, we determined that cytochrome c is released
after treatment of CA-OV-3 cells with CD437 but not after treatment of
CA-CD437R cells.
Caspase-9 has been shown to be released after mitochondrial
depolarization in conjunction with cytochrome c and APAF-1,
prior to activation of caspase-3. Parental CA-OV-3 ovarian carcinoma cells treated with a caspase-9 inhibitor (LEHD-cho) 1 h prior to
the addition of CD437 or 4-HPR blocked apoptosis induced by these
retinoids. Measurements of the activity of the caspase-9 proteases
using the substrate LEHD-pNA mixed with lysates from either CD437- or
4-HPR-treated CA-OV-3 cells but not CA-CD437R cells indicates that both
retinoids induce caspase-9 protease activity. Treatment of CA-CD437R
cells with LEHD-cmk protected these cells from 4-HPR-induced apoptosis,
caspase-9 does not appear to be altered in the CA-CD437R cell line and
therefore is not responsible for resistance to CD437.
Our studies show that the CA-CD437R cell line is resistant to apoptosis
induced by TNF-. This suggests that CD437-induced apoptosis utilizes
a similar mechanism as TNF-. Cross-resistance of ovarian tumor cell
lines to multiple apoptotic agents has been reported previously.
Kumar et al. (58) found that a cell line selected for
resistance to paclitaxel was also resistant to CD437. The fact that
CA-CD437R cells contain functional caspase-3-like and caspase-9
protease activation and activity suggests that the alteration
responsible for CD437 resistance lies upstream of caspase-3-like and
caspase-9 activation. A recent report suggests that resistance to CD437
of a leukemia cell line mapped upstream of cytochrome c
release and activation of caspases (49). The release of cytochrome c is dependent on release from the mitochondria often
through a depolarization of the mitochondrial membrane (55, 59). CMXRos staining demonstrated that mitochondria were depolarized in CA-OV-3 cells but not CA-CD437R cells after CD437 treatment. Likewise, cytochrome c was found to be released in CD437-treated
CA-OV-3 cells but not in CA-CD437R cells. However, because 4-HPR
treatment of CA-CD437R cells did result in mitochondrial depolarization and release of cytochrome c, this step in the apoptotic
pathway is not defective and thus not responsible for CD437 resistance.
TR3 has been associated with the depolarization of mitochondria through
increased expression, translocation to the cytosol, and targeting of
the mitochondria (8, 31, 43-45, 60-62). In light of these findings it
was logical to determine whether a lack of TR3 expression could be the
cause of resistance to the effects of CD437 in the CA-CD437R cell line.
Our data indicate that both cell lines express TR3 and that there was a
comparable response in the increase of TR3 expression occurring in both
the parental CA-OV-3 and the CA-CD437R cell lines after CD437
treatment. Whereas we did not examine translocation of TR3, it would
appear, at least based on our expression analysis, that a TR3 defect is not likely to be responsible for CA-CD437R cell line resistance to
CD437.
Hail et al. (29) reported that resistance to CD437 treatment
occurs in cutaneous squamous carcinoma cells if mitochondria numbers
are reduced. Our studies do not show that the CA-CD437R cell line has a
reduced number of mitochondria compared with parental CA-OV-3 cells but
do exhibit significantly lower oxygen consumption after CD437
treatment. This suggests that the resistance seen in the CA-CD437R cell
line might involve a defect in the electron transport chain.
TNF- has been reported to activate tyrosine kinases of cellular
stress-induced pathways consequently leading to activation of c-Jun
NH2-terminal kinase (JNK) and p38 MAPK (63-65). Recent reports suggest that activated p38 MAPK is associated with apoptosis and regulates the release of cytochrome c from the
mitochondria (39, 46, 48). Because CD437 has been reported to activate JNK, this suggests that the JNK pathway may be a common link for apoptosis induced by TNF- and CD437. Our in vivo p38 MAP
kinase activity assays show that activity occurred in both the CA-OV-3 and CA-CD437R cell lines after CD437 treatment. These data indicate that the resistance to CD437 in the CA-CD437R cell line maps downstream of MAP kinase activation.
A model for the pathway by which CD437 induces apoptosis in CA-OV-3
cells is shown in Fig. 12. We propose
that CD437 induces apoptosis by a similar mechanism as has been
reported for TNF-. As indicated in our model (Fig. 12), CD437 is
able to induce the activation of the p38 MAPK after 6 h treatment.
As reported in the literature, p38 MAPK can activate the MEF2
transcription factor (66-69). Because it has been reported that a MEF2
site is located on the TR3 gene promoter (66), MEF2 could be
responsible for the increase in TR3 expression that we observed 8 h after treatment with CD437. The translocation of TR3 has been
reported to occur after CD437 treatment. TR3 translocation can induce
the depolarization of mitochondrial membranes (8, 31, 43-45, 60-62).
We demonstrate that CD437 treatment results in mitochondrial
depolarization. This occurs after activation of p38 and is followed by
the release of cytochrome c (55, 59). Depolarization of the
mitochondrial membrane is associated with the release of cytochrome
c, APAF-1, and procaspase-9. These proteins when released
from the mitochondria will form a complex that results in the
activation of caspase-9. Active caspase-9 will cleave procaspase-3 and
result in the activation of caspase-3. Caspase-3 activity is indicative
of the final stages of apoptosis induction. This is consistent with our
findings that both caspase-3 and caspase-9 activity is induced by CD437
and that apoptosis occurs after this activation. Future studies will focus on identification of molecular events, which follow activation of
p38 MAPK and precede mitochondrial depolarization.
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Fig. 12.
A model for CD437-induced apoptosis in
ovarian carcinoma cells. The model depicts the molecular pathway
activated in the parental CA-OV-3 ovarian carcinoma cell line after
treatment with CD437. Our results show that the CA-CD437R cell line has
increased levels of TR3 after CD437 treatment but no mitochondrial
depolarization. This indicates that the alteration in the pathway
exhibited by the CA-CD437R cell line maps downstream of TR3 mRNA
induction and upstream of mitochondrial depolarization. Our results
also show that, in contrast to CD437, no p38 MAPK activity was induced
after 4-HPR treatments. In addition MAP kinase inhibitors failed to
protect CA-OV-3 cells from 4-HPR-induced apoptosis. Thus, it would
appear that the difference between 4-HPR-induced apoptosis and
CD437-induced apoptosis occurs upstream of mitochondrial depolarization
and activation of p38 MAP kinase.
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§,
, and
TOP
ABSTRACT
INTRODUCTION
