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J. Biol. Chem., Vol. 277, Issue 46, 43763-43770, November 15, 2002
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From the
Received for publication, July 30, 2002, and in revised form, September 9, 2002
The objective of this article is to dissect the
mechanisms by which the down-regulation of c-Myc induces programmed
cell death in melanoma cells. In stable and doxycycline-inducible M14
melanoma cells, down-regulation of c-Myc induced apoptosis subsequent
to a decrease in the intracellular reduced glutathione content and a
concomitant accumulation of its oxidized form. This redox alteration was associated with a decrease of the enzyme activities of
Maintenance of normal function of cells and tissues is dependent
on precise regulation of multiple signaling pathways that control
cellular decisions to either proliferate, differentiate, arrest cell
growth, or initiate programmed cell death. Genes of the myc
family, including c-myc, have been implicated in the
regulation of many cellular processes such as proliferation,
differentiation, and transformation (1, 2). Deregulated expression of
c-myc accelerates apoptosis in myeloid cells
(interleukin-3-dependent) deprived of interleukin-3 (3) and in
serum-deprived fibroblasts (4). Over-expression of c-myc
induced apoptosis under certain conditions such as viral infection and
treatment with tumor necrosis factors and chemotherapeutic agents
(5-7). Despite intensive research, the molecular mechanisms underlying
apoptosis mediated by c-Myc are yet to be understood. It has been
proposed that c-Myc induces apoptosis by up-regulating its target
genes, such as those expressing ornitine decarboxylase (8, 9), lactate
dehydrogenase (10, 11), cyclin A, cdc25A (12, 13), or p53 (14, 15). Moreover, at least in tumor cells, not only the over-expression of
c-myc but also its down-regulation induces apoptosis
(16-18). In this context, we previously demonstrated that treatment
with c-myc antisense oligodeoxynucleotides caused a
significant inhibition of cell proliferation and induced apoptosis in
several human melanoma cell lines (19-21). By using stable
transfectants, we also demonstrated that apoptosis following
down-regulation of c-Myc (22) was associated with an increase in
the production of ROS1 (23).
In fact, oxidative stress is a well known inducer of apoptosis (24,
25). In particular, it has been demonstrated extensively that apoptosis
is stimulated by cell depletion of GSH (26, 27), the low molecular
weight thiol that is crucial for antioxidant defense (28). In this
context we demonstrated that following apoptogenic stimuli that
do not directly elicit an oxidative stress, GSH is extruded from the
cells at very early stages of the process (29) and that the inhibition
of the extrusion of GSH prevents apoptosis (30). Moreover, we evidenced
that the release of cytochrome c from mitochondria followed
cell depletion of GSH, independently of the destiny of the cells,
i.e. apoptosis or survival (31).
The present study aims at dissecting the molecular mechanism by which
down-regulation of c-Myc induces apoptosis, with the expectation of
identifying new molecular factors that may represent therapeutic
targets in reestablishing apoptotic pathways in cancer cells. We
demonstrate that down-regulation of c-Myc triggers apoptosis in stable
and doxycycline-inducible clones of M14 melanoma cells through a
canonical redox-mediated pathway involving depletion of GSH and release
of cytochrome c as early events, whereas production of ROS
and activation of caspases are late effectors. Decrease of GSH results
from an impairment of both its synthesis and GSSG reduction. The key
role of GSH in the apoptosis induced by down-regulation of c-Myc is
further supported by the ability of Cys-NAc or GSH ester to suppress
the commitment of cells to death.
Cell lines, Culture Conditions, and Treatments--
The stable
M14 melanoma transfectants (MAS51 and MAS53 c-Myc low expressing clones
and MN2 control clone) were obtained by transfection with an expression
vector carrying antisense c-myc cDNA and/or a selection
marker gene (23). The M14-derived doxycycline-inducible clones
expressing low c-Myc (MAS IND1 and MAS IND18) were obtained by a double
transfection with a commercial inducible TET-ON gene expression system
(Clontech, Florence, Italy) consisting of two expression vectors, a regulator and a response one carrying
c-myc cDNA (exon 2 + exon 3) in the antisense
orientation. Doxycycline (1 µg/ml, administered every 24 h)
down-regulates c-Myc protein in about 72 h by 50-60% in both
clones as compared with uninduced transfectants.
Materials--
DTT, EDTA, EGTA, PIPES disodium salt,
potassium borohydride, GSH ethyl ester, L-glutamate,
L- ROS, Apoptosis, and Caspase Activity--
For ROS content,
adherent cells were first assayed for viability and then incubated with
4 µM dihydroethidium (DHE, Molecular Probes, Eugene, OR)
for 45 min at 37 °C. After incubation, the cells were analyzed by
flow cytometry.
Apoptosis was evaluated by annexin V versus PI assay. Cells
were harvested, suspended in annexin-binding buffer (1 × 106 cells/ml), incubated with fluorescein
isothiocyanate-annexin V and PI (Molecular Probes) for 15 min at room
temperature in the dark, and then immediately analyzed by flow
cytometry. The annexin V positive/PI negative (annexin V+/PI
Activity levels of caspase-9 and -3 were evaluated using a
CaspaTagTM Kit according to the manufacturer's instruction
(Intergen, Oxford, UK). Cells were harvested and resuspended in
fresh medium (1 × 106 cells/ml). The specific
fluorescent caspase substrate was added directly to the cell suspension
and left for 1 h at 37 °C under 5% CO2 and
protected from the light. After washing, cells were stained with PI and
analyzed by flow cytometry.
Determination of Cytosolic Cytochrome c--
Cells were washed
with phosphate-buffered saline and collected by centrifugation at
700 × g for 7 min at 4 °C. The cell pellet was
resuspended in extraction buffer containing 220 mM
mannitol, 68 mM sucrose, 50 mM PIPES-NaOH, pH
7.4, 50 mM KCl, 5 mM EGTA, 2 mM
MgCl2, 1 mM dithiothreitol, and protease
inhibitors. After a 30-min incubation on ice, cells were homogenized
with a glass Dounce homogenizer. Cell homogenates were spun at
14,000 × g for 15 min at 4 °C, and supernatants
were removed and stored at Western Blotting--
40 µg of total proteins were loaded from
each sample on denaturing 12% SDS-PAGE. Immunodetection of PARP,
Determination of Glutathione--
For intracellular glutathione
determination, cell monolayers were washed with phosphate-buffered
saline, resuspended, and lysed by repeated cycles of freezing and
thawing under liquid nitrogen. Lysates were acidified with 5%
meta-phosphoric acid and centrifuged at 22,300 × g for 15 min at 4 °C. Low molecular weight free thiols
were derivatized to S-carboxymethyl derivatives upon
treatment of supernatants with iodoacetic acid. GSH and GSSG concentrations were determined by the conversion of free amino groups
to 2,4-dinitrophenyl derivatives by the reaction with
1-fluoro-2,4-dinitrobenzene. Low molecular weight thiols were, finally,
separated by HPLC using a µBondapak NH2 column (Waters)
as described by Reed et al. (32). GSH and GSSG were used as
external standards. Data were expressed as nmol/mg protein. For the
extracellular glutathione assay, culture media were collected and
centrifuged at 700 × g for 10 min at 4 °C in order
to discard detached cells. Media were then acidified and treated as
described before. Data were expressed in nmol/ml.
Macroarray Analysis of Gene Expression--
Total RNA was
isolated from exponentially growing cells using Trizol reagent
(Invitrogen) following standard protocols and quantified
spectrophotometrically. Poly(A)+ RNA was extracted from 50 µg of total RNA using the Oligotex mRNA purification system
(Qiagen, Milano, Italy) and retrotranscribed to cDNAs in the
presence of [32P]dATP (Amersham Biosciences) using a
mixture of specific oligonucleotides (Clontech).
Equal amounts of labeled cDNAs were hybridized to filters
containing 1200 genes (ATLAS 1.2 human Cancer,
Clontech). After being washed, the filters were
autoradiographed, and gene expression patterns were evaluated using the
ATALS Image software (Clontech). Expression
intensities were considered comparable only if at least one of the two
samples (c-Myc low expressing clone and control clone) had intensity
greater than 2-fold the background value. Three macroarrays were
performed for MN2 control clone, and the values for each gene were averaged.
Enzyme Activities--
Statistical Analysis--
The results are presented as
means ± S.D. Significant changes were assessed using Student's
t test for unpaired data, and p values < 0.05 were considered significant.
Apoptosis Mediated by Down-regulation of c-Myc Is Associated with
Cell Depletion of GSH--
Two M14 melanoma transfectants (MAS51 and
MAS53) expressing 6-10 times less c-Myc protein than the control
clone (MN2) and M14 parental line (23) were employed. By means of the
annexin V versus PI staining assay (Fig.
1A), we demonstrated that
MAS51 and MAS53 clones showed spontaneous apoptosis, which increased from about 6% on day 2 to about 40% on day 6. On the contrary, no
apoptosis was observed in either of the control lines for up to 6 days
of growth. On day 4 of growth, when about 15% of apoptosis was evident
in both MAS51 and MAS53 transfectants, the values of GSH were
significantly lower (p < 0.01) than both the M14 and MN2 control cells, with a concomitant significant increase
(p < 0.05) in the GSSG form of the tripeptide, whereas
mixed disulfides between glutathione and cysteine residues of proteins
were undetectable (Fig. 1B). Fig.
2A shows that GSH content was
significantly different as early as 2 and 3 days of growth (*,
p < 0.05; **, p < 0.01), far ahead of
the time when the apoptotic process was activated, indicating
that its decrease represents an upstream event in the induction of
apoptosis.
The steady state level of GSH in the cell results from a balance
between the rates of synthesis and loss of the tripeptide via oxidation
or excretion. Under our experimental conditions we detected an increase
in intracellular GSSG content as early as 2 days of growth (Fig.
2B); this increase is commonly observed when cells
are oxidatively stressed. This evidence could, at least in part,
provide an explanation for the lower levels of GSH present in MAS51 and
MAS53 clones. On the other hand, a significant increase in GSH content
was determined in the culture media of these clones (Fig.
2C) on days 3 and 4 of growth (p < 0.01and
0.001, respectively).
Down-regulation of c-Myc Reduces
The dependence of gene expression profiles on c-Myc was analyzed by
macroarray technology (Fig. 4). The
figure shows that many genes are down-regulated in the clones
expressing low c-Myc with respect to control, including
c-myc itself and many genes already known as targets of
c-Myc. In particular, down-regulation of c-Myc induced a significant
reduction of genes of enzymes related to the metabolism of glutathione:
Down-regulation of c-Myc Commits M14 Melanoma Cells to Apoptosis
via the Mitochondrial Pathway--
Production of ROS (Fig.
5A) progressively increased in
the clones expressing low c-Myc after 4 days of growth, reaching
significant values (35% more than in control cells) on day 6 of
culture. On the contrary, no detectable ROS were produced in the M14
and MN2 control clones during the days of culture. Neither caspase-9
nor caspase-3 was activated until day 4 of growth in all of the cell lines analyzed, regardless of c-Myc expression (Fig. 5B).
The proteolytic cleavage of the 116-kDa PARP to an 89-kDa product appeared only in MAS51 and MAS53 cells on day 6 of culture,
concomitantly with the activation of both caspases (Fig.
5C). On the contrary, cytochrome c release from
mitochondria was detectable as early as day 2 of growth (Fig.
5D). The release was concomitant with a decrease in GSH and
preceded the surface exposure of phosphatidylserine, as specifically
detected by annexin V staining (see Fig. 1A).
Activation of Apoptosis in Clones Expressing Low Amount of c-Myc
Depends on GSH--
When Cys-NAc (5 mM), which is known to
support GSH synthesis, or GSH ester (5 mM) was added to
transfectants with low c-Myc on day 1 of growth and left in the medium
for the following 24 h, the intracellular GSH content was
increased to levels as high as those assayed in M14 cells on day 2 of
growth (Fig. 6A). This effect
was maintained for the following days of growth (data not shown) and
caused a reduction of apoptosis (Fig. 6B). The reversing ability of Cys-NAc and GSH ester was not due to increased
expression of c-Myc (Fig. 6C). As soon as the GSH
concentration rose following both treatments, cytochrome c
was not efficiently accumulated in the cytosol (Fig. 6D).
Although both treatments partially reversed the induction of apoptosis
in both clones with low c-Myc, the Cys-NAc treatment only was able to
completely abolish the intracellular ROS production, whereas the
administration of GSH ester reduced it by about 50%. The activities of
caspase-9 and caspase-3 were significantly reduced following Cys-NAc or
GSH ester treatment, the proteolytic activity being less than 10%.
Transient Down-regulation of c-Myc Induces Apoptosis through a
Decrease in GSH--
To ascertain whether cell depletion of GSH and
induction of the apoptotic program were actually specific events of
down-regulation of c-Myc, we generated M14 doxycycline-inducible clones
(named MAS IND 1 and MAS IND 18). Both clones, following doxycycline administration, showed a reduction in the protein levels of c-Myc (50-60%) (Fig. 7A) and a
concomitant decrease in GSH content (Fig. 7B). Moreover, a
time-dependent increase in the percentage of annexin V+
cells was detected starting from 24 h after c-Myc down-regulation (Fig. 7B). Subsequent to cell depletion of glutathione,
cytochrome c was released from mitochondria, leading to the
induction of the downstream events of apoptosis such as production of
ROS and activation of caspase-9 and -3 (data not shown).
The biological effects of c-Myc are clearly due to its ability to
affect gene transcription both positively and negatively. Current
evidence is in agreement with the notion that deregulation of cell
growth and proliferation is critically affected in c-Myc-related tumorigenesis. It is also clear that c-Myc controls or affects other
processes that may be highly relevant to its tumorigenic action.
In the present report we demonstrate that down-regulation of c-Myc
commits melanoma cells to apoptosis via alteration of the GSH balance
in the cell at two key points: GSH biosynthesis and GSH regeneration
from GSSG reduction. In fact, by using stable and doxycycline-inducible
M14 melanoma cells, we found that the down-regulation of c-Myc induced
a decrease in the intracellular GSH content with a concomitant
accumulation of its oxidized form. The process is strictly related to
down-regulation of c-Myc and not to GSH loss from the dying cells, as
clearly evidenced by the kinetics of the appearance of apoptotic cells.
We and other authors have demonstrated previously that GSH is
efficiently extruded from cells undergoing programmed cell death (26,
27, 29, 35, 36) and that supplementation with GSH precursors or
inhibition of GSH efflux leads to inhibition or delay of the death
program (30, 37-39).
The observed imbalance in the redox equilibrium of GSH is due to
down-regulation of two key enzymes of glutathione metabolism, along
with changes observed in other gene transcription, that, as expected,
are more related to cell cycle regulators (40). In this context, it
should be remembered that another gene involved in redox regulation has
recently been reported to be a target of c-Myc, i.e.
PRDX3, encoding a mitochondrial protein of the peroxiredoxin
family (41). As far as Cell depletion of GSH caused by down-regulation of c-Myc is linked
closely to mitochondrial dysfunction. In fact, we found evidence that
release of cytochrome c from the mitochondria to the cytosol
was concomitant with the decrease of GSH. These results are consistent
with our previous data demonstrating that a diminution of intracellular
GSH content, obtained either by chemical inhibition of its neosynthesis
or by eliciting the extrusion of GSH, causes cytochrome c
release from mitochondria (31). However, we previously demonstrated
that cytochrome c release is a cellular response to lack of
GSH, which can occur even in the absence of cell commitment to
apoptosis; thus, cytochrome c release per se is
not sufficient to trigger apoptosis, but other events have to occur as
well (31). In our model, cytochrome c release is an early
event that is followed by ROS production and proteolytic activation of
caspase-9 and -3. The increased ROS production in cells deprived of GSH
can be ascribed to an alteration of the redox equilibrium following the
impairment of systems able to scavenge or detoxify the various reactive
oxygen intermediates generated by normal cell metabolism.
In summary, in this study we have outlined the mechanisms by which
down-regulation of c-Myc commits melanoma cells to apoptosis. In
particular, we have demonstrated that inhibition of the expression of
c-myc causes deregulation of the biosynthesis of GSH and of the GSH/GSSG ratio. A decrease in GSH and consequent cytochrome c release are early events in the apoptotic process, because
they precede the surface exposure of phosphatidylserine. On the
contrary, ROS production and activation of caspase-9 and -3, occurring
later in the apoptotic process, appear as consequences of redox
imbalance. On the basis of these results, We thank Adele Petricca for her assistance in
typing the manuscript.
*
Supported by grants from the Italian Association for Cancer
Research (AIRC), Ministero della Sanità, and Consiglio
Nazionale delle Ricerche-Ministero dell'Istruzione,
dell'Università e della Ricerca.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Experimental
Chemotherapy Laboratory, Regina Elena Cancer Institute, Via delle Messi
d'Oro 156, 00158 Rome, Italy. Tel.: 39-06-52662569; Fax: 39-06-52662505; E-mail: biroccio@ifo.it.
¶
Recipients of a fellowship from the Italian Foundation for
Cancer Research (FIRC).
Published, JBC Papers in Press, September 10, 2002, DOI 10.1074/jbc.M207684200
The abbreviations used are:
ROS, reactive oxygen
species;
Glutathione Influences c-Myc-induced Apoptosis in M14 Human
Melanoma Cells*
§,¶,
,¶,,, and
Experimental Chemotherapy Laboratory, Regina
Elena Cancer Institute, Via delle Messi d'Oro, 00158 Rome, the
Department of Biology, "Tor Vergata" University, Via della
Ricerca Scientifica 00133 Rome, the
Department of Biomedical Sciences, "G.
D'Annunzio" University, Via dei Vestini, 66013 Chieti, and the
** Department of Oncology, "Mario Negri" Research
Institute, via Eritrea, 20154 Milan, Italy
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glutamyl-cysteine synthetase and NADPH-dependent GSSG
reductase, as well as a consequent glutathione release in the
extracellular medium. Cytochrome c was released into the
cytosol at very early stages of apoptosis induction, long before
detectable production of reactive oxygen species and activation of
caspase-9 and -3. Macroarray analysis revealed that down-regulation of
c-Myc produced striking changes in gene expression in the section
related to metabolism, where the expression of -glutamyl-cysteine
synthetase and GSSG reductase was found to be significantly reduced.
The addition of N-acetyl-L-cysteine or
glutathione ethyl ester inhibited the apoptotic process, thus confirming the key role of glutathione in programmed cell death induced
by c-Myc.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-aminobutyrate, and Cys-NAc were obtained from
Sigma. GSH, GSSG, ATP disodium salt, phosphoenolpyruvate, NADH, NADPH, and pyruvate kinase/lactate dehydrogenase were purchased from Roche Molecular Biochemicals. All other chemicals were obtained from Merck.
) cells
were considered apoptotic.
80 °C until analysis by gel
electrophoresis. 20 µg of cytosolic protein extracts were loaded onto
each lane of a 12% SDS-PAGE, separated, and then blotted to
nitrocellulose membrane (Bio-Rad). Purified mouse anti-cytochrome
c antibody (clone 7H8.2c12, Pharmingen) was used as primary
antibody (1: 5,000). The specific protein complex, formed upon
anti-mouse secondary antibody treatment (1:10,000), was identified
using SuperSignal substrate chemiluminescence reagent.
-GCS, c-Myc, and 
-actin were performed using rabbit anti-PARP
(1:2000, Roche Molecular Biochemicals), rabbit anti--GCS (1:1000;
provided by Dr. A. Cantin, Service de pneumologie, CHUS-Fleurimont),
mouse anti-c-Myc (1:1000, clone 9E-10, Santa Cruz
Biotechnology), and mouse anti-actin (1:1000, clone AC-40, Sigma)
antibodies. ECL was used for detection.
-GCS was assayed as described by
Seelig and Meister (33). The cell pellet was lysed in 0.1 M
Tris-HCl, pH 8, containing 5 mM MgCl2 and 2 mM dithiothreitol and centrifuged at 12,550 × g for 30 min. Supernantants were used for -GCS
determination following oxidation of NADH at 340 nm in 0.1 M Tris-HCl, pH 8, containing 150 mM KCl, 5 mM Na2-ATP, 2 mM
phosphoenolpyruvate, 10 mM L-glutamate, 10 mM L--aminobutyrate, 20 mM
MgCl2, 2 mM Na2-EDTA, 0.2 mM NADH and 17 µg of pyruvate kinase/lactate
dehydrogenase. Data were expressed as nmol of NADH oxidized/mg of
protein. GSSG reductase activity was monitored spectrophotometrically,
as described previously (34), by following the changes in absorbance at
340 nm due to the oxidation of NADPH and using as substrates 0.17 mM NADPH and 2.2 mM GSSG in 100 mM
phosphate buffer, 0.5 mM EDTA, pH 7.2, at 37 °C. Data
were expressed as nmol of NADPH oxidized/mg of protein.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Apoptosis induced by down-regulation of c-Myc
is associated with cell depletion of GSH. A,
cytofluorimetric analysis of the annexin V versus PI
staining assay performed in M14 (a), MN2 (b),
MAS51 (c), and MAS53 (d) cell lines on days 2, 4, and 6 of culture; (n = 5). The percentage reported in
the annexin V+/PI region of each histogram represents the
apoptotic cells. B, intracellular GSH (slashed
columns) and GSSG (black columns) content was measured
in M14 (a), MN2 (b), MAS51 (c), and
MAS53 (d) transfectants on day 4 of culture as described
under "Experimental Procedures" (n = 10); *,
p < 0.05; **, p < 0.01.
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Fig. 2.
The decrease of GSH is an early event in the
induction of apoptosis. HPLC analysis of intracellular GSH
(A), intracellular GSSG (B), and extracellular
GSH content (C) was performed in M14 (black
columns), MN2 (dark gray columns), MAS51 (light
gray columns), and MAS53 (white columns) cells. On the
indicated days, cells and media were treated for glutathione
determinations as described under "Experimental Procedures"
(n = 10); *, p < 0.05; **,
p < 0.01; ***, p < 0.001.
-GCS and GSSG Reductase
Expression and Activity Levels--
In search of a mechanism
responsible for the early alterations observed in GSH content following
down-regulation of c-Myc, we monitored two key enzymes determining the
balance of glutathione forms in the cells: -GCS, which is the
rate-limiting enzyme in GSH synthesis, and the
NADPH-dependent GSSG reductase, which is responsible for
efficient back-reduction of GSSG to GSH. Fig. 3 shows the activity (panel A)
and protein levels (panel B) of -GCS. The results show a
significant decrease in activity (about 40%) as early as day 2 of
growth, with no further decrement in either the MAS51 or MAS53
transfectant when compared with the MN2 and M14 parental line.
These data are in line with the protein concentration of the two
subunits of the synthetase, the heavy catalytic (-GCSH)
and the light regulatory (-GCSL) subunit, as
assayed by Western blot analysis (Fig. 3B). Similarly, the activity of GSSG reductase showed a significant decrease in both of the
clones expressing low c-Myc (Fig. 3C), thus correlating well
with the changes in GSSG content observed in the MAS51 and MAS53
transfectants as compared with control lines.
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Fig. 3.
Down-regulation of c-Myc reduces the
activities of -GCS and GSSG reductase.
Enzymatic activity (A) and immunoblot assay (B)
of -GCS and enzymatic activity of NADPH-dependent GSSG
reductase (C) were evaluated in M14 (black
columns), MN2 (dark gray columns), MAS51 (light
gray columns), and MAS53 (white columns) transfectants.
On the indicated days, cells were treated for spectrophotometric
determinations of enzyme activities as described under "Experimental
Procedures" (n = 6); **, p < 0.01. The blot shown is from a typical experiment of three separate
experiments with comparable results.
-GCS, NADPH-dependent GSSG reductase, glutathione
S-transferase-µ1, -
1, and -A1, and microsomal glutathione S-transferase II.
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Fig. 4.
Gene expression of
-GCS (GCS) and GSSG reductase
(GSS-Red) is reduced in clones expressing low amount
of c-Myc. Scatter plots of log50-transformed
expression data are shown for MAS51 (left panel) and MAS53
(right panel) versus control transfectant MN2.
Points placed below or above the
solid line represent down- or up-regulated genes in the two
clones expressing low c-Myc levels with respect to the control
transfectant.
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Fig. 5.
Down-regulation of c-Myc triggers apoptosis
via the mitochondrial pathway. The following are shown:
flow cytometric analysis of ROS content (A) and caspase-9
and caspase-3 activity (B); Western blot analysis of PARP
cleavage (C) and cytosolic cytochrome c
(D) evaluated in M14 (a), MN2 (b),
MAS51 (c), and MAS53 (d) cells on the indicated
days (n = 5). Each blot is from a typical experiment of
three separate experiments with comparable results.
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Fig. 6.
Induction of apoptosis depends on cell
depletion of GSH. A, intracellular GSH content
evaluated in untreated M14, MAS51, and MAS53 or in Cys-NAc-treated
(NAC) or GSH ester (GSHest)-treated cells on day
2 of growth; (n = 4). B, percentage of
apoptosis evaluated by an annexin V/PI assay in M14 ( 
), MAS51 and
MAS53 untreated (
, 
), Cys-NAc-treated (
, 
), and GSH
ester-treated (
, 
) transfectants at 2, 4, and 6 days of growth;
(n = 4). C, immunoblot analysis of c-Myc
protein expression evaluated in M14 and MAS51 untreated,
Cys-NAc-treated, and GSH ester-treated cells on day 2, 4, and 6 of
culture. D, immunoblot analysis of cytosolic cytochrome
c performed in M14, MAS51, and MAS53 untreated and Cys-NAc-
and GSH ester-treated cells on day 2 of growth. E,
percentage of ROS production and caspase-9 and -3 activity calculated
by flow cytometry in M14, MAS51, and MAS53 untreated and Cys-NAc- and
GSH ester-treated cells on day 6 of growth (n = 4).
Each blot shown is from a typical experiment of three separate
experiments with comparable results.
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Fig. 7.
Transient down-regulation of c-Myc induces
apoptosis through the decrease of GSH. A, Western blot
analysis of c-Myc expression levels performed in uninduced and
doxycycline-induced (Dox) MAS IND1 and MAS IND18 clones.
B, intracellular GSH content (n = 4) and
percentage of apoptosis performed by annexin V/PI assay
(n = 4) evaluated in uninduced MAS IND1 and MAS IND18
( , ) cells and doxycycline-induced MAS IND1 and MAS IND18
(, ) cells. Upon doxycycline administration, both clones
down-regulate c-Myc protein in about 72 h by 50-60% compared
with the same uninduced transfectants. This time is conventionally
reported as time 0. The blot shown is from a typical experiment of
three separate experiments with comparable results.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-GCS and NADPH-dependent GSSG
reductase enzyme regulation is concerned, there are no data demonstrating a direct transcriptional regulation of these genes by c-Myc. The results obtained with transient down-regulation of c-Myc
(Fig. 7) strongly favor this hypothesis. A rapid decrease of
intracellular GSH following deregulation of GSH biosynthesis has
recently been demonstrated to be the result of cleavage of the
catalytic subunit of -GCS by caspase-3 during apoptotic cell death
(42). However, under our experimental conditions, the activation of
caspase-3 was significant only after 6 days of growth, whereas a
decrement in both the protein and activity levels of -GCS was
detectable as early as 2 days of growth, thus indicating that
these decreases are probably a direct consequence of
down-regulation of c-Myc. Moreover, the reduction of intracellular GSH
content is aggravated by efficient extrusion of GSH, observed as early as 2 days of growth, before the execution of apoptosis. These results
are in agreement with previous data demonstrating that cells stimulated
to undergo apoptosis get rid of their GSH to allow apoptosis to take
place (30). However, we previously found that GSH loss may be necessary
but not sufficient for triggering apoptosis, because in lowering the
GSH content of the cell by L-buthionine-sulfoximine
and diethyl maleate we were not able to induce apoptosis in U937 or
HepG2 cells (30). This discrepancy may depend on cell context or on the
modality by which GSH is lost; some cell types may adapt slowly to a
situation of GSH deprivation by setting up other ways of maintaining a
correct redox equilibrium, whereas other types, including
melanomas, are particularly sensitive to depletion of GSH (43, 44). In
our experimental model, the decrease in GSH is strictly associated to
apoptosis, because supplementation with Cys-NAc or with GSH ester
inhibited apoptosis.
-GCS and GSSG reductase
appear to be possible therapeutic targets that can be modulated
specifically to reestablish the apoptotic pathway in cancer cells.
Moreover, because elevated levels of GSH have also been reported to
play an important role in mediating tumor cell resistance to
chemotherapy, the effect of down-regulation of c-Myc on GSH may
contribute to increase drug susceptibility.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
-GCS, -glutamyl-cysteine synthetase;
PARP, poly(ADP-ribose) polymerase;
Cys-NAc, N-acetyl-L-cysteine;
PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid);
PI, propidium
iodide;
HPLC, high pressure liquid chromatography.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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