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Interferon-cbolstersCD95/Fas-mediatedapoptosis of
astroglioma cells
Anna M. Giammarioli
1
, Rosa Vona
1
, Lucrezia Gambardella
1
, Barbara Ascione
1
, Angela Maselli
1
,
Cristiana Barbati
2
, Antonella Tinari
3
and Walter Malorni
1
1 Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanita’, Rome, Italy
2 Department of Cell Biology and Neurosciences, Istituto Superiore di Sanita’, Rome, Italy
3 Department of Technology and Health, Istituto Superiore di Sanita’, Rome, Italy
Introduction
Astroglioma represents the most common primary
brain tumor in humans. The prognosis of patients with
the most severe form of astrocytoma is poor and only
marginally improved by chemotherapy. Astrogliomas
are generally resistant to drug and radiation therapy
and this greatly limits the available treatment options.
The induction of apoptosis, the program for cell sui-
cide, is the main goal of anti-tumor treatments. This is
normally induced by chemotherapic drugs or by spe-
cific stimuli, such as the interaction of death receptors
with their ligands. Well-defined signal transduction
pathways and effector mechanisms have been described
that finally lead to cell demise [1,2]. Among these, a
key role is played by Bcl-2 family molecules, by the
mitochondria, which can release apoptogenic factors,
and by a family of cysteine proteases (caspases) that
act in cascade, initiating and executing programmed
cell death [3,4].
Apoptosis induced by CD95 ⁄ Fas receptor was origi-
nally identified as the main mechanism for inducing
apoptosis in lymphoid cells, but it has also been shown
to trigger programmed cell death in other cell types,
including nervous system cells [5–8]. In particular,
astroglioma cells constitutively express CD95 ⁄ Fas but
are usually resistant to cell death as a result of the
ligation of this receptor [9].
Interferons (IFNs) constitute a large family of multi-
functional proteins that share antiviral, antitumor and
Keywords
apoptosis; astrocytoma; CD95 ⁄ Fas;
interferon-c; proteasome
Correspondence
A. M. Giammarioli, Department of
Therapeutic Research and Medicines
Evaluation, Section of Cell Aging and
Degeneration, Istituto Superiore di Sanita’,
viale Regina Elena 299, 00161 Rome, Italy
Fax: +39 06 49903691
Tel: +39 06 49902443
E-mail: anna.giammarioli@iss.it
(Received 31 March 2009, revised 21 July
2009, accepted 11 August 2009)
doi:10.1111/j.1742-4658.2009.07271.x
In the present study, we investigated the mechanisms of the resistance to
CD95-mediated cell death and the effects ofinterferon-c in modulating the
susceptibility to CD95-induced apoptosisof human astroglioma cells. We
found that interferon-c administration sensitized cancer cells to CD95-med-
iated apoptosis. The mechanism underlying this sensitization appeared to
be associated with a framework of cell changes, including up-regulation of
death receptor at the cell surface, pro-apoptotic molecule Bax and Bak
over-expression and mitochondria hyperpolarization, as is known to be
associated with cell sensitization to apoptosis. An involvement of the pro-
teasome activity in the mechanism of sensitization by interferon-c was also
detected, probably as a result of the differing expression of catalytic protea-
some subunits. Taken together, these findings suggest that interferon-c
could represent a promising candidate for modulating astroglioma cell
apoptotic susceptibility.
Abbreviations
BrdUrd, bromodeoxyuridine; CD95R, CD95 receptor; CisPt, cisplatin; cyt c, cytochrome c; FasL, Fas ligand; FITC, fluorescein isothiocyanate;
IFN, interferon; I-jB, NF-jB inhibitor; LMP, low molecular weight protein; MMP, mitochondrial membrane potential; NF-jB, nuclear factor
kappa B; PKR, RNA-dependent protein kinase; STAT, signal transducer and activator of transcription.
5920 FEBS Journal 276 (2009) 5920–5935 ª 2009 The Authors Journal compilation ª 2009 FEBS
immunomodulatory activities [10]. They have also been
implicated in the modulation ofapoptosis [11–14].
Interferons are included in therapeutic protocols against
human malignancies either as single agents or, more
frequently, in combination with chemotherapeutic
drugs [15,16]. For example, IFN-c, a type II inter-
feron, has been suggested to increase apoptotic suscep-
tibility of some tumor cell types (e.g. melanoma cells)
[17–19]. In the present study, we show that IFN-c
sensitizes astrogliomacells to CD95-mediated apopto-
sis, facilitating the induction of biochemical events of
relevance in cell death, such as an increased expression
of CD95 receptor at the cell surface, the modulation of
Bcl-2 family proteins and mitochondrial hyperpolariza-
tion, as is known to be associated with cell sensiti-
zation to apoptosis [20,21]. Finally, we also suggest
that IFN-c could sensitize astrogliomacells to CD95-
induced apoptosis by modulating catalytic proteasome
activity.
Results
Sensitization to apoptosis by IFN-c
To determine the susceptibility ofastrogliomacells to
apoptosis, ADF, LN229 and U373 cell lines were
treated with monoclonal CD95 serum (clone CH11)
and IFN-c, or a combination of these. Astroglioma
cell lines were also treated with a chemotherapic com-
pound of widespread use in the clinical practice (cis-
platin; CisPt), a drug that provided some beneficial
effect in the treatment of different astrocytic tumors
[22]. Because it was previously reported that sensitiza-
tion by IFN-c in other tumor cell lines occurred after
at least 48–72 h of treatment [23], astroglioma cells
were pre-treated with this cytokine for 72 h before the
addition of monoclonal anti-CD95 serum or CisPt.
The optimal dose of IFN-c (500 IU) was determined
after a dose–response curve assay (see Experimental
procedures). As shown in Fig. 1, cell treatment with
monoclonal anti-CD95 serum (CH11) for 24 h or
IFN-c given alone did not exert any significant pro-
apoptotic effect in ADF, LN229 and U373 cell lines,
whereas the combined treatment of IFN-c with mono-
clonal anti-CD95 serum (CH11) cooperated to trigger
apoptosis (Fig. 1A). When we analyzed the effect of
CisPt (i.e. cells were treated as indicated in Experimen-
tal procedures), we observed a significant increase of
apoptotic cells in combined treatment with IFN-c in
ADF cells, but not in LN229 and U373 astroglioma
cell lines (Fig. 1B). In brief, sensitization to apoptosis
by IFN-c occurred in anti-CD95-treated cells in all
astroglioma cell lines (although by different degrees:
ADF > LN229 > U373) (Fig. 1A), whereas increased
apoptotic rates as a result of CisPt were observed in
ADF astrogliomacells only (Fig. 1B). On the basis of
these results, we decided to investigate the mechanisms
underlying IFN-c-mediated sensitization to CD95-
mediated apoptosis in the ADF astroglioma cell line.
Caspase activity
To identify the CD95-mediated apoptotic pathway in
IFN-c pre-treated cells, caspase activity was analyzed
in ADF cells by both flow cytometry and western blot-
ting. In particular, caspase 8 and 10 (mainly involved
in receptor-mediated apoptosis) and caspase 9 (mainly
involved in mitochondria-mediated apoptosis) were
considered. After 72 h of IFN-c treatment followed by
monoclonal anti-CD95 serum for a further 24 h, we
demonstrated the powerful activation of apical caspas-
es (caspase 8 and 10) (Fig. 2A,B). An increase of
caspase 9 activity was also evident (Fig. 2C). Western
blotting confirmed the results obtained by flow cytom-
etry (Fig. 2, right).
Accordingly, specific caspase inhibitors significantly
impaired apoptotic execution (Fig. 3). In detail, ADF
cells pre-treated with IFN-c were pre-incubated with
the pan-caspase inhibitor Z-VADfmk and with selec-
tive inhibitors of caspase 8 (Z-IETDfmk), caspase 10
(Z-AEVDfmk) or caspase 9 (Z-LEHDfmk) for 2 h
before the administration of monoclonal anti-CD95
serum. Notably, inhibition of caspase 10 and 9 was
highly effective in impairing the apoptotic process
(Fig. 3C,D).
The role of mitochondria
To further characterize the apoptotic pathway trig-
gered by IFN-c ⁄ anti-CD95 in ADF cells, we also
focused our attention on the possible role of mito-
chondria, which are well known regulators of cell
death [24]. In particular, previous studies suggested
that a significant decrease of mitochondrial mem-
brane potential (MMP) and cytochrome c (cyt c)
release into the cytosol can be considered as a hall-
mark ofapoptosis execution [21,25]. According to
the results reported above (Fig. 1), we observed a
MMP loss in IFN-c ⁄ anti-CD95-treated cells,
whereas no changes were detected with IFN-c
and ⁄ or monoclonal anti-CD95 serum given alone. In
Fig. 4, graphs of MMP loss from a representative
experiment are reported (Fig. 4A). Parallel analyses
were carried out on cytosolic cyt c released from
mitochondria. Cyt c localization was analyzed by
immunofluorescence microscopy after double-labeling
A. M. Giammarioli et al. IFN-c sensitizes cells to apoptosis
FEBS Journal 276 (2009) 5920–5935 ª 2009 The Authors Journal compilation ª 2009 FEBS 5921
of cyt c and mitochondria (Fig. 4B). In particular,
to visualize mitochondria, ADF cells were loaded
with Mitotracker Green, a fluorescent dye that loads
into mitochondria (see Experimental procedures). As
shown in Fig. 4B, treatments that did not induce
apoptosis display a yellow punctuate pattern result-
ing from the overlap of green and red fluorescence
and consistent with cyt c (red fluorescence) localiza-
tion into the mitochondrial compartment (green
fluorescence). By contrast, after IFN-c ⁄ anti-CD95
treatment, cyt c exhibited a more diffuse red fluores-
cent staining that corresponded to the release of cyt
c from the mitochondria into the cytosol (Fig. 4B).
As shown in Fig. 4C, quantitative analysis of cyt c
release was performed by using a commercial ELISA
Kit (note the significant cyt c release in IFN-c ⁄ anti-
CD95-treated cells with respect to cells treated with
anti-CD95 only). In Fig. 4D, the results of morpho-
metric analyses carried out in samples shown in
Fig. 4B and obtained by counting at least 300
stained cells for each condition are reported (mean
values from three different experiments performed in
triplicate are shown). Note the high percentage of
cells showing the diffuse red fluorescence.
Effects of IFN-c
Because the combined treatment of IFN-c and mono-
clonal anti-CD95 serum strongly enhanced the induc-
tion ofapoptosis in ADF cells, and because this
80
A
B
Control
C
*
*
*
30
40
50
60
70
Anti-CD95
IFN-γ
IFN-γ/anti-CD95
Apoptotic cells (%)
ADF LN229
U373
0
10
20
IFN-γ
IFN-γ/CisPt
Apoptotic cells (%)
40
50
60
70
80
*
CisPt
Control
0
10
20
30
ADF LN229 U373
Control
4.1%
2.1%
6.5%
2.5%
Control
4.5%3.2%
4.3%
PI
45%
7.3%
21%
7.5%
4.0%
53%
9.8%
10.5%
3.6%
Ann
e
xin
V
-FIT
C
IFN-γ IFN-γ
IFN-γ/anti-CD95 IFN-γ/CisPt
CisPt Anti-CD95
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
10
1
10
0
10
2
10
3
10
4
10
1
10
0
10
2
10
3
10
4
10
1
10
0
10
2
10
3
10
4
10
1
10
0
10
2
10
3
10
4
10
1
10
0
10
2
10
3
10
4
10
1
10
0
10
2
10
3
10
4
10
1
10
0
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
Fig. 1. Evaluation ofapoptosis in astrogli-
oma cell lines. Histograms show the per-
centage of AnnexinV positive cells after
anti-CD95 (A) or CisPt (B) administration in
the absence or presence of IFN-c in ADF,
LN229 and U373 cells. Data were obtained
from four different experiments. Note the
significant increase ofapoptosis in cells pre-
treated with IFN-c (*P < 0.01). (C) A repre-
sentative flow cytometry experiment with
ADF cells is shown. Numbers reported in
the bottom and top right quadrants
represent the percentage of annexin V
single-positive cells and annexin V ⁄ PI
double-positive cells, respectively.
IFN-c sensitizes cells to apoptosis A. M. Giammarioli et al.
5922 FEBS Journal 276 (2009) 5920–5935 ª 2009 The Authors Journal compilation ª 2009 FEBS
increase was the result of a simple additive effect of
single agents, we further evaluated the effects of IFN-c
administration per se. The aim was to assess the mech-
anism involved in the sensitization to apoptosis
described above.
Up-regulation of CD95 receptor and the release of Fas
ligand (FasL)
IFN-c has previously been shown to mediate up-regula-
tion of CD95 receptor (CD95R) in various types of
Caspase 8 activity
Caspase 10 activity
Caspase 9 activity
50
A
B
C
Positive cells (%)
0
10
20
30
40
*
Control
IFN-γ
IFN-γ /anti-CD95
Anti-CD95
Pro-caspase 8
Caspase 8
α-actin
Control
IFN-γ
IFN-γ /anti-CD95
Anti-CD95
50
0
Control
IFN-γ
IFN-γ /anti-CD95
Anti-CD95
10
20
30
40
*
Positive cells (%)
Positive cells (%)
Pro-caspase 10
Caspase 10
α-actin
Control
IFN-γ
IFN-γ /anti-CD95
Anti-CD95
Control
IFN-γ
IFN-γ /anti-CD95
Anti-CD95
20
30
40
50
*
0
10
Pro-caspase 9
Caspase 9
α-actin
Control
IFN-γ
IFN-γ /anti-CD95
Anti-CD95
Control
4.9%
5.6%
Counts
Fluorescence intensity
7.2%
38.7%
IFN-γ
Anti-CD95
IFN-γ/anti-CD95
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
0
300
0
300
0
0
300
300
4%
Control
5.2%
Fluorescence intensity
Counts
5.8%
33.8%
IFN-γ
Anti-CD95
IFN-γ/anti-CD95
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
0
300
0
300
0
0
300
300
IFN-γ
Control
Counts
28.7%
8.7%
6.7%
Anti-CD95
4.5%
IFN-γ/anti-CD95
Fluorescence intensity
0
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
300
0
300
0
300
0
300
Fig. 2. Caspase activity. Left: caspase 8 (A), caspase 10 (B) and caspase 9 (C) activity were measured by a colorimetric assay. Data are
expressed as the mean ± SD of four independent experiments (*P < 0.01 statistically significant compared to control cells). Middle panel: a
representative experiment is shown with respect to the activity of caspase 8, caspase 10 and caspase 9, respectively. Right: caspase 8,
caspase 10 and caspase 9 activity by western blotting. Note cleaved caspase 8 (18 kDa), caspase 10 (30 kDa) and caspases 9 (35 kDa) in
IFN-c ⁄ anti-CD95-treated cells. Blots were reprobed for a-actin to establish equivalent loading. A representative blot is shown.
A. M. Giammarioli et al. IFN-c sensitizes cells to apoptosis
FEBS Journal 276 (2009) 5920–5935 ª 2009 The Authors Journal compilation ª 2009 FEBS 5923
tumors. Up-regulation of CD95R was demonstrated to
be related to an increased susceptibility to CD95-depen-
dent apoptosis in neuroblastoma, leukemia, melanoma,
colon adenocarcinoma and cervical cancer cells [6,26–28].
To evaluate the possible role of up-regulation of
CD95R in CD95-mediated cell death, we investigated the
constitutive and IFN-c induced expression of CD95R in
the ADF cell line. Under our experimental conditions,
ADF cells showed a constitutively low expression of
CD95R, whereas IFN-c treatment leads to a time-dependent
increase of surface receptor expression (Fig. 5A). More-
over, time-dependent analysis of FasL released into the
medium from IFN-c-treated cells showed only a slight
increase of FasL release at all time points (Fig. 5B).
Bcl-2 family proteins
Sensitization activity of IFN-c has also been corre-
lated with Bcl-2 family protein modulation [11]. To
investigate the possibility that IFN-c treatment could
sensitize ADF cells to CD95 apoptosis through the
regulation of Bcl-2 family proteins, we also performed
flow cytometry and western blotting analyses to evalu-
ate the expression of some important Bcl-2 proteins.
Both pro-apoptotic and anti-apoptotic Bcl-2-family
proteins were considered. We found that IFN-c admin-
istration did not result in any significant change of the
anti-apoptotic molecules Bcl-
XL
and Bcl-2 (Fig. 6A).
By contrast, the expression of pro-apoptotic molecules
Bax and Bak was found to be significantly increased
(Fig. 6B,C).
Mitochondria analysis
In consideration of the key role of mitochondria in
apoptosis execution and on the basis of the above
results (Figs 2 and 4), we investigated the effects of
IFN-c administration on this organelle. We found
A
45
60
75
90
*
0
Control
IFN-γ
IFN-γ/anti-CD95
Anti-CD95
Anti-CD95
IFN-γ/anti-CD95
IFN-γ + z-VAD
z-VAD
+ z-VAD
+ z-VAD
15
30
Apoptotic cells (%)
B
45
60
75
90
Control
IFN-γ
IFN-γ/anti-CD95
Anti-CD95
Anti-CD95
IFN-γ/anti-CD95
IFN-γ + z-IETD
z-IETD
+ z-IETD
+ z-IETD
Apoptotic cells (%)
0
15
30
Control
IFN-γ
IFN-γ/anti-CD95
Anti-CD95
Anti-CD95
IFN-γ/anti-CD95
IFN-γ + z-AEVD
z-AEVD
+ z-AEVD
+ z-AEVD
Apoptotic cells (%)
C
45
60
75
90
*
0
15
30
Control
IFN-γ
IFN-γ/anti-CD95
Anti-CD95
Anti-CD95
IFN-γ/anti-CD95
IFN-γ + z-LEHD
z-LEHD
+ z-LEHD
+ z-LEHD
Apoptotic cells (%)
45
60
75
90
*
D
0
15
30
Fig. 3. Inhibition of CD95-mediated apoptosis by different caspase inhibitors. ADF cells pre-treated with IFN-c were preincubated with selec-
tive caspase inhibitors for 2 h before the administration of monoclonal anti-CD95 serum. Histograms show the percentage of Annexin V posi-
tive cells treated in the absence or presence of caspase inhibitors: (A) z-VAD-fmk, pan-caspase inhibitor; (B) z-IETD-fmk, caspase 8 inhibitor;
(C) z-EAVD-fmk, caspase 10 inhibitor; (D) z-LEHD-fmk, caspase-9 inhibitor. Data are expressed as the mean ± SD of four independent experi-
ments using flow cytometry. Note that inhibitors significantly impair the apoptosisof IFN-c ⁄ CD95 treated cells (*P < 0.01).
IFN-c sensitizes cells to apoptosis A. M. Giammarioli et al.
5924 FEBS Journal 276 (2009) 5920–5935 ª 2009 The Authors Journal compilation ª 2009 FEBS
that cell treatment with IFN-c induced a profound
mitochondrial remodeling that was characterized by
ultrastructural modifications (Fig. 7A) and re-distribu-
tion (Fig. 7B). Moreover, it was recently demon-
strated in lymphoid cells that an elevation of MMP is
associated with an increased sensitivity to apoptosis
and that this hyperpolarization of mitochondria
occurs before any activation of caspases and before
phosphatidylserine externalization [20,21,29]. Interest-
ingly, a marked increase of MMP was detected
in IFN-c-treated astrogliomacells (mean values
from four different experiments: 24 ± 6% of cells
with hyperpolarized mitochondria versus 67 ± 7%
detected in IFN- c-treated cells). The results obtained
from a representative flow cytometry experiment are
shown in Fig. 7C.
6%
8.5%
43%
8.9%
J-monomers
J-aggregates
IFN-γ IFN-γ/anti-CD95
Anti-CD95
Control
IFN-γ
IFN-γ/anti-CD95
Anti-CD95
Control
IFN-γ
IFN-γ/anti-CD95
Anti-CD95
Control
IFN-γ
IFN-γ/anti-CD95
Anti-CD95
Control
Cyt C
Mitochondria
Merged
*
Release cyt c (ng·mL
–1
)
40
30
20
10
0
50
40
30
20
10
0
*
% ofcells with
diffuse red staining
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
A
B
CD
Fig. 4. Analysis of MMP and cyt c release.
(A) Cytofluorometric analysis of the MMP
performed using the JC-1 probe. The num-
bers in the bottom quadrants represent the
percentages ofcells with depolarized mito-
chondria (MMP). Note that, after 72 h of
IFN-c plus monoclonal anti-CD95 serum for
a further 24 h, the mitochondrial depolariza-
tion becomes evident as a drop in the
MMP. The results obtained from a repre-
sentative flow cytometry experiment are
shown. (B) Fluorescence microscopy analy-
sis of cyt c and mitochondria. The release of
cyt c from mitochondria is evident in cells
treated with IFN-c and monoclonal anti-
CD95 serum. Note that yellow staining in
the merged images is lost in IFN-c ⁄ anti-
CD95 treated cells where green (mitochon-
dria) and red (cyt c) fluorescence are obser-
vable. The results obtained from a
representative experiment are reported.
(C) The release of cyt c is evaluated by a
sensitive and specific immunoassay using a
commercial ELISA Kit. Data are expressed
as the mean ± SD of three independent
experiments. (D) Morphometric analyses.
The percentage ofcells with diffuse red
staining have been calculated. Data are
expressed as the mean ± SD of four
independent florescence microscopy
experiments. Note that the treatment with
IFN-c ⁄ anti-CD95 significantly increased the
release of cyt c. All the analyses were
performed 72 h after treatment with
IFN-c (*P < 0.01).
A. M. Giammarioli et al. IFN-c sensitizes cells to apoptosis
FEBS Journal 276 (2009) 5920–5935 ª 2009 The Authors Journal compilation ª 2009 FEBS 5925
IFN-c and cell signaling
Several molecules with important signaling functions
are known to be induced by IFN-c. Among these is
the double-stranded RNA-dependent protein kinase
(PKR), which has also been implicated in the apop-
totic process. PKR can provoke apoptosis through
its ability to control protein translation in part by
activating nuclear factor kappa B (NF-jB) by phos-
phorylating its inhibitor, IjB, (23). Moreover, PKR
inhibits protein synthesis by phosphorylating eukary-
otic translational initiation factor 2 [30]. To deter-
mine whether sensitization by IFN-c could be
mediated by these pathways, further analyses were
carried out. However, flow cytometry analysis of
IFN-c-treated cells showed a slight but not signifi-
cant increase of phosphorylated eukaryotic transla-
tional initiation factor 2 (Fig. 8A). Strikingly, the
total protein content, evaluated as described previ-
ously [31], was found to be significantly modified
(mean values from three different experiments:
310 ± 25 lgÆlL
)1
in control cells versus 585 ± 30
lgÆlL
)1
in IFN-c-treated cells; P < 0.01). With
respect to NF-jB, IFN-c was shown to stimulate a
specific signaling that requires components of the
NF-jB pathway [32–34]. However, the relationship
between IFN-c and NF-jB activation concerning the
modulation of tumor cell apoptosis remains contro-
versial [35–37]. We therefore decided to evaluate the
role of NF-jB in ADF cell death and its possible
regulation by IFN-c. We studied the nuclear translo-
cation of NF-jB by immunofluorescence analysis in
ADF cells treated with IFN-c for 72 h. Control and
IFN-c-treated cells were negative for nuclear translo-
cation of NF-jB, with both exhibiting a clear cyto-
plasmic staining (data not shown). Because the
activation ⁄ translocation of NF-jB to the nucleus is
regulated by a ubiquitin–proteasome pathway thro-
ugh the cytosolic degradation of I-jB, we analyzed
the level of the I-jB in IFN-c-treated ADF cells. A
significant up regulation of I-jB was detected in
IFN-c-treated cells compared to control ADF cells
(Fig. 8B, left). This suggests a probable inhibition of
proteasome by IFN-c. A recent study has also
shown that constitutive proteasome catalytic subun its
(b1, b5, b2) can be replaced by their IFN-c inducible
counterparts, namely low molecular weight proteins
(LMP2 and LMP7) and multicatalytic endopeptidase
complex-like 1, resulting in alternatively assembled
immunoproteasomes [38]. To characterize the sensi-
tivity to apoptosis induced by IFN-c in astrocytoma
cells, the expression of proteasome catalytic subunits
was then evaluated. We found an increase in LMP2
and LMP7 protease subunits (Fig. 8B, middle and
right, respectively). Western blotting confirmed the
results obtained by flow cytometry (Fig. 8C). These
changes may favor the assembly of immunoprotea-
somes, thus modifying the proteasome activity. To
better elucidate this point, the effects of a specific
proteasome inhibitor such as lactacystin, was also
evaluated. Different concentrations of lactacystin (10,
5, 2.5 and 1.25 lm) were given alone or in combina-
tion with IFN-c for 72 h and ⁄ or monoclonal anti-
CD95 serum for further 24 h to trigger apoptosis.
Samples treated with lactacystin alone or in combi-
nation with IFN-c did not show any significant
change in the apoptotic rates compared to control
cells. Conversely, a significant dose-dependent
increase of apoptotic cells was evident in lactacy-
stin ⁄ anti-CD95-treated cells (Fig. 8D). Interestingly,
the sensitizing effect was more evident on adding
IFN-c to lactacystin ⁄ anti-CD95.
4
5
6
7
A
B
Control
IFN-γ
CD95 expression
FasL release
0
1
2
3
0
24 h 48 h 72 h
Control
IFN-γ
15
20
5
10
24 h 48 h 72 h
0
Fig. 5. CD95 and FasL expression in IFN-c-treated cells. (A) Time
course analysis of cell surface expression of CD95 by flow cytome-
try on ADF cells treated with IFN-c. Note that surface expression
of CD95 was found to be significantly increased in IFN-c-treated
cells, starting from 24 h, with respect to control cells. (B) Time-
dependent analysis of FasL released into the medium from IFN-c-
treated cells. A slight increase of FasL release was detected at all
time points studied. The release of FasL into the culture medium
was evaluated by a sensitive and specific immunoassay using a
commercial ELISA Kit. Data are expressed as the mean ± SD of
three independent experiments.
IFN-c sensitizes cells to apoptosis A. M. Giammarioli et al.
5926 FEBS Journal 276 (2009) 5920–5935 ª 2009 The Authors Journal compilation ª 2009 FEBS
Cell cycle analysis
On the basis of these results, and to better define the
therapeutic potential of IFN-c in the treatment of as-
troglioma cells, the antiproliferative activity of the
cytokine was also evaluated. Cell growth was evaluated
in terms of viable cell counts after 24, 48 and 72 h of
culture. As shown in Fig. 9A, a time-dependent cell
growth inhibition was detectable in IFN-c cultured
cells. This effect was detectable after 24 h of IFN-c
culture and gradually rose, reaching values of 50%
and 60% of cell growth inhibition at 72 h. To further
investigate the mechanism underlying cell growth
inhibition, IFN-c -treated cells were analyzed for cell
cycle distribution: subconfluent ADF cultures were
treated with IFN-c and harvested after 72 h of culture.
IFN-c induced an increased percentage ofcells in the
G0 ⁄ G1 and a corresponding decrease ofcells in the S
and G2-M phases. Representative plots of bromodeoxy-
uridine (BrdU) versus propidium iodide (PI) are shown
in Fig. 9B,C.
Discussion
In the present study, we demonstrated the ability of
IFN-c to significantly increase the sensitivity to
Counts
Counts
30
60
90
120
150
Bax expression (a.u.)
Bcl-XI expression
Bcl-2 expression
Bax expression
Bak expression
*
Counts
0
90
120
150
*
Control
Bak
α-actin
IFN-γ
Control
IFN-γ
Counts
0
30
60
Bak expression (a.u.)
Control
Control M = 102
Bak
α-actin
IFN-γ
IFN-γ M = 108
Control
IFN-γ
Control M = 9
IFN-γ M = 8.3
Control M = 54
IFN-γ M = 126
Control M = 55
IFN-γ M = 112
10
0
0 200
10
1
10
2
10
3
10
4
0 200
0 200
0 200
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
A
B
C
Fig. 6. IFN-c effects on pro- and anti-apoptotic proteins. (A) Flow cytometry analysis shows that IFN-c treatment (72 h) did not modify the
expression of anti-apoptotic molecules Bcl-2 and Bcl-
XL
. Conversely, the expression of Bax (B) and Bak (C) were found to be significantly
increased by both flow cytometry (left) and western blotting (right). Histograms obtained from four independent experiments (mean ± SD)
are shown (*P < 0.01).
A. M. Giammarioli et al. IFN-c sensitizes cells to apoptosis
FEBS Journal 276 (2009) 5920–5935 ª 2009 The Authors Journal compilation ª 2009 FEBS 5927
CD95-mediated apoptosisofastroglioma cells. This
appears to occur via a mechanism that brings into
play: (a) the over-expression of death receptor CD95R;
(b) the over-expression of pro-apoptotic proteins Bax
and Bak; and (c) mitochondrial changes and remodel-
ing. The first is a phenomenon that was already
observed in different types of tumors. Up-regulation of
CD95R has been related to an increased susceptibility
to CD95-dependent apoptosis in leukemia, mela-
noma, colon adenocarcinoma and cervical cancer cells
[6,26,27,39]. Accordingly, in the present study, apopto-
sis induced by the combined treatment IFN-c ⁄ anti-
CD95 correlates with cell surface expression of
CD95R. By contrast, the weak release of FasL by
astroglioma cells did not provoke apoptosis per se.
The results obtained also indicate that, in addition to
its effect on death receptor expression, IFN-c might
regulate the intracellular apoptotic machinery in ADF
cells. Indeed, induction of caspase expression by IFN-c
resulted in activation of caspase 8 upon anti-CD95
treatment, leading to activation of downstream apop-
totic pathway. In the present study, IFN-c treatment
Control
IFN-γ
Control
IFN-γ
Control
C
28%
J-monomers
J-aggregates
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
68%
IFN-γ
B
A
Fig. 7. Effects of IFN-c on mitochondria (A)
Transmission electron microscopy analysis
indicates that ultrastructural features of
mitochondrial network appear modified by
72 h of IFN-c (exposure magnification
· 20 000). (B) Fluorescence microscopy
analysis clearly shows a redistribution and
polarization of mitochondria in IFN-c -treated
cells. (C) Quantitative cytofluorimetric analy-
ses of the MMP. Note a significant mito-
chondrial hyperpolarization in IFN-c-treated
cells: numbers in the boxed areas represent
the percentage ofcells with hyperpolarized
mitochondria.
IFN-c sensitizes cells to apoptosis A. M. Giammarioli et al.
5928 FEBS Journal 276 (2009) 5920–5935 ª 2009 The Authors Journal compilation ª 2009 FEBS
also lead to an alteration of Bcl-2 family pro-apoptotic
proteins. This is in accordance with a previous study
suggesting that IFN-c, by inducing signal transducer
and activator of transcription (STAT)-1 binding to the
c-activated sequence of the target gene, could result in
an increased Bax : Bcl-2 ratio [15]. Members of the
Bcl-2 family proteins play a major role in governing
the mitochondria-dependent pathway of apoptosis,
with proteins such as Bax functioning as inducers of
apoptosis and proteins such as Bcl-2 functioning as
suppressors of apoptosis. In this regard, it is interest-
ing to consider the involvement of mitochondrial
changes in the sensitization ofastrogliomacells by
IFN-c. We found a hyperpolarization of mitochondria
10
0
0
50
100
150
A
B
C
D
p-eIF2α expression
I-kB expression
I-kBα
Control
Percentage of annexin V
positive cells
Lactacystin
Control IFN IFNγ/Lact Lact Lact/anti-CD95 Lact/IFN/anti-CD95
100
80
60
40
20
0
1.25 µ
M 2.5 µM 5 µM 10 µM
LMP2 LMP7
α-actin α-actin α-actin
LMP2 expression LMP7 expression
p-eIF2α expression
(a.u.)
IFN-γ M = 85
IFN-γ
Control
Control M = 65
IFN-γ M = 16
IFN-γ
Control M = 6
Control
IFN-γ
Control
IFN-γ
Control
IFN-γ M = 45
Control M = 19
IFN-γ M = 27
Control M = 10
0 200
Counts
0
0
200
Counts
200
Counts
0 200
Counts
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
Fig. 8. IFN-c and cell signaling. (A, left and right) Modulation of phosphorylated eIF2a by IFN-c. Flow cytometry analyses of phosphorylated
eIF2a are expressed as histograms of the mean ± SD of four independent experiments (A, left) A representative experiment of four is
shown (right). Flow cytometry (B) and western blotting (C) analysis show that IFN-c treatment increased the expression of I-jB, LMP2 and
LMP7 with respect to control cells. (D) Different concentrations of lactacystin (10, 5, 2.5 and 1.25 l
M) were given alone or in combination
with IFN-c for 72 h and ⁄ or monoclonal anti-CD95 serum for a further 24 h to trigger apoptosis. Note a significant dose-dependent increase
of apoptotic cells in lactacystin ⁄ anti-CD95 treated cells and in a combination of IFN-c and lactacystin plus anti-CD95.
A. M. Giammarioli et al. IFN-c sensitizes cells to apoptosis
FEBS Journal 276 (2009) 5920–5935 ª 2009 The Authors Journal compilation ª 2009 FEBS 5929
[...]... bolster astroglioma cell apoptosis by inducing a type of cellular activation, including mitochondria hyperpolarization and the formation of the immunoproteasome When considering the importance of the induction of cell death in cancer treatment, the results obtained also indicate IFN-c as a possible new candidate for the management of an IFN-c sensitizes cells to apoptosis untreatable form of cancer... FITCconjugated Annexin V ⁄ PI apoptosis detection kit (Marine Biological Laboratory, Woods Hole, MA, USA) in accordance with the manufacturer’s instructions FITC-conjugated Annexin V-positive cells were considered as cells in the early stages ofapoptosisCells distinguished by their ability to take up both FITC-Annexin V and PI were considered as cells in the later stages ofapoptosis Live cells were considered... Recently, it was also shown that the induction of immunoproteasome by IFN-c is extremely rapid, transient and self-controlled [38,42] Moreover, in some tumor cells, the induction of immunoproteasome by IFN-c causes an up-regulation of apoptosis- related genes and an increase in early apoptotic cells [11,15,16] For example, Ceruti et al [43] demonstrated that, in ADF cells normally resistant to mitochondriotropic... anti-phosphorylated form of eIF2a (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at 37 °C After washing, cells were incubated for 30 min at 37 °C with FITC-labeled anti-mouse or antirabbit sera (Alexa fluor 488-coniugated IgG; Molecular Probes, Leiden, The Netherlands) Evaluation ofapoptosis Quantitative evaluation ofapoptosis was performed in the total population (adhering + detached cells) using... activation of several transcription factors, such as STATs and NF-jB, and it also leads to the increase of several apoptosis- related molecules, including CD95 receptor, different members of the Bcl-2 family (in particular Bax and Bak) and caspases [46] With respect to the phosphorylation of eIF2a, which provides a fundamental mechanism to couple the rate of protein synthesis with the capacity of fold... Clin Oncol 23, 630–639 45 Awasthi N & Wagner BJ (2004) Interferon-gamma induces apoptosisof lens alphaTN4-1 cells and proteasome inhibition has an antiapoptotic effect Invest Ophthalmol Vis Sci 45, 222–229 46 Gil J & Esteban M (2000) Induction ofapoptosis by the dsRNA-dependent protein kinase (PKR): mechanism of action Apoptosis 5, 107–114 47 Pierdominici M, Giammarioli AM, Gambardella L, De Felice... were poorly active in apoptosis sensitization, whereas higher doses (1000 and 2000 IU), although effective, were toxic to the cells (and far from possible clinical use) On the basis of these results, and in accordance with previous studies [6], a dose of 500 UI was chosen as the experimental dose for the present study Drug treatments were started 24 h after cells were plated ADF cells were exposed to:... described previously [48] In brief, cells were incubated in complete medium for 10 min at room temperature (25 °C) in the dark with 10 lgÆmL)1 JC-1 probe At the end of the incubation period, cells were washed twice in ice-cold NaCl ⁄ Pi, resuspended in a total volume of 400 lL and analyzed by flow cytometry Detection of caspase-8, -10 and -9 activity Activation state of the caspases 8 and 9 was evaluated... (2004) IFNgamma enhances TRAIL-induced apoptosis through IRF-1 Eur J Biochem 271, 4222–4228 14 Thyrell L, Erickson S, Zhivotovsky B, Pokrovskaja K, ´ Sangfelt O, Castro J, Einhorn S & Grander D (2002) Mechanisms of interferon-alpha induced apoptosis in malignant cells Oncogene 21, 1251–1262 15 Das A, Banik NL & Ray SK (2009) Molecular mechanisms of the combination of retinoid and interferongamma for inducing... induces necrosis of L929 tumor cells via a p38MAPK ⁄ NF-kappaB ⁄ NO-dependent mechanism Oncogene 27, 3780– 3788 Shultz DB, Fuller JD, Yang Y, Sizemore N, Rani MR & Stark GR (2007) Activation of a subset of genes by IFN-gamma requires IKKbeta but not interferondependent activation of NF-kappaB J Interferon Cytokine Res 27, 875–884 Moitreyee CK, Suraksha A & Swarup AS (1998) Potential role of NF-kB and RXR . mechanisms of the resistance to CD95-mediated cell death and the effects of interferon-c in modulating the susceptibility to CD95-induced apoptosis of human astroglioma cells. We found that interferon-c. considered as cells in the early stages of apoptosis. Cells distinguished by their ability to take up both FITC-Annexin V and PI were con- sidered as cells in the later stages of apoptosis. Live cells were. Interferon-c bolsters CD95/Fas-mediated apoptosis of astroglioma cells Anna M. Giammarioli 1 , Rosa Vona 1 , Lucrezia Gambardella 1 ,