ARTICLE
Auteur(s) :, S Wittnebel1, A
Jalil1, J Thiery1, S DaRocha1, E
Viey1, B Escudier2, S Chouaib1, A Caignard1,*
1INSERM Unité 487, Cytokines et immunologie des
tumeurs humaines, Institut Gustave-Roussy, 39 rue
Camille-Desmoulins, 94805 Villejuif Cedex
2Unité des thérapies innovantes, Institut
Gustave-Roussy, 39 rue Camille-Desmoulins, 94805 Villejuif
Cedex
Renal cell carcinoma (RCC) is the most common renal tumor. It
accounts for 2-3% of all adult malignancies and the incidence is
rising. The most important prognostic factor for the clinical
outcome is tumor stage, with a 5-year survival from 50-90% for
localized disease, decreasing to 0-13% for metastatic disease
[1].Up to now, only surgical resection (mainly by radical
nephrectomy) of localized disease (Stage I-II) offers a reasonable
chance of curing the disease. Once the tumor has reached the
metastatic stage, the treatment options remain limited.
Chemotherapeutic drugs or γ-irradiation are not effective in RCC
(response rate 2-6%) [2, 3].At date, the standard treatment for
metastatic renal cell carcinoma is based on the administration of
interleukin-2 (IL-2) and IFN-α, alone or in combination, with
response rates ranging between 10-15% [4, 5]. The mechanisms of
action of IL-2 comprise, beside other effects, the stimulation of
NK and T-cells, differentiation of lymphokine-activated killer
(LAK) cells and maturation of antigen-presenting cells (APC), thus,
a general stimulation of the immune system [6, 7]. Although IFN-α
has been shown to be active in the treatment of RCC, its mode of
action remains poorly understood. However, it has been reported to
stimulate cell-mediated cytotoxicity, to exert a direct
antiproliferative activity on tumours and to have antiangiogenic
effects [8, 9].Recently it has been shown that IFN-α increased the
expression of p53 thereby boosting its responses to a variety of
stress stimuli [10, 11]. It is well established that this tumor
suppressor gene plays multiple roles in cell cycle control,
differentiation, genomic stability, angiogenesis and apoptosis
[12]. As a transcription factor, the protein becomes activated in
response to DNA damage and to a variety of other stress signals
including hypoxia, nucleotide depletion, hyperoxia, and activated
oncogenes. It executes its function mainly by transactivating other
genes, which are implicated in the control of the cell cycle and/or
apoptosis (e.g. Bcl-2, Bax, Bcl-XL, p21WAF1, GADD45, etc.) [12,
13].Evidence has been provided indicating that tumor cells bearing
a mutant p53 are highly resistant to chemo- and radiotherapy and
that the sensitivity to these agents could be increased upon
restoration of p53-function. Furthermore, recent studies point to
the implication of p53 to CTL-mediated cytotoxicitiy [12, 14].In
RCC, in contrast to other malignancies, p53- mutations are rare,
with a frequency of 10-30%, but the proapoptotic function of p53
seems to be repressed by a yet unknown mechanisms [15]. In this
study, we demonstrate that IFN-α induces p53 expression in a RCC
cell line, which could be correlated with an intensification of its
death-inducing response to genotoxic stress. Furthermore, we show
that cell death is preceded by a shift of the p53 target Bax to the
mitochondria. Our results reveal a role of p53 and Bax in the
sensitivity of RCC to IFN-α.
Materials and methods
Cells
Tumor cell lines derived from primary RCC were maintained in
Dulbecco’s modified Eagle’s medium/Ham F12 1:1 with Glutamax
(Invitrogen, Cergy, France) medium supplemented with 10% fetal
bovine serum (FBS) and 1% Ultroser G (Gibco BRL, Scotland).
Cytokine treatment
IFN-α was purchased from Cell Signaling Technology. Cells were
treated for the indicated time. Unless otherwise stated, the
concentration of IFN-α was 750 U/mL.
Phenotypic analysis
RCC cells were treated with IFN-α (750 U/mL), controls were
grown in medium without IFN-α. After 48 hours cells were
harvested, washed twice with phosphate-buffered saline (PBS) and
then incubated for 20 minutes at 4 °C with the first mAb
directed against HLA class I (W6/32, IgG2a), washed twice with
PBS followed by incubation with FITC-conjugated goat antimouse
immunoglobulin, washed twice with PBS, and fixed before analysis on
a FACS-Sort (Becton Dickinson, San Jose, CA, USA). Background
levels were measured using isotypic controls. Low forward-scatter
elements (dead cells or debris) were excluded from the analysis and
10 000 events were collected and analyzed using the Cellquest
software (Becton Dickinson).
Western blot analysis
Total cellular extracts were prepared by lysing cells in ice cold
buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100,
1mM PMSF, 10 μg/mL aprotinin and 10 μg/mL leupeptin).
Equivalent protein extracts (25-50 μg) were denatured by
boiling in sodium dodecyl sulfate (SDS) and β-mercaptoethanol,
separated by SDS-PAGE and transferred onto HybondTM
membranes (Amersham, Biosciences). The efficiency of the protein
transfer was assessed by Ponceau Red staining of the membranes.
Blots were blocked overnight with TBS containing 5% non-fat dried
milk, 0.1% Tween and probed with the following Ab: p53 (AB-2,
Oncogene, Boston, MA, USA), p21WAF1 (AB-1, Oncogene, Boston, MA,
USA), BcL-2 and Bax (Santa Cruz Biotechnology, Santa Cruz, CA,
USA). After washing, blots were incubated with appropriate
secondary, conjugated Ab-HRPO. The complexes were detected using an
ECLTM detection kit (Amersham, Biosciences).
Densitometric analysis including correction for background was
performed by using the Bioprofil Bio1D Windows application V99-04
software.
Apoptosis assay – propidium iodide (PI) staining
Flow cytometry analysis of PI-stained cells was performed to
analyze the effect of IFN-α treatment on cell viability. Renal
tumor cells were pretreated with INF-α and/or submitted to
γ-irradiation and cultured for an additional 72 h. Cells were
then harvested, washed, and fixed in 70% ethanol. They were washed
with PBS and stained with 1 mL of PI (20 μg/mL)
containing 100 μg/mL RNase and 20 mM EDTA. DNA content
was determined using a FACSCalibur flow cytometer (Becton
Dickinson, San Jose, CA, USA) and the proportion of cells in a
particular phase of the cell cycle was determined by CellQuest
software (Becton Dickinson, San Jose, CA, USA). Induced apoptotic
cell death was determined by measuring the proportion of
subG1 cells.
Confocal scanning immunofluorescence microcopy
Renal tumor cells were grown on sterile coverslips and subjected to
treatments as described above. Coverslips were then washed once
with PBS and fixed for 30 min in 4% paraformaldehyde (PFA) solution
in PBS, and washed 3 times with PBS. After 5 min incubation
with methanol, slides were washed 3 times with PBS, and cell
membranes were permabilized for 10 min with 0,1% SDS in PBS and
washed 3 times with PBS. Non-specific sites were blocked for
20 min with 10% FBS in PBS and washed once with PBS. The cells
were incubated for 1 hour with anti-Bax polyclonal rabbit
antiserum (N-20, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and
anti-cytochrome-c mouse monoclonal antibody (Becton Dickinson, San
Jose, CA, USA). After three washes with PBS, coverslips were
incubated with Alexa 546 conjugated antimouse antibody (Molecular
Probes, Eugene, OR, USA) and Alexa 488 conjugated anti-rabbit
antibody (Molecular Probes, Eugene, OR, USA) for 1 hour. After
washing with PBS, nuclei were stained using To-Pro®-3
iodide (Molecular Probes, Eugene, OR, USA). The coverslips were
mounted with anti-fading Vectashield from (Vector, Burlingame, CA,
USA). Confocal microscopy analysis was performed on a Zeiss LSM 510
microscope.
Results
HLA class I induction in renal cell carcinoma cell lines in
response to IFN-α
We selected two of our RCC-cell lines (RCC5 and RCC7) on the basis
of their capacity to express functional p53 as assessed by their
capacity to induce p21 after γ-irradiation (data not shown). In
order to examine the global responsiveness of these cell lines to
IFN-α, we measured the HLA-class I-induction following treatment
with this cytokine. The cell lines were treated for 48 hours with
IFN-α (750 U/mL) and then submitted to FACS-analysis for HLA-I
expression. As shown in ( figure 1 ), a significant
increase in HLA-I expression after exposure to IFN-α was detectable
in both cell lines in a comparable manner.
Differential p53 expression in RCC during IFN-α treatment
On the basis of their global responsiveness to the IFN-α treatment,
we then explored the influence of IFN-α on p53-expression. The
cells were treated with IFN-α (750 U/mL) and the expression of
p53 was probed by westernblot at different time points. As
demonstrated in ( figure 2 ), a notable
increase in p53 was detected in RCC7, reaching a maximum after 8
hours, whereas no changes were detected in RCC5.
IFN-α-induced p53 expression correlates with increased
sensitivity to genotoxic stress
One major role of p53 besides regulation of the cell cycle is the
induction of cell death after genotoxic stress stimuli such as
γ-irradiation. To investigate, whether the increase in p53-protein
level in RCC7 resulted in a greater susceptibility to
γ-irradiation, cells were treated with IFN-α (750 U/mL) before
γ-irradiation. After an additional 72h, the cells were probed for
cell death by FACS, using PI-staining. As outlined in ( figure 3 ), IFN-α
increases the sensitivity of RCC7 to γ-irradiation (5Gy), a dose,
which by itself does not induce cell death in RCC7. In contrast, no
increase in cell death is observed in RCC5, in which IFN-α-induced
p53 expression is not observed.
Expression of Bax and Bcl-2 is not affected by IFN-α-induced
p53 expression
It is thought that p53 influences the balance between the pro- and
anti-apoptotic members of the Bcl-2 family by means of
transcriptional control. Furthermore, evidence has been provided
indicating that the ratio of the pro-apoptotic protein Bax and the
anti-apoptotic protein Bcl-2 plays a crucial role in the control of
the intrinsic pathway of apoptosis. We therefore reasoned that the
enforced cell death in RCC7 treated with IFN-α and γ-irradiation
might be due to changes in this ratio. The expression of Bcl-2 and
Bax was probed by Western blot in cells treated with IFN-α and/or
γ-irradiation. As shown in ( figure 4 ), no change
was observed in the Bax/Bcl-2 ratio in either of the cell lines.
Enforced Bax-shift to the mitochondria following IFN-α
treatment and γ-irradiation
Recently it has been shown that p53, besides its action as
transcription factor, can activate Bax through a mechanism that is
transcription-independent. To evaluate, whether treatment with
IFN-α could enhance the translocation of Bax to the mitochondria,
we performed confocal microscopy with intracellular staining of Bax
and cytochrome c. As shown in ( figure 5 ), such
pretreatment enhances Bax translocation to the mitochondria after
γ-irradiation in RCC7, but not in RCC5. Interestingly, in RCC5, Bax
co-localized with the mitochondria even in non-treated cells,
without an obvious release of cytochrome c.
Discussion
IFN-α is used in the treatment of metastatic renal cell carcinoma
[16]. However, the way in which tumor cell growth is suppressed by
IFN-α is not well understood [17]. Recently it has been reported
that IFN-α may execute its anti-neoplastic action at least in part
through induction of the tumor suppressor protein p53, thereby
boosting the cellular response to a variety of stress stimuli
activating p53, including γ-irradiation and chemotherapeutic drugs
[10, 11].
The present study was performed to explore the impact of the
tumor suppressor protein p53 on the action of IFN-α in renal cell
carcinoma using two, well characterized RCC cell lines (RCC5 and
RCC7). Although both cell lines express a functional p53, we have
shown that IFN-α induced p53 expression only in RCC7, whereas the
p53-level in RCC5 remained unaffected. The mechanisms underlying
the deficit in the p53 induction in RCC5 are not clear. It has been
reported that resistance of renal cell carcinoma to IFN-α treatment
may be due to defects in the signal transduction, mainly through
defective induction of Stat1 [18]. However, the interferon
signaling, as assessed by the induction of HLA-class I upon
treatment with IFN-α is not disturbed in our cell lines.
Furthermore, the over-expression of the multidrug resistance (MDR)
gene and the MDR gene product, P-glycoprotein (Pgp) frequently
observed in RCC, has been associated with resistance of these
tumors to IFN-α [19]. Whether this or other mechanisms account for
the deficit in p53 induction of RCC5 needs further
investigation.
The treatment of our RCC cell lines with IFN-α did not affect
cell viability by itself but it potentiated cell death following
γ-irradiation, a stimulus that typically induces p53-regulated
responses such as cell cycle arrest and/or apoptosis [12], in RCC7
but not in RCC5. Up to now, the mode of action of IFN-α in RCC has
been poorly understood, but is presumed to be a combination of the
stimulation of cell-mediated cytotoxicity, a direct
antiproliferative activity, and antiangiogenic effects [9]. Our
findings indicate that p53 may be implicated in the effects of
IFN-α in the treatment of RCC taking into account the crucial role
of p53 in controlling cell growth, apoptosis and angiogenesis [12].
Furthermore it has been demonstrated in other tumor models that the
p53 function is important with respect to tissue target
susceptibility to specific effector killer cells [14]. Although it
has been hypothesized that the p53-pathway is repressed in RCC by a
factor that remains to be defined [15], there is evidence that the
activation of this pathway can sensitize RCC to additional
treatments such as anti-Fas or anti-TRAIL Ab [20, 21].
It is well established that p53 engages the so-called
“intrinsic”, mitochondrial apoptotic pathway, mainly by regulating
the transcription of the pro- and anti-apoptotic proteins of the
Bcl-2 family such as Bcl-2 and Bax [13, 22]. This results in the
permeabilizing of the mitochondrial membrane and subsequent release
of apoptogenic factors including SMAC/DIABOLO, HtrA2/Omi, AIF and
cytochrome c, which leads to effector caspase activation [13,
22].
In our experimental system, despite the increased cell death, we
were not able to demonstrate changes in the expression of Bcl-2 or
Bax after combination of IFN-α and γ-irradiation in RCC7. This may
be explained by the rather short period of observation, as
up-regulation of Bax 24 hours after p53 induction in RCC has been
reported [20]. On the other hand, it has been shown, that p53 can
induce cell death through the mitochondrial pathway in the absence
of transcription [23, 24]. In fact, the intracellular staining
revealed a pronounced shift of Bax to the mitochondria after
combination of IFN-α with γ-irradiation in RCC7. Interestingly, in
RCC5, Bax co-localized with the mitochondria regardless of the
treatment, without obvious signs of ongoing apoptosis. We therefore
hypothesise, that the dynamics in the localization of the
pro-apoptotic protein Bax may be important in the response to
genotoxic stress stimuli and that the enforced expression of p53
may be implicated in this process even though the exact mechanistic
connection requires further investigation.
Taken together, our results indicate a role for p53 in the
control of renal cell carcinoma sensitivity to IFN-α by mechanisms
implicating Bax translocation and mitochondrial apoptosis pathway
activation. Whether and how this can contribute to new treatment
strategies needs further experimental studies.
Acknowledgements
This work was supported by a grant from the ARC to AC (grant 4613)
and to SC (grant 4744). SW was supported by a fellowship from the
Dr Mildred Scheel Stiftung.
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