WO2005075677A2 - Identification of compounds susceptible to induce pidd in cells lacking p53 and their use in combination with genotoxins for the treatment of cancer - Google Patents

Abstract

The present invention relates to the identification of compounds susceptible to induce PIDD in cells lacking p53 and their use in combination with genotoxins for the treatment of cancer.

Description

Identification of compounds susceptible to induce PIDD in cells lacking p53 and their use in combination with genotoxins for the treatment of cancer

The present invention relates to the identification of compounds susceptible to induce PIDD in cells lacking p53 and their use in combination with genotoxins for the treatment of cancer. These compounds may be known compounds but their PIDD inducing properties being unknown. These compounds may also be new compounds identified and selected for their PIDD inducing properties. It is known that genotoxins used for the treatment of cancer may loose their genotoxic properties, the tumor cells losing their sensitivity to these toxins and the treatment of the disease evolving through a therapeutic failure. There is therefore a need to restore the cells sensitivity to the genotoxins, with the identification and use of new compounds inducing or restoring cell sensitivity, to be used in combination with the genotoxins. Moreover, such compounds inducing cells sensitivity to genotoxins may be used in combination to genotoxins in order to lower the doses of said genotoxins generally used for the treatment of cancer. It has now been found that compounds susceptible to induce PIDD in cells lacking p53 are such compounds susceptible to induce and restore cells sensitivity to genotoxins. Genotoxins are known in the art (USP Dictionary of USA and International Drag Names, United States Pharmacopoeia, Rockville MD, Last Edition), including the following: Etoposide, Alkeran, Ara-C, BiCNU, Busulfan, CCNU, Carboplatinum, Cisplatinum, Cytoxan, Daunorubicin, DTIC, 5-FU, Fludarabine, Hydrea, Idarubicin, Ifosfamide ,Methotrexate ,Mithramycin, Mitomycin, Mitoxantrone, Nelban, Nincristine, NP-16 and gamma-irradiation. Compounds susceptible to induce PIDD in cells lacking p53, also called PIDD Inducers, may be used simultaneously or sequentially with the genotoxin. When used simultaneously, the PIDD Inducer and the selected genotoxin may be used in the same pharmaceutical composition, or may be used in two distinct pharmaceutical compositions. When present in the same composition, the PIDD Inducer and the genotoxin may me mixed together or eventually separated in order to avoid contact between the two component prior administrations. The two components, PIDD Inducer on the one side and the selected genotoxin on the other side, may be coated by an appropriate layer prior mixing in the pharmaceutical composition. The coating layer may be selected in order to regulate the bioavailability of the said component. For instance, the PIDD Inducer may be coated with a layer appropriate for its immediate release when the genotoxin may be coated with a layer appropriate for a delayed release in order to maximize the effect of the PIDD Inducer. When present in two distinct pharmaceutical compositions, each of the components, PIDD Inducer on the one side and the selected genotoxin on the other side, may be administered through the same or distinct administration routes. Pharmaceutical compositions and their preparation are well known in the art. Pharmaceutical compositions and combinations thereof are also within the scope of the present invention. PIDD Inducer may be selected within known compounds, which PIDD inducing properties are unknown, or may be new compounds selected for their specific PIDD inducing properties. PIDD Inducers may be selected by known screening methods in order to distinguish among the screened compounds, those inducing PIDD expression. In a preferred embodiment, PIDD Inducers may be selected using PIDD Inducer identification procedure, comprising exposition of a chemical library with a PIDD Inducer identification test selected among the following procedures. Chemical libraries and how to prepare such libraries are known in the art, some libraries being available commercially. According to the invention, a chemical library comprises at least one individual chemical compound, preferably more than hundred individual chemical compounds, more preferably more than thousand individual chemical compounds. For a library comprising more than one individual chemical compound, each of the compounds may be isolated and characterized. In a preferred embodiment, the chemical library is a norined chemical library. Procedure 1 The first procedure according to the present invention comprises the step of contacting cells lines expressing a marker or reporter gene under control of the PIDD-promoter with the chemical library and analysing the expression of the said marker or reporter gene. Comparison of expression level in absence or in presence of the chemical library allows identification of such compounds of the library inducing PIDD-promoter activity and PIDD expression in the cell. The promotor region of PIDD is identified in the human genome (from the ensemble databank). A promoter sequence is a sequence of 1-2 kb of bases upstream of the trancription initation site. The human PIDD sequence (AF274972) is disclosed by Lin & al. (Nat. Genet. 26 (1), 122-127 (2000)). A method for the identification and cloning of its promoter is disclosed in Nucleic Acids Res. 2003 Jul l;31(13):3540-5 (Solovyev , Shahmuradov IA., PromH: Promoters identification using orthologous genomic sequences) or in Genome Res. 2003 Jul; 13(7): 1765-74 (Khambata-Ford S, Liu Y, Gleason C, Dickson M, Airman RB, Batzoglou S, Myers RM. Identification of promoter regions in the human genome by using a retroviral plasmid library-based functional reporter gene assay). The promotor region is then cloned in a reporter Vector, for example pGL3-Basic Vector from Promega. The pGL3-Basic Vector lacks eukaryotic promoter and enhancer sequences, allowing maximum flexibility in cloning putative regulatory sequences. Expression of luciferase activity in cells transfected with this plasmid depends on insertion and proper orientation of a functional promoter upstream from luc+, in this case the PIDD promotor. The expression vectors carrying the PIDD-promotor is co-transfected with a vector carrying a resistance gene (such as ampicillin) into p53-negative SAOS2 cells by calcium phosphate or lipofectamine. Ampicillin-resistance cells are selected and the expression of PIDD-promotor-Luc vector tested by transfecting the cells with a p53 containing vector. PIDD-promotor-Luc vector expressing cell lines are then exposed to a chemical library. In order to avoid cell death, a caspase inhibitor such as zVAD-fink may be included in the assay. Luciferase activity can be measured on samples containing equivalent amounts of protein using a luminometer and the luciferase assay reagent (Promega). Other marker/reporter genes known in the art, including commercially available genes, may be used in place of the luciferase gene. Procedure 2 p53-negative cells (for example SAOS2) are exposed to a chemical library, in the presence of a caspase inhibitor, and the upregulation of PIDD mRNA assayed by isolating the rαRNA of the cells after treatment. mRNA will be reverse transcribed and the presence of PIDD message determined by the Polymerase chain reaction (PCR) using appropriate primers selected from the PIDD sequence (AF274972). The two sequences below may be examples of primers susceptible to be used in this procedure: Forward: gcacctgaagaatgtgaaggag Reverse: gatgcgctgcacctcccggtag

The present invention is based on the finding that increased levels of PIDD expression result in spontaneous caspase-2 activation and sensitization to apoptosis by genotoxic stimuli. Caspase-2 is the most conserved caspase across different species (_). Its role in apoptosis, however, still remains enigmatic and controversial, despite the fact that it was the second mammalian caspase to be identified (2, 3). Although a role for caspase-2 in apoptosis initiated by β-amyloid cytotoxicity (4), trophic factor deprivation (5) and granzyme B (6) has been proposed, the interest in this caspase has been dampened due to the lack of an overt phenotype of caspase-2 deficient mice and limited knowledge of its mode of activation and downstream targets. Nevertheless, recent evidence clearly established a requirement for caspase-2 in stress- induced apoptosis (7). in support of results that indicated resistance of caspase-2 deficient germ cells and oocytes to cell death after treatment with chemotherapeutic drugs (<5). Caspase-2 appears to trigger apoptosis upstream of mitochondria, by inducing Bid cleavage, Bax translocation to mitochondria and subsequent cytochrome-c release (7-9). m accord with this are the observations that caspase-2-induced apoptosis requires caspase-9 (9) and that recombinant caspase-2, when added to purified mitochondria, is the only caspase that directly causes the release of cytochrome c (8, 9). Caspase-2 is expressed in two forms. Both have a Caspase-Recruitment Domain (CARD) pro-domain which, when overexpressed, associates with RAIDD, an adaptor protein containing a CARD and a Death Domain (DD) (10). RAIDD, via its DD was proposed to interact with TNF-receeptor 1 (TNF-R1), thereby linking TNF-R1 activation with casρase-2- mediated apoptosis (10, 11). However, this notion could not be confirmed when assembly of the endogenous TNF-R1 signaling complex (12) or the TNF-sensitivity of caspase-2 deficient cells were studied (6). Usually, caspases that possess a CARD pro-domain are activated by CARD-containing proteins, as exemplified by Apaf and caspase-9. We therefore expressed numerous CARD containing proteins and tested their possible interaction with the CARD of caspase-2. The unique interaction partner identified was RAIDD (data not shown), in agreement with previous results (10, 11). The CARD of RAIDD interacted specifically with caspase-2 and not with any of the other CARD-containing initiator caspases. Using a similar approach, we identified the upstream DD-containing interaction partner that bound the DD of RAIDD. Of the more than 20 different proteins analyzed, PIDD was found to be the only protein interacting with RAIDD. Through overexpression, a PIDD-RAIDD-caspase-2-trimolecular complex could be reconstituted. Interestingly, the presence of PIDD appeared to considerably stabilize caspase-2-RAIDD interaction. PIDD (p53-induced protein with a DD) was initially identified as a prime p53-target gene in an erythroleukaemia cell line that undergoes Gl phase arrest and subsequent apoptosis following p53 expression (13). Independently, PIDD was also described as a DD-containing protein with unknown function (14). The amino terminal region of PIDD contains 7 leucine- rich repeats (LRR), a protein interaction motif found in a variety of proteins with diverse function, followed by 2 ZU-5 domains and a C-teπninal DD. Prototypical ZU-5 interaction domains are present in the tight junction protein ZO-1, ankyrin and Unc5-like netrin receptors. Expression of PIDD is widespread, but is highest in the spleen (13). Expression is dramatically induced by p53 (13). The 910 amino-acid containing full-length protein, migrating at approximately 90 kDa, was frequently found to be cleaved into two fragments of 48 kDa ( -teπninal region) and 51 kDa (C-terminal region) (14). The size of the fragment predicts constitutive cleavage between the two ZU-5 domains by an unknown protease. Processing does not result in the dissociation of the two fragments (data not shown). We next investigated whether the PIDD-RAIDD-Caspase-2 complex (the PIDDosome) also forms under more physiological conditions. Recently, the characterization of a caspase-2- containing complex that is distinct from the Apaf- 1 -caspase-9 apoptosome was described (15). Formation of the caspase-2 containing complex occurred spontaneously in cell lysates upon incubation at 37°C for 1 h (15), which is in marked contrast to apoptosome formation that requires the addition of dATP and cytochrome c. In agreement with the published data, spontaneous caspase-2 complex formation was detectable in cell extracts from HeLa cells, Jurkat T cells, 293T cells, or Ramos B lymphocytes. When cell extracts incubated at 0°C were analyzed by gel filtration chromatography, caspase-2 eluted mainly in fractions corresponding to the monomeric protease (50 kDa), while prior incubation at 37°C caused a substantial portion to elute in fractions corresponding to a molecular mass of >670 kDa. The same shift was observed for RAIDD. Read et al. previously failed to observe complex formation of RAIDD under identical conditions (15). Because of this discrepancy and to corroborate our data, 293T cells were stably transfected with a Flag-tagged version of RAIDD. Independent of the antibody used (anti-RAIDD or anti-Flag), the analysis of cell extracts revealed that RAIDD was incorporated in a complex in a temperature-dependent manner. Complex formation was also observed when only the PIDD-interacting DD of RAIDD was expressed, but not with the CARD of RAIDD, suggesting that complex formation of RAIDD was PIDD- dependent. Unlike caspase-2 and RAIDD, PIDD was already present in a complex of approximately 550kDa in untreated cell extracts. The size of the PIDD-complex further increased in response to a temperature rise. Taken together, these data suggested that PIDD, RAIDD and caspase-2 spontaneously assemble into a high molecular weight complex in cell extracts. Caspase-2 is an initiator caspase for which proteolytic processing is not absolutely required for activation (16, 17). In order to detect unequivocally active caspase-2, we incubated cell extracts of 293T cells overexpressing various PIDDosome components with biotinylated NAD-frnk, which irreversibly binds to the exposed active site of caspases. Subsequent streptavidin precipitates revealed that overexpression of either RAIDD or PIDD resulted in the generation of enzymatically active caspase-2. In contrast to PIDD or RAIDD, overexpression of other apoptosis inducers, such as RIPl or caspase-9, resulted in strong activation of caspase-3 but not caspase-2. Thus, PIDD-induced caspase-2 activation is not relying on caspase-3, a conclusion, which was supported by the observation that PIDD- mediated caspase-2 processing was not impaired in caspase-3 deficient MCF7 cells. We next examined whether caspase-2 activation occurs not only upon artificially increasing the concentration of PIDD but also in the spontaneously assembling PIDDosome in cell extracts of 293T cells. Even though NAD-fink is not caspase-2 specific, the remaining initiator caspases are not spontaneously activated in Jurkat T cells under the conditions of cell rapture used ((15) and see below), and thus it was feasible to use biotin-NAI)-fink to specifically immunoprecipitate the active form of caspase-2. Caspase-2 labeled with biotin- NAD-increased over time, reaching a plateau after 60 min. In agreement with the fact that cleavage of initiator caspases is not required for activity (see above) and that further processing is inhibited by NAD-fink in cell extracts, the totality of caspase-2 was found in its precursor form. Along with caspase-2, RAIDD and PIDD were co-precipitated, indicating that a functional active site within caspase-2 is generated upon complex formation. Recruitment of PIDD and RAIDD was not detectable in murine embryonic fibroblasts deficient in caspase-2, confirming that caspase-2 was the only caspase able to spontaneously interact with RAIDD and PIDD upon cell rupture. Spontaneous assembly of PIDD, RAIDD and caspase-2 was also observed when PIDD instead of caspase-2 was immunoprecipitated. Recruitment of RAIDD and caspase-2 was observed in a time dependent manner. Unlike with biotin-NAD-finJ , which blocks processing of caspase-2 in cell extracts, the processed form of caspase-2 was now detected in the complex. What is the physiological role of PIDDosome-induced caspase-2 activation? In agreement with previous results, transient overexpression of high amounts of PIDD resulted in slow cell death (13), associated with caspase-2 activation (data not shown). Despite this, we succeeded in the generation of HeLa and B cell Ramos cells that stably expressed PIDD. Interestingly, in these PIDD-expressing cells, almost 50% of caspase-2 was processed, indicating that a mere increase of PIDD concentration led to caspase activation. Spontaneous activation of caspase-2 in PIDD expressing cells was confirmed when binding of biotin-NAD was analyzed. In contrast, cells expressing PIDD lacking the DD behaved like wild-type cells, and no caspase-2 activation was detectable, indicating that Ihe RAIDD-interacting DD of PIDD was essential for caspase-2 activation. If an increase of PIDD levels triggers the activation of caspase-2 in living cells, we have to postulate that PIDDosome complexes are present in PIDD-transduced cells. Indeed, a fraction of caspase-2 and RAIDD were found to be in high molecular weight complexes without the need of prior incubation of cell extracts at 37°C. A striking difference between PIDD-expressing cells and wild-type cells became apparent when cells were exposed to genotoxic stress. Within 5h after exposure to the topoisomerase inhibitors doxorubicin (Fig. 1) or etoposide (data not shown), 60% of the cells were dead, while in wild-type cells or cells expressing PIDDΔDD, most of the cells remained viable. This difference was also reflected at the level of caspase-activation. In PIDD- expressing cells, the remaining, non-processed caspase-2 was almost completely processed down to the pi 9 fragment, and the activity of caspase-3 was highly increased as evidenced by the cleavage of two of its substrates, DEND-AFC and PARP1 (18). Inhibition of PIDD expression was previously shown to attenuate p53 -induced apoptosis, while overexpression of PIDD inhibited cell growth (13). Thus, PIDD appears to be a crucial target gene of a signaling pathway that is triggered upon p53 activation and which ultimately leads to apoptosis. Recruitment of PIDD into a RAIDD complex and subsequent caspase-2 activation appears to be possible by two mechanisms. The first mechanism involves the mere increase of PIDD concentration and the second is triggered under stress conditions at low PIDD concentrations, for example upon cell rupture. We therefore have to propose a model whereby PIDD has a high propensity to oligomerize, but where the ultimate trigger leading to assembly is given by a stress-related signal whose nature remains to be discovered. Perhaps a cytoplasmic protein whose conformation is altered under stress conditions binds to the LRR-domain of PIDD, thereby inducing complex formation (at high PIDD-concentrations, as found in the transfected cells, this trigger may no longer be required). If true, the proposed model of PIDDosome assembly is similar to the one proposed for assembly of the apoptosome and the inflammasome (19, 20). Unlike with the apoptosome, where the ensuing caspase-9 activation leads to apoptosis in most cases, PIDDosome-based activation of caspase-2 is not toxic and cells survive even if an important fraction of the caspase-2 pool is activated. The caspase-2 activating complex therefore more resembles the caspase- 1 -activating inflammasome complex (21). An additional stress signal is required to activate the latent caspase-2-mediated death signal. Genotoxins, such as the topoisomerase inhibitor used in this study, are known to activate several signaling cascades including those leading to p53, ΝF-kB, JNK or ERK. These signals in combination with the presence of active caspase-2 may ultimately be sufficient to kill cells. How the active PIDDosome or active caspase-2 sensitizes to apoptosis induced by genotoxins is poorly understood. As supported by several reports (7-9), active caspase-2 may induce translocation of Bax to the mitochondria and/or the cleavage of Bid, resulting in subsequent release of cytochrome c, AIF, and Smac/Diablo and ensuing Apaf-caspase-9 dependent cell death. This caspase-2-mediated cell death pathway may be efficiently blocked by members of the anti-apoptotic Bcl-2 family and thus concurrent repression of the inhibitors, for example under genotoxic stress through activating BH3 proteins, would be required. This model would explain why genotoxic stress induced cell death was found not only be dependent on the presence of caspase-2 (6, 7), caspase-9 (7) and Bax (22), but also on the two p53-inducible BH3 members Noxa and Puma (22, 23). With the identification of the caspase- 2 activating platform, the testing of this model is now highly facilitated. Figure Legend

Fig. 1. Increased PIDD expression results in caspase-2 activation and sensitizes cells to genotoxin-induced cell death. HeLa cells transiently expressing equivalent levels of PIDD or a mutant PIDD lacking the DD (PIDDΔDD) were treated or not with the pan- caspase inhibitor zNAD (50μM), lysed and cell extracts incubated with biotin-VAD- fink. Note that the concentrations of zNAD that completely block the proteolytic activity of most caspases, is unable to block the PIDD-induced generation of the pi 9 fragment of caspase-2 when added to intact cells. Expression levels of PIDD and PIDDΔDD were similar (data not shown). evileleCytoplasmic extracts from wild-type (WT) HeLa cells or HeLa cells expressing PIDD were subjected to size exclusion chromatography after prior incubation (1 h) at 4°C and 37°C, respectively. Fractions were analyzed for the presence of caspase-2, RAIDD and FADD (control). HeLa cells subjected to doxorubicin (lOμg/ml) were analyzed after different periods of time for cell death, caspase-2 processing and caspase-3 activity (DENDase activity and PARP1 cleavage). Material and Methods

Caspase-2 forms a complex with RAIDD and PIDD RAIDD specifically interacts with caspase-2. CARD-containing Flag-tagged caspases were overexpressed in 293T cells along with NSN-tagged RAIDD and anti-Flag-caspase immunoprecipitates analyzed for the presence of RAIDD by western blot analysis. ASC represents control of a CAIlD-containing, non caspase protein. The RAIDD-DD interacts with the DD of PIDD. Flag-tagged Death Domains (DD) containing proteins were overexpressed in 293 T cells along with the NSN-tagged DD of RAIDD and anti-Flag immunoprecipitates analyzed for the presence of RAIDD . Increased binding of RAIDD to caspase-2 in the presence of PIDD The indicated proteins were overexpressed in 293T cells and proteins interacting with the CARD of caspase-2 analyzed. Domain structure of PIDD. LRR, Leucine-Rich-Repeats; ZU5; ZU5 domain; DD, Death Domain. Within cells, PIDD is processed between the two ZU5 domains. Left panel: Western blot of HeLa cells stably expressing tagged PIDD (Ν-terminus NSN, C-terminus Flag). Analysis of the NSN-PIDD-Flag construct expressing Jurkat cells. PIDD-C was detected using an antibody detecting the DD of PIDD. Note that this antibody detects two additional bands which are absent in non-transfected cells. Spontaneous complex formation of PIDD. RAIDD and caspase-2 293T cell lysates were subjected to size exclusion chromatography after prior incubation (1 h) at 4°C and 37°C, respectively. Fractions were analyzed for the presence of caspase-2, RAIDD and FADD (control). Experiments shown in the two lower panels were done with cell extracts from 293T cells stably expressing RAIDD, the DD or the CARD of RAIDD, respectively. The elution positions of the molecular weight markers are indicated. Cell extracts of Jurkat cells were analyzed for the presence of Caspase-2, RAIDD, and PIDD. Activation of caspase-2 during PIDDosome assembly Cell extracts of 293T cells transiently expressing the indicated activators of caspases were incubated with Biotin-VAD and strepatavidin immunoprecipitates analyzed for the presence of caspase-2 and cleaved caspase-3. Recruitment of RAIDD and PIDD to active caspase-2 PIDDosome assembly was spontaneously induced in cell extracts from Jurkat cells and active caspase-2 immunoprecipitated using biotin-NAD. The presence of RAIDD, PIDD and caspase-3 was determined in immunoblots. The anti-PIDD antibody used cross-reacts with two proteins that co-migrate close to processed PIDD (51 kDa fragment). The lower panel shows PIDD incorporation into the assembling caspase-2 complex in Ramos B cells. Right panel: analysis of cell extracts. Recruitment of RAIDD by biotin-NAD is caspase-2 dependent. In MEFs deficient for caspase-2, no RAIDD is recruited to the caspase-active site interacting biotinylated-NAD, indicating that RAIDD-recruitment is dependent on the presence of caspase-2. Recruitment of caspase-2 and RAIDD into the assembling PIDD complex PIDD in extracts from 293T cells stably expressing Flag-tagged PIDD, was immunoprecipitated at various time points and association with caspase-2, RAIDD, FADD, and cytochrome c analyzed in immunoblots. Cloning PIDD was cloned from two ESTs: IMAGE 3908102 and IMAGE 4852968. The first EST was used to amplify the 5' region of the cDΝA and the second EST to amplify the 3' region. Pidd was cloned with a 5' NSN-tag and a 3' Flag-tag. RAIDD-DD (101-199) was obtained by PCR using the primer 5'-gaattcatggacagattgactgggatccc-3' containing an EcoRI site using full-length RAIDD cDΝA as a template. Likewise, CARD-caspase-2 (1-104) was generated with the reverse primer 5'-ggattcggtggtgagcaacatatcc-3' from full-length caspase-2. All constructs were cloned in a PCR-3 based vector (Invitrogen). Antibodies The following antibodies were used: anti-RAIDD (MBL); anti-caspase-2 11B4 (Apotech); anti-caspase-2 C-20 (Santa Cruz); anti-cleaved caspase-3 (Cell Signaling); anti-

FADD and anti-caspase-3 (Transduction Laboratories); anti-Flag and anti-NSN (Sigma);

AL233 is a rabbit polyclonal antibody raised against PIDD-DD (776-910) by Eurogentec

(Belgium). Cell culture 293T cells and caspase-2 -/- MEF cells were maintained in Dulbecco's modified

Eagles medium (DMEM) (Life Technologies) supplemented with 10% fetal calf serum and lOOμg/ml penicillin/neomycin/streptomycin (PNS. Ramos and Jurkat cell lines were maintained in RPMI 1640 supplemented with 10% fetal calf serum, PNS and lOμM β- mercaptoethanol. Transfection and immunoyrecivitation Transfection of 293T cells was performed with the calcium phosphate method. MCF-7 were transfected with Lipofectamine2000 (Invitrogen) according to manufacturer's protocol. Cells were lysed in 1% NP-40 lysis buffer on ice. Preclearing was performed with Sepharose- 6B beads (Sigma) for 1 hour and M2 anti-Flag -agarose beads (Sigma) were then added. In order to detect active caspase-2, biotin-NAD was added to the lysates and incubated overnight with streptavidin-Sepharose beads (Amersham) on a rotating wheel at 4°C. Preparation of the cell lysates for in vitro activation Cells were washed twice with PBS and resuspended in hypotonic buffer (20 mM

Hepes-KOH, 10 mM KC1, 1 mM MgCl₂, ImM EDTA, 1 mM EGTA, 1 mM DTT, pH 7.5) supplemented with protease inhibitors (Complete™; Roche). Resuspended cells were subjected to three rounds of freeze thawing using liquid nitrogen. Cellular debris were removed by centrifugation at 10,000xg for 20 min at 4°C, followed by filtration at 0.4μm. In vitro activation and immunoprecipitation Clear lysates were incubated at 37°C for the indicated times or left untreated on ice.

After incubation, an equivalent volume of hypotonic buffer containing 0.1% ΝP-40 and 300mM NaCl was added. The mix was incubated overnight with Biotin-NAD (Apotech,

Lausen, Switzerland) and streptavidin-Sepharose beads or M2-beads at 4°C on a rotating wheel. Gel filtration Treated lysates were fractionated using FPLC protein purification system on a Superdex 200 column (Amersham Life Science) at 4°C. The column was equilibrated with hypotonic buffer, and lysates (2 mg) were applied and eluted from the column with the same buffer at a flow rate of 0.4 ml/min and 400μl fractions were collected. The column was calibrated with Amersham gel filtration standards containing bovine thyroglobulin (670 kD), horse gamma globulin (158 kD), Bovine Serum albumin (67kD), chicken ovalbumin (43 kD), and chymotrypsinogen A (25kD). Western Blot Samples in loading buffer were loaded on an acrylamide gel and blotted on Hybond ECL nitrocellulose membrane (Amersham Life Science). The membrane was blocked in PBS- 0.5% Tween-20 with 5% milk. The membrane was incubated lhr at RT or o/n at 4°C with the primary antibody diluted 1/1000 in the blocking solution, then incubated with the horseradish- peroxidase (HRP)-conjugated secondary antibody diluted 1/5000 in the blocking solution for 45 minutes at RT and revealed with a chemiluminescent substrate (ECL, Amersham). Generation of cells stably expressing PIDD Cell populations stably expressing PIDD were obtained by subcloning PIDD into the MSCN puromycin-selectable retroviral vector (Clontech). The recombinant virus was obtained following transfections of 293T in combination with a vector encoding the viral structural genes (NSN-G pseudoryping vector). Cells were infected, selected with puromycin

(0.5 μg/ml) for 2 weeks. Cell death assay Cell death was analysed using Trypan blue staining. DEVDase activity was measured using DEVD-AFC (Apoptech) according to manufacturer's instruction.

References and Notes

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Claims

What is Claimed is:

1. A method for the identification of PIDD Inducers using a PIDD Inducer identification procedure comprising the step consisting in the exposition of a chemical library with a PIDD

Inducer identification test.

2. The method of claim 1, wherein the PIDD Inducer identification test comprises the steps of contacting cells lines expressing a marker or reporter gene under control of the PIDD- promoter with the chemical library and analysing the expression of the said marker or reporter gene.

3. The method of claim 1, herein the PIDD Inducer identification test comprises the steps of exposing p53-negative cells to a chemical library, in the presence of a caspase inhibitor, and assaying the PIDD mRNA upregulation by isolating the mRNA of the cells after treatment.

4. A compound susceptible to induce PIDD in cells lacking p53 identified using the procedure of one of claims 1 to 3.

5. A method for the treatment of cancer comprising the use of a PIDD Inducer compound according to claim 4 in combination with genotoxins.

6. A method according to claim 5, wherein the genotoxin is selected among Etoposide, Alkeran, Ara-C, BiCNU, Busulfan, CCNU, Carboplatinum, Cisplatinum, Cytoxan, Daunorubicin, DTIC, 5-FU, Fludarabine, Hydrea, Idarabicin, Ifosfamide, Methotrexate, Mithramycin, Mitomycin, Mitoxantrone, Velban, Vincristine, VP-16 and gamma-irradiation.