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WO2007121576A1 - Méthodes pour induire l'apoptose de cellules cancéreuses au moyen de dérivés de l'apoptine - Google Patents

Méthodes pour induire l'apoptose de cellules cancéreuses au moyen de dérivés de l'apoptine Download PDF

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WO2007121576A1
WO2007121576A1 PCT/CA2007/000681 CA2007000681W WO2007121576A1 WO 2007121576 A1 WO2007121576 A1 WO 2007121576A1 CA 2007000681 W CA2007000681 W CA 2007000681W WO 2007121576 A1 WO2007121576 A1 WO 2007121576A1
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apoptin
akt
cells
cdk2
seq
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Los Marek
Ted Paranjothy
Subbareddy Maddika
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University of Manitoba
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University of Manitoba
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/162—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/10011—Circoviridae
    • C12N2750/10022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the Phosphatidylinositol 3'-k ⁇ nase is a lipid kinase that catalyses phosphorylation of the Inositol ring of phosphoinositides [Pl, PI(4)P and Pl(4,5) P 2 ] at the D3 position [1 , 2]
  • Three classes of PI3-K have been identified which differ it, their primary structure, regulation and substrate specificity (reviewed in [3])
  • Class I PI3-Ks have been the major focus of PI3-K studies and are heterodimers composed of a catalytic subunit (p110) and a regulatory subunit (p85)
  • Four isoforms of the p110 subunit have been described ( ⁇ , ⁇ , ⁇ and ⁇ ), and three mammalian genes encode the adapter subunits p85 ⁇ , p85 ⁇ and p55 ⁇
  • the p85 subunit contains an N-terminal SH3 domain followed by a proline- rich domain
  • Apoptin is a 14 kDa protein derived from the chicken anemia virus Apoptin selectively induces apoptosis in cancer cells but not in primary cells (reviewed in [31 32]) In primary cells, apoptin remains in the cytoplasm, whereas in transformed cells it migrates into the nucleus and ultimately kills the cell by activation of the mitochondrial death pathway, in a Nur77-dependent manner independently of death receptors [33-36] However, targeted translocation of apoptin into the nuclei of primary cells is not sufficient for apoptm's toxicity Thus, additional interaction partners or specific activation of other signaling pathways in the cancer cells preceding nuclear accumulation might be necessary for apoptm's tumor specific toxicity Nuclear accumulation is strongly linked to phosphorylation of apoptin at the threon ⁇ ne-108 (ThM 8) residue by an unknown kinase [37 38] specifically in cancer cells but not normal cells Thus, identifying the pathways responsible for the phosphorylation of apoptin
  • a method of inducing apoptosis in a cancerous cell comprising administering an effective amount of an isolated or purified peptide comprising PKPPSK (SEQ ID NO 3)
  • a method of manufacturing a pharmaceutical composition comprising mixing an effective amount of an isolated or purified peptide comprising PKPPSK (SEQ ID NO 3) with a suitable excipient
  • a purified or isolated peptide comprising amino acids PKPPSK (SEQ ID NO 3)
  • Figure 1 Apoptin selectively kills cancer cells via the interaction conferred by the SH3 domain of the PI3-Kinase p85 subunit and apoptin's proline rich motif.
  • A B-cells from either normal Peripheral Blood Lymphocytes (PBLs) or the CLL PBLs were double stained for CD5/CD19 surface markers using FITC-conjugated CD5 and Per CP-conjugated CD19 antibodies at different time points after either TAT-GFP or TAT- Apoptin treatment The samples were then analyzed by flow cytometry and the number of CD5/CD19 double-positive cells were plotted
  • B GST-pull down assay performed with MCF-7 lysate using either GST control or GST-Apoptin and the proteins (p85 pl3 K and Akt) specific for apoptin interaction identified by mass spectrometry are indicated
  • C GST pull down assay performed with PC-3, MCF-7, 293 and L929 cell lysates with either GST or GST-apoptin The presence of the p85 subunit of PI3-K and Akt in apoptin complexes was determined by immunoblotting with the respective antibodies
  • D Co-immuno
  • PI3-K activity was measured by an ELISA-based assay after immuno-precipitating PI3-K from the lysates of PC-3 and MCF-7 cells transfected to express apoptin (time points indicate time post-transfection), as described in the methods section, and the fold induction was calculated PI3-K activity in non-transfected cells was considered as a basal level (1 x)
  • PC-3 cells were either transfected with apoptin alone, pretreated with wortmannin, LY294002 followed by apoptin transfection, or transfected to co-express apoptin and a PI3-K dominant negative vector The PI3-K activity was measured 24 hours post-transfection as described in 2A
  • C PI3-K activity was measured 24 hours after transfecting the cells to express the different apoptin deletion mutants
  • D PI3-K-mutants described in Fig 1 G were expressed after transfection in PC-3 cells (see main text for
  • FIG. 3 Akt translocates to the nucleus during apoptin-induced cell death.
  • A The activation of Akt by apoptin was detected in PC-3 cells by immunoblotting using an antibody against Akt-phosphorylated at Ser-473 at different time points after transfection to express apoptin Total Akt was detected by immunoblotting
  • B PC-3 cells were transfected to express apoptin in the absence or presence of treatment with wortmannin or LY294002, and phosphorylated Akt, then detected by immunoblotting Akt activation was also determined by immunoblotting in lysates from cells transfected to express apoptin alone or apoptin together with either dominant negative PI3-K (DN), PDK1 -DN, wild type PTEN, or phosphatase deficient C124S-PTEN mutant (C) The effect of Akt inhibition on apoptin toxicity was assessed in PC-3 cells by flow cytometry (Nicoletti method) either 24 hours or 48 hours after transfection to
  • CDK2 is the tumor specific apoptin kinase.
  • A In vitro kinase assay performed with GST-Apoptin and TAT-Apoptin as substrates using active CDK1/Cycl ⁇ n B 1 CDK2/Cycl ⁇ n E or CDK2/Cycl ⁇ n A Apoptin phosphorylation was detected by immunoblotting using an antibody against phosphor-threonme-proline Total apoptin levels were detected by anti-apoptin antibody Histone was used as a positive control (B) Active CDK2, CDK1 , Cyclin B, Cyclin E, Cyclin A were immuno-precipitated using their respective antibodies and the CDK2 T160A mutant was immuno-precipitated using anti- HA antibody, and used in a kinase assay with TAT-GFP, TAT-apoptin or H1 as substrates Phosphorylation was monitored as in (A) (C) PC-3 cells were transfected to express G
  • Figure 7 Model for the role of PI3-K/Akt pathway activation during cell death induced by apoptin, and other cell death stimuli.
  • PI3-K is constitutively activated, which leads to PDK1-dependent Akt activation and its nuclear translocation, probably through a piggy-back transport mechanism
  • Nuclear Akt phosphorylates and downregulates p27 k ⁇ p1 , which leads to aberrant CDK2 activation and propagation of the cell death signal
  • Activated CDK2 phosphorylates, among other substrates, apoptin at ThM 08 site and regulates its nuclear accumulation in cancer cells
  • Phosphorylated CDK2 translocates to the cytoplasm and phosphorylates Bcl2, which is then targeted for proteosome-dependent degradation
  • the resulting imbalance in the levels of the cell's anti-apoptotic factors leads to apoptosis DESCRIPTION OF THE PREFERRED EMBODIMENTS
  • an isolated or purified peptide comprising an amino acid sequence of PX 1 X 2 PX 3 (RZK) (SEQ ID NO 2) wherein X 1 X 2 and X 3 are any ammo acid
  • 'consisting essentially of means that the small peptide may be fused to a carrier or may be otherwise presented to the cell as part of a larger molecule which retains the anticancer activity of the small peptide
  • the peptide comprises an amino acid sequence of
  • PKPPSK (amino acids 81 -86 of apoptin (SEQ ID NO 1 ), SEQ ID NO 3)
  • PKPPSK comprises an isolated or purified peptide consisting of or consisting essentially of an amino acid sequence of PKPPSK
  • 'consisting essentially of means that the small peptide may be fused to a carrier or may be otherwise presented to the cell as part of a larger molecule which retains the anti-cancer activity of the small peptide
  • the peptide comprises an amino acid sequence of
  • PKPPSKKR (amino acids 81 -88 of apoptin (SEQ ID NO 1 ), SEQ ID NO 4)
  • PKPPSKKR comprises an isolated or purified peptide consisting of or consisting essentially of an amino acid sequence of PKPPSKKR
  • 'consisting essentially of means that the small peptide may be fused to a carrier or may be otherwise presented to the cell as part of a larger molecule which retains the anti-cancer activity of the small peptide
  • the peptide may comprise or may consist of or may consist essentially of amino acids 1-1 1 1 (SEQ ID NO 5), 74-121 (SEQ ID NO 6), 74-1 1 1 (SEQ ID NO 7) or 74-104 (SEQ ID NO 8) of apoptin
  • the apoptin-de ⁇ ved peptide is a recombinant peptide and comprises an amino acid sequence of at least one of SEQ ID NO 2-8
  • 'recombinant' is distinct from 'isolated or purified 1 and refers to the amino acid sequence in question being presented in a non- native context
  • the recombinant peptide comprises an amino acid sequence of at least one of SEQ ID NO 2-8 and the recombinant peptide is not full-length apoptin (SEQ ID NO 1 )
  • the recombinant peptide may comprise a truncated apoptin peptide that includes at least one of SEQ ID NO 2-8 or may comprise SEQ ID NO 2-8 inserted in a non-native peptide for example a carrier peptide
  • the apoptin-de ⁇ ved peptide consists of or consists essentially of
  • an apoptin-de ⁇ ved peptide as described above is used to induce or trigger cell death in a cancerous cell, to induce or trigger apoptosis in a cancerous cell, to direct the PI3-K pathway from cell survival to cell death or to activate PI3-K, thereby inducing apoptosis
  • these methods involve administering an effective amount of an apoptin-de ⁇ ved peptide as described above to an individual in need of such treatment
  • an 'effective amount' is an amount sufficient to achieve the desired result and will of course depend on many factors including but by no means limited to the age, weight and condition of the patient
  • the desired result is an amount sufficient to direct the PI3-K pathway from cell survival to cell death or to trigger or induce apoptosis in a cancerous cell as discussed herein
  • a method for manufacturing a pharmaceutical composition comprising mixing a purified or isolated apoptin-de ⁇ ved peptide as described above with a suitable excipient
  • the pharmaceutical composition may be for treating cancer, for inducing apoptosis or cell death in a cancerous cell, for directing the PI3-K pathway from cell survival to cell death or for activating PI3-K, as discussed herein
  • cyclin A-associated CDK2 is constitutively activated by apoptin
  • CDK2 is a crucial player in the ce.l cycle during the progression from G1 to S phase [66]
  • apoptosis [67, 68]
  • CDK2 inactivation by either pharmacological inhibitors or by siRNA severely impairs apoptin-induced cell death
  • Fig 5C the levels of CDK2 protein did not change with apoptin expression
  • cyclin A increased, this did not appear to occur at the transcriptional level, suggesting that it is rather due to the prevention of protein degradation This may be partially due to the inhibition of the C-subunit of the Anaphase-Promoting-Complex (APC/C) by apoptin as reported earlier [39]
  • APC/C Anaphase-Promoting-Complex
  • PI3-K/Akt pathway Different components of the PI3-K/Akt pathway are involved in tumorigenesis and are highly active in various types of cancers compared to normal cells (reviewed in [70, 71 ]) Furthermore PTEN, a phosphatase that counteracts PI3-K's action, is the second most commonly mutated tumor suppressor gene after p53 [71 ] Both CDK2 and Cyclin A are reported to be highly over-expressed in several tumors compared to the normal tissues (reviewed in [72, 73]) Hyper-activation of the above pathways leads to a poor clinical prognosis and also contributes to drug resistance during cancer treatment Thus, apoptin s targeting of these very pathways may explain its unique properties of tumor specific toxicity Our data strongly indicate that apoptin "hijacks" these survival pathways and redirects them from their normal survival/prohferatory action towards the activation of the cell death Importantly, the discovery here of the novel mechanism of the redirection of survival signaling into death pathways, will very likely
  • PI3-K deletion mutants were tagged with haemagglutinin tag (HA) at their N-terminus (Fig 1 G) Both full length PI3-K and the mutant lacking the ⁇ SH2 domain were immuno-precipitated by ant ⁇ -p85 antibody, while other mutants were immuno-precipitated using ant ⁇ -HA antibodies Immuno-detection of apoptin in the immune complexes of PI3-K and its deletion mutant derivatives implied that apoptin interacts with the intact SH3 domain of PI3-K (Fig 1 G)
  • PI3-K is constitutively activated during apoptin-induced apoptosis
  • PI3-K activity was increased nearly four-fold in apoptin treated MCF-7 cells and up to six-fold in PC-3 cells, compared to the GFP-transfected control PI3-K activation was seen around six hours after transfection, consistent with the interaction data (Fig 1 FG), with activation retained at a similar level for up to ⁇ 40 hours
  • various PI3-K inhibitors prevented apoptin-t ⁇ ggered generation of PIP 3 (Fig 2B), whilst co-transfection of apoptin with a dominant negative PI3-K vector reduced apoptin-induced PI3
  • PI3-K inhibition affects apoptin sub-cellular localization
  • Apoptin is mainly localized in the nucleus of PC-3 and MCF-7 cells, but in the presence of wortmannin, apoptin was found mainly distributed in the cytoplasm as shown either by the immuno- stainmg followed by confocal laser microscopic imaging (Fig 2G) or by sub-cellular fractionation followed by Western blotting (Fig 21)
  • the effect of PI3-K inhibition on apoptin's localization was further studied using p85 siRNA Upon the inhibition of p85 expression, nuclear localization of apoptin was almost completely abrogated (Fig 2H) indicating that PI3-K activity is not only necessary for apoptin-induced cell death but also for the localization of the protein during its apoptotic action in cells Previous reports indicated that the tumor-specific phosphorylation of the Thr-108
  • Akt translocates to the nucleus during apoptin induced cell death
  • Akt Akt phosphorylates and downregulates p27 k ⁇ p1 in the nucleus
  • p27 k p1 a cell cycle inhibitor
  • phosphorylation was monitored using anti- phospho-se ⁇ ne or anti-phospho-threonine antibodies p27 k ⁇ p1 threonine phosphorylation levels increased in the presence of apoptin, but apoptin had no effect on serine phosphorylation (Fig 4A)
  • CDK2/cyclin A activity is elevated and is required during apoptin-induced cell death
  • CDK2 is localized mainly in the nucleus during the execution of its normal cell cycle regulatory function, but in the presence of apoptin, CDK2 was observed predominantly in the cytoplasm (Fig 5H, see also Fig 6D)
  • Bcl2-phosphorylat ⁇ on is known to facilitate its degradation via the proteosome pathway [41 , 42], to determine the effect of CDK2 on Bcl2, we tested the levels of Bcl2 phosphorylated either at serine- or threonine residues after immuno-precipitation from lysates from cells transfected to express apoptin Increased Bcl2 phosphorylation at threonine
  • Activated cyclin A-associated CDK2 is the apoptin kinase that regulates its nuclear localization in cancer cells Apoptin phosphorylation at Thr-108 has been previously reported to be critical for its activity and tumor cell-specific nuclear localization [37, 38]
  • CDK2 may directly phosphorylate apoptin
  • in vitro kinase assay using recombinant GST-apoptin and TAT-apoptin as substrates
  • detection of phosphorylated apoptin using a phospho-threonine-proline specific antibody revealed that apoptin can be phosphorylated by active, recombinant CDK2/cycl ⁇ n
  • active, recombinant CDK2/Cycl ⁇ n E was not able to phosphorylate apoptin in vitro, nor was CDK1/Cycl ⁇ n B, although both were able to phosphorylate Histone H1 in vitro
  • CDK1/Cycl ⁇ n B
  • MCF-7 PC-3, 293 and L929 cells were grown in RPMI-1640 medium supplemented with 10% FBS (Hyclone), 100 ⁇ g/ml penicillin and 0 1 ⁇ g/ml streptomycin (Gibco BRL) The cells were grown at 37°C with 5% CO 2 in a humidified incubator
  • the peripheral blood lymphocytes were isolated from Chronic Lymphocytic Leukemia (CLL) patients or normal healthy individuals by ficoll gradient fractionation, as described previously [74] and maintained in RPMI medium
  • the following antibodies were used murine ant ⁇ -PI3-K (p85), anti-mouse IgG-HRP, anti-rabbit IgG-HRP (all from Upstate Cell Signaling), goat anti-Akt, rabbit ant ⁇ -p27 K p1 , murine anti-tubulin, rabbit anti-GFP, rabbit ant ⁇ -phospho-p27 k ⁇ p1 -Thr-187, rabbit ant ⁇ -CDK2, ant ⁇ -CDK1
  • Peripheral blood lymphocytes from normal individuals and CLL patients either left untreated or TAT-Apoptin treated for the indicated times were washed twice with ice cold PBS and then incubated with both CD5-FITC and CD19-PerCP antibodies (each 0 5 ⁇ g per 10 6 cells) for 30 minutes at 4 0 C in dark The cells were then washed twice with cold PBS and resuspended in 300 ⁇ l of PBS Samples were analysed by flow cytometry by using both FL1 (FITC) and FL2 (PerCP) channels and percentage of B-cells obtained by gating the double positive cells compared to unstained control
  • the TAT-GFP and TAT-Apoptin proteins were purified as previously [75] GST and GST-apoptin were purified by using glutathione sepharose high performance beads (Amersham Biosciences) according to the manufacturer's protocol
  • the GST-pull down assay was performed to detect apoptm's interacting partners Briefly, either purified GST or GST-apoptin along with total PC-3 cell lysate was immobilized on glutathione sepharose beads overnight at 4°C The beads were washed thrice with ice-cold lysis buffer and the bound proteins were isolated on SDS-PAGE The proteins specific for apoptin were subjected to in-gel digestion and further identified by MALDI-TOF mass spectometry at the proteomics centre at the University of Manitoba Finally, proteins from the GST-pull down assay were identified by immunoblotting
  • the following plasmid were used GFP-apoptin (apoptin cloned into pEGFP-C1 vector, clonetech), GST-apoptin (apoptin cloned into PGEX-2T vector, Amersham biosciences), PI3-K dominant negative vector, Akt wild type vector (J Downward, UK, [49]), Apoptin mutant plasmids (Fig 1 EF) [37]), p85 deletion mutants (Fig 1G) (T Mustelin, [76]) PDK1 dominant negative and constitutively active vectors (A Halayko, Winnipeg) PTEN wt and PTEN C124S phosphatase dead mutant (D H Anderson, Saskatchewan Cancer Agency, Saskatchewan), PKC dominant negative vector (E Kardami, Winnipeg), CDK2 T160A mutant (D O Morgan, San Franscisco, [77], Ad-NLS- Akt (M A Sussman, San Diego, [78], and Ad-Akt-dominant negative vector
  • PI3-kinase ELISA A non-radioactive competitive ELISA based assay was used to assess the PI3- kinase activity under different conditions (Fig 2A-D) according to the manufacturers protocol (Echleon Biosciences) Briefly, equal amounts of PI3-K from the PC-3 cell lysates were immuno-precipitated with ant ⁇ -p85 antibodies overnight at 4 0 C and then incubated with protein A-Sepharose beads for 1 hour at 4°C The bead-bound enzymes were incubated with 100 pM of phosphatidylmositol (4, 5) bisphosphate (Pl(4, 5)P2) substrate in kinase reaction buffer (4 mM MgCI 2 , 20 mM Tris, pH 7 4, 10 mM NaCI, and 25 ⁇ M ATP) for 2 h at room temperature The mixtures were then incubated with phosphatidylmositol (3,4,5) triphosphate (PI(3,4,5)P
  • RNA interference The plasmids coding for PI3-K siRNA (pKD-PI3 Kinase, p85-V3), CDK2 siRNA
  • pKD-CDK2-v6 pKD-CDK2-v6
  • the negative control siRNA was purchased from Upstate cell signaling
  • the p27 k ⁇ p1 siRNA was obtained commercially from Santacruz Biotechnologies
  • the described plasmids or the siRNA sequences were transfected into the cells grown to 70% confluency using Lipofectamine (Invitrogen) according to the manufacturer's protocol
  • the expression of the proteins was analysed by Western blotting after 48 hours of transfection
  • MCF-7 and PC-3 cells were transfected to express apoptin in the absence or presence of various treatments and fixed 24 h later in 4% Paraformaldehyde in PBS, permeabilized in 0 2% Triton X-100 and stained with either ant ⁇ -p85-, anti-Akt-, or anti- CDK2 antibodies followed by their respective secondary antibodies conjugated to Cy3
  • the fluorescent images were then analysed by a confocal microscopy
  • Cancer-specific toxicity of apoptin is independent of death receptors but involves the loss of mitochondrial membrane potential and the release of mitochondrial cell-death
  • BCL-2 is phosphorylated and inactivated by an ASK1/Jun N-terminal protein kinase pathway normally activated at G(2)/M MoI Cell Biol 19, 8469-8478
  • Granzyme B induces apoptosis and cyclin A-associated cyclin-dependent kinase activity in all stages of the cell cycle J Immunol 157, 2381-2385
  • TAT-apoptin is efficiently delivered and induces apoptosis in cancer cells Oncogene 23,

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Abstract

L'apoptine est une protéine dérivé du virus de l'anémie du poulet qui tue spécifiquement des cellules cancéreuses sans toucher aux cellules normales. Nos expériences ont été réalisées dans le but d'étudier les mécanismes d'apoptose tumorale spécifique induite par l'apoptine. Cette connaissance des mécanismes est utilisée par la suite pour concevoir des peptides de l'apoptine plus courts qui seront beaucoup plus efficaces pour induire l'apoptose tout en conservant leur toxicité spécifique vis-à-vis du cancer.
PCT/CA2007/000681 2006-04-21 2007-04-23 Méthodes pour induire l'apoptose de cellules cancéreuses au moyen de dérivés de l'apoptine Ceased WO2007121576A1 (fr)

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EP07719608A EP2013228A1 (fr) 2006-04-21 2007-04-23 Méthodes pour induire l'apoptose de cellules cancéreuses au moyen de dérivés de l'apoptine
CA002648558A CA2648558A1 (fr) 2006-04-21 2007-04-23 Methodes pour induire l'apoptose de cellules cancereuses au moyen de derives de l'apoptine

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002032954A2 (fr) * 2000-10-20 2002-04-25 Leadd B.V. Modifications de l'apoptine$m(3)
WO2003089467A1 (fr) * 2002-04-19 2003-10-30 Leadd B.V. Fragments d'apoptine
US20060057652A1 (en) * 2004-08-13 2006-03-16 Michael Green Methods for identifying therapeutic agents and for treating disease

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002032954A2 (fr) * 2000-10-20 2002-04-25 Leadd B.V. Modifications de l'apoptine$m(3)
WO2003089467A1 (fr) * 2002-04-19 2003-10-30 Leadd B.V. Fragments d'apoptine
US20060057652A1 (en) * 2004-08-13 2006-03-16 Michael Green Methods for identifying therapeutic agents and for treating disease

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DANEN-VAN OORSCHOT A.A.A.M. ET AL.: "The chicken anemia virus-derived protein apoptin requires activation of caspases for induction of apoptosis in human tumor cells", JOURNAL OF VIROLOGY, vol. 74, August 2000 (2000-08-01), pages 7072 - 7078 *

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