EP2013228A1 - Methods of inducing apoptosis of cancerous cells using apoptin derivatives - Google Patents

Methods of inducing apoptosis of cancerous cells using apoptin derivatives

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Publication number
EP2013228A1
EP2013228A1 EP07719608A EP07719608A EP2013228A1 EP 2013228 A1 EP2013228 A1 EP 2013228A1 EP 07719608 A EP07719608 A EP 07719608A EP 07719608 A EP07719608 A EP 07719608A EP 2013228 A1 EP2013228 A1 EP 2013228A1
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Prior art keywords
apoptin
akt
cells
cdk2
seq
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German (de)
French (fr)
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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
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    • 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

This application relates to methods of inducing apoptosis in a cancerous cell, which comprise administering isolated or purified apoptin- derived peptides comprising the amino acids sequence PKPPSK.

Description

Molecular Dissection of the Role of the PI3-Kinase/Akt Pathway; Redirection from Pro- Survival Signaling to a Death Pathway through Nuclear Relocation of Akt.
PRIOR APPLICATION INFORMATION This application claims the benefit of US Provisional Patent Application
60/793,654, filed April 21 , 2006
BACKGROUND OF THE INVENTION
The Phosphatidylinositol 3'-kιnase (PI3-K) is a lipid kinase that catalyses phosphorylation of the Inositol ring of phosphoinositides [Pl, PI(4)P and Pl(4,5) P2] 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 (α, β, γ and δ), and three mammalian genes encode the adapter subunits p85α, p85β and p55γ The p85 subunit contains an N-terminal SH3 domain followed by a proline- rich domain, a breakpoint cluster region homology domain, a second proline-rich domain and two SH2 domains It also has a putative coiled coil domain between the two SH2 domains (ιSH2 domains) that mediates the stable dimeπzation with the p1 10 catalytic subunit The p1 10 subunit contains a closely-spaced N-terminal binding site for p85 and a c-terminal kinase domain [4, 5] Regulation of activity of p85/p1 10 PI3-K is a complex process In general, p85 inhibits p110-assocιated catalytic activity The binding of the N- terminal SH2 domain of p85 with the phosphorylated tyrosine residues within the specific docking sites (YxxM) in the cellular domain of receptor tyrosine kinases [6 7] or in other intracellular non-receptor tyrosine kinase adaptors, leads to a conformational change in p85 which alleviates p1 10 inhibition and further activates PI3-K [8-1 1 ] Interaction of the nSH2 domain with the tyrosine-phosphorylated proteins is universally accepted to be responsible for PI3-K activation, but there are other studies reported the activation of PI3- K via alternate mechanisms For example, the conformational switch within the p85-p1 10 holoenzyme can also occur via the interactions of SH3 domain/prohne rich sequences, BCR-homology domain/GTP loaded adaptor proteins and others [12-15] Akt/PKB, a serine/threonine kinase, is a crucial kinase downstream of the PDK1 recruitment to the Ptdlns(3 4 5)P3 lipid messenger produced via the PI3-K activation [16] Activated Akt modulates the function of numerous substrates related to the regulation of cell proliferation, such as glycogen syntase kιnase-3 (GSK-3), Cyclin dependent kinase inhibitors, P21 Cip1/Waf1 , p27kιp1 , and mammalian target of rapamycin (mTOR) [17, 18] Another important function of activated PI3-K/Akt in cells is maintaining the cell survival via inhibition of apoptosis The targets for Akt during this process include Bad phosphorylation (a pro-apoptotic Bcl-2 family member), FKHRL1 inactivation (a transcription factor for FasL and Bim) and NF-κB activation [19, 20] Several molecules in the literature have been assigned a dual role in both cell survival and cell death mechanisms For instance the oncogenes Myc [21 , 22], NF-κB [23], molecules of Ras/Map kinase pathway [24, 25], Bcl-2 [26, 27], caspases [28], and even an orphan nuclear receptor Nur77 [29] are all involved in promoting either cell proliferation or cell death, depending on the context and the stimulus So far, the PI3-K/Akt pathway has been implicated only in the promotion of cell survival, proliferation, growth, transcription and translation (reviewed in [30]) The specific role of the PI3-K/Akt pathway in a pro-cell death pathway has thus far not been clarified
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 threonιne-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 might provide clues about its tumor specific toxicity These pathways may also represent attractive targets for the development of new highly selective anticancer drugs
In this study, we have investigated the role of the PI3-K/Akt pathway in apoptin- induced cell death Our results intriguingly demonstrate that apoptin interacts with the regulatory p85pl3 K subunit, which leads to the constitutive activation of PI3-K, inhibition of this activation process severely impairs apoptin-induced cell death We also demonstrate nuclear translocation of Akt during apoptin-induced cell death, apparently through an apoptin-mediated piggy-back effect, and further, show the cell cycle regulators p27kιp1 and CDK2 to be nuclear Akt targets during the apoptotic process Nuclear translocation of Akt alone does not have a toxic effect, but it strongly potentiates the toxicity of apoptin and also some anticancer drugs Finally, we have identified CDK2 as the previously unidentified kinase phosphorylating apoptin, which regulates its tumor cell-specific nuclear localization
SUMMARY OF THE INVENTION According to a first aspect of the invention there is provided 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)
According to a second aspect of the invention, there is provided 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
According to a third aspect of the invention, there is provided a purified or isolated peptide comprising amino acids PKPPSK (SEQ ID NO 3)
BRIEF DESCRIPTION OF THE DRAWINGS
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 (p85pl3 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-precipitation indicates the interaction of apoptin with p85 and Akt PC-3 cell lysates were immuno- precipitated with anti-GFP antibody (GFP-apoptin) at different time-points upon transfection with GFP-apoptin Co-immuno-precipitated p85 and Akt were detected by Western blot Reciprocal immuno-precipitation with either antι-p85- or anti-Akt antibodies and detection of apoptin by immunoblotting is shown at the bottom (E) Schematic representation of apoptin deletion mutants tagged with N-terminal GFP The deletion mutants were transfected into PC-3 cells, immuno-precipitated with anti-GFP antibody after 18 hours post transfection and the presence (association) of p85 detected by immunoblotting (F) The percentage of cell death induced by different apoptin mutants assessed by flow cytometry (Nicoletti method) 24 hours post-transfection (G) Schematic representation of the PI3-K p85 deletion mutants highlighting the structural domains The wild type and p85ΔiSH2 were not tagged, with the remaining four mutants possessing a HA tag The p85 deletion mutants were co-transfected together with apoptin into PC-3 cells 28 hours post-transfection, cells were lysed, and p85 immuno-precipitated using either antι-p85 antibody (the wt, and ΔιSH2 mutants) or antι-HA antibody for other mutants Apoptin interaction with the mutants was detected by immunoblotting using anti- apoptin antibodies and the expression of deletion mutants detected by antι-p85 or antι-HA antibodies Figure 2: PI3-K is constitutively activated during apoptin-induced apoptosis.
(A) The 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) (B) 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 details) Cells were lysed 24 hours later, and PI3-K forms immuno-precipitated as described in the legend to figure 1 G Kinase activity was then measured by ELISA as per (A) above Similar results were obtained with MCF7 cells (E) The effect of PI3-K inhibition on apoptin-induced cell death was assessed by flow cytometry (Nicoletti) The cells were either transfected to express GFP (control) or GFP-apoptin in the absence of presence of treatment with wortmannin or LY294002, or transfection to coexpress the dominant negative PI3-K PI3-K-DN Apoptosis was then measured 24 and 48 hours post transfection with results compared to those for control treatments of wortmannin or LY294002 alone or PI3-K-DN Expression alone (F) Cells were either transfected with p85 siRNA plasmid (Upstate) or control siRNA plasmid for 48 hours and p85 expression in the presence or absence of siRNA detected by immunoblotting, tubulin was used a loading control (top panel) PC-3 cells were transfected to express apoptin alone or transfected with or without p85-ιnhιbιtory siRNA, followed by transfection to after 48 hours, to express apoptin After a further 24- or 48 hours, cell death was measured by flow cytometry (Nicoletti) (G) The localization of GFP-apoptin in both MCF-7 and PC-3 cells was detected by confocal microscopy after transfection in the absence or presence of wortmannin (H) The effect of p85 inhibition on apoptin localization was determined by confocal microscopy, 24 hours upon transfection of cells to express GFP-apoptin either without or with co-transfected p85-targettιng siRNA plasmid The expression of p85 was detected by antι-p85 antibody followed by Cy-3 conjugated secondary antibody (I) The nuclear/cytoplasmic distribution of apoptin in the presence of wortmannin or p85-ιnhιbιtory siRNA in both MCF-7 and PC-3 cells was investigated by sub-cellular fractionation followed by Western blotting
Figure 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 express apoptin either alone or together with PDK1 -DN, Akt-DN (adenoviral vector), wild type PTEN, or C124S-PTEN (D) The localization of Akt either in the absence or presence of apoptin in MCF-7 and PC-3 cells was detected by confocal microscopy, after immuno-staining with anti-Akt antibody followed by Cy3 conjugated secondary antibody DAPI was used as counter-stain for the nuclei, and the images overlaid (right panels) (E) The MCF-7 cells were transfected to express the indicated apoptin derivatives (see Fig 1 G for details), and Akt immuno- detected as per D (F) The cells were infected with either adenoviral Myc-tagged NLS-Akt alone or co-transfected to express apoptin and the localization of NLS-Akt was detected using anti-Myc-tag antibody followed by Cy3-marked secondary antibody The arrows indicate representative cells undergoing apoptosis that show chromatin condensation (G) Quantitative assessment of apoptin-tπggered apoptosis in NLS-Akt expressing cells as compared to controls Cells were infected with adenovirus encoding NLS-Akt, and/or transfected to express GFP-apoptin Apoptotic cell death was detected by flow cytometry (Nicoletti) 24 or 48 hours after transfection (H) MCF-7 and PC-3 cells were infected with adenovirus coding for NLS-Akt 2 hours later, cells were treated with anticancer drugs (methotrexate 10 μM, docetaxel 50 nM, doxorubicin 50 μg/ml, cisplatin 75 μg/ml, staurosporine 2 5 μM, antι-CD95 50 ng/ml), and 24 hours later apoptosis was assessed by flow cytometry (Nicoletti) Data represent the average of three independent experiments Figure 4: Akt phosphorylates- and mediates p27kιp1 downregulation in the nucleus. (A) Phosphorylation of p27kιp1 at serine and threonine residues either in the presence of GFP control plasmid or GFP-apoptin was detected by specific phospho-seπne or phospho-Thr-specific antibodies after immuno-precipitation using a antι-p27kιp1 antibody Threonine phosphorylation was also detected using antibody against p27kιp1 phosphorylated at Thr-157 (B) p27kιp1 phosphorylation after transfection to express apoptin at different time points after transfection was detected by immunoblotting using antι-phospho-p27kιp1-Thr-157 antibody, with tubulin as a loading control (C) The effect of PI3-K inhibition on p27kιp1 phosphorylation at Thr-157 was detected by immunoblotting 18 hours after transfecting the cells to express apoptin with or without treatment with wortmannin, or PD98059 (upper panel) The effect of PI3-K, PDK1 , Akt and PKC on p27k p1 phosphorylation was also determined by immunoblotting Cells were transfected to express apoptin alone or together with either 'wild type' PI3-K, constitutively active PDK1 (CA), dominant-negative PI3-K (DN), PDK1-DN, Akt-DN and PKC-DN After 16 hours, cells were lysed and phosphorylated p27kιp1 detected by Western blotting (D) Co-ιmmuno- precipitation of Akt and p27kιp1 PC-3 cells were transfected with apoptin and 12 hours later Akt was immuno-precipitated The precipitates were then resolved by SDS-PAGE and p27kιp1 detected using a specific antibody The association between Akt and p27k p1 could be detected over 12-24 hours after transfection to express apoptin Similar results were observed in MCF-7 cells (E) The levels of p27kιp1 in GFP-apoptin transfected PC-3 cells were followed within 24 hours post-transfection by Western blotting, with tubulin as a loading control A control experiment with cells transfected to express GFP showed no decrease of p27kιp1 in the cells (not shown) (F) The effect of selected kinases on p27kιp1 protein levels, in the presence of apoptin p27kip1 was detected by immunoblotting in lysates of PC-3 cells 24 hours after transfection to express GFP (Control), or GFP-apoptin either alone or together with PI3-K-DN, PDK1-DN, Akt-DN and PKC-DN Where indicated, cells were treated with the kinase inhibitors PD98059, wortmannin, or MG-1 15 Similar results were also obtained in MCF-7 cells (G) The effect of the inhibition of p27kιp1 expression on apoptin-tπggered apoptosis PC-3 cells were transfected with either p27kιp1 siRNA or scrambled (control) siRNA for 36 hours and the p27kιp1 expression was controlled by immunoblotting PC-3 cells were then transfected to express GFP (control) or GFP-apoptin (AP), or transfected with p27k p1-sιRNA alone, with or without GFP-apoptin encoding plasmid Apoptosis was measured by flow cytometry (Nicoletti) at the indicated time points The data represent results from three independent experiments, with similar experimental outcome obtained using MCF-7 cells Figure 5: CDK2 is activated during apoptin-induced cell death. (A) CDK2, CDK1 , Cyclin E and Cyclin A immuno-precipitated from the cells transfected to express GFP or GFP-apoptin 24 hours post-transfection, were used in an in vitro kinase assay on Histone H1 substrate The levels of Histone H1 phosphorylation were detected by immunoblotting with an antibody against the phosphorylated Histone (B) The kinase activity of CDK1 and CDK2 at different times of post-transfection to express GFP or GFP- apoptin as indicated was measured as per (A) and the kinase activity, determined by quantifying the immuno-blot signals against the non-transfected control, plotted (C) PC-3 cells were transfected to express apoptin either alone or together with the indicated dominant negative (DN) kinases, treated without or with PI3-K inhibitors as indicated, and CDK2 activity measured as in (A) Total CDK2 levels (bottom) are indicated by immunoblotting (D) The effect of CDK2 inhibitor on apoptin toxicity was determined by transfecting cells to express apoptin with or without treatment with CDK2 inhibitor Cell death was measured by flow cytometry (Nicoletti method) after 24 and 48 hours respectively, with results compared to those for cells treated with CDK2 inhibitor alone (E) PC-3 cells were transfected with either CDK2 siRNA plasmid or control siRNA for 48 hours and CDK2 expression determined by immunoblotting The level of apoptosis in cells transfected to express GFP (control) or GFP-apoptin in the absence or presence of CDK2- targetting siRNA at 24 and 48 hours was determined by flow cytometry (F) CDK2 kinase activity was measured 24 hours after transfection to express apoptin either without or with treatment with CDK2 inhibitor, caspase inhibitor zVAD-fmk or Bcl2-over-expressιon prior to transfection (G) The levels of cytosolic cytochrome c were detected by cellular fractionation followed by immunoblotting with cells transfected to express apoptin without or with treatment with CDK2 inhibitor or Bcl2 over-expression Indicated samples were lipofected with Bcl2-expressιon vector 24 hours before apoptin transfection (H) The nuclear or cytoplasmic localization of CDK2 was detected by cellular fractionation followed by Western blotting at different time-points after apoptin transfection (I) PC-3 cells were transfected with apoptin, and 20 hours later CDK2 was immuno-precipitated The precipitates were then resolved on SDS-PAGE, and Bcl2 was detected in the immune complexes a by specific antibody (J) The phosphorylation levels of Bcl2 were detected, at indicated time-points after transfection with apoptin, by immunoblotting of Bcl2- preacipitates, with anti-phospho-Ser, anti-phospho-Thr, and antι-Bcl2-phopho-Thr-56 specific antibodies The total Bcl2 levels were detected by immunoblotting with antι-Bcl2 antibody (K) PC-3 cells were either transfected with apoptin alone, co-transfected with apoptin PI3-K siRNA, and/or CDK2 siRNA or pre-treated with a proteosome inhibitor MG- 1 15 20 hours later, the level of Bcl2 phosphorylation at Thr-56 residue and the total Bcl2 were detected by immunoblotting with antι-Bcl2-phospho-Thr-56 antibody and antι-Bcl2 antibody respectively
Figure 6: CDK2 is the tumor specific apoptin kinase. (A) In vitro kinase assay performed with GST-Apoptin and TAT-Apoptin as substrates using active CDK1/Cyclιn B1 CDK2/Cyclιn E or CDK2/Cyclιn 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 GFP- apoptin, in the presence or absence of CDK2 inhibitor or CDK2-targettιng siRNA and GFP-apoptin was immuno-precipitated 24 hours later using anti-GFP antibodies Apoptin phosphorylation in the immuno-precipitates was detected by anti-phospho-Thr-Pro antibodies and immunoblotting Total apoptin and CDK2 levels are indicated (D) The effect of CDK2 inhibition on apoptin localization in PC-3 and MCF-7 cells Cells were transfected to express GFP-apoptin in the presence or absence of CDK2-targettιng siRNA plasmid followed by confocal imaging CDK2 expression was detected using antι-CDK2 antibody and Cy-3 conjugated secondary antibodies (E) The localization of apoptin in the presence of co-expressed active PDK1 (PDK1-CA), PDK1-DN, wild type Akt or Akt-DN in PC-3 cells Confocal imaging was performed 24 hours after transfection (F) The level of phosphorylation of apoptin in PC3 cells in the presence of the indicated co-expressed dominant negative (DN) kinases was determined as per as described in figure 6C Total apoptin was detected with anti-apoptin antibodies Similar results were observed in MCF7 cells
Figure 7: Model for the role of PI3-K/Akt pathway activation during cell death induced by apoptin, and other cell death stimuli. During apoptin-induced cell death, 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 p27kιp1 , 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
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described All publications mentioned hereunder are incorporated herein by reference
Apoptin is a chicken anemia virus derived protein that kills cancer cells specifically without affecting normal cells Our experiments were designed to study the mechanisms of tumor specific apoptosis induced by Apoptin This knowledge of mechanism is subsequently utilized in designing the shorter Apoptin peptides that will be much more effective in inducing apoptosis and at the same time retaining the cancer specific toxicity The amino acid sequence of apoptin is
MNALQEDTPP GPSTVFRPPT SSRPLETPHC RE IRIGIAGI TITLSLCGCA NARAPTLRSA TADNSESTGF KNVPDLRTDQ PKPPSKKRSC DPSEYRVSE L
KESLITTTPS RPRTAKRR IR L (SEQ ID NO 1 )
Described herein is an isolated or purified peptide comprising an amino acid sequence of PX1X2PX3(RZK) (SEQ ID NO 2) wherein X1 X2 and X3 are any ammo acid In alternative embodiments, comprises an isolated or purified peptide consisting of or consisting essentially of an amino acid sequence of PX1X2PX3(RZK) wherein X1 X2 and X3 are any amino acid As will be appreciated by one of skill in the art, as used herein, '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 In an alternative embodiment, the peptide comprises an amino acid sequence of
PKPPSK (amino acids 81 -86 of apoptin (SEQ ID NO 1 ), SEQ ID NO 3) In alternative embodiments, comprises an isolated or purified peptide consisting of or consisting essentially of an amino acid sequence of PKPPSK As will be appreciated by one of skill in the art, as used herein, '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
In an alternative embodiment, the peptide comprises an amino acid sequence of
PKPPSKKR (amino acids 81 -88 of apoptin (SEQ ID NO 1 ), SEQ ID NO 4) In alternative embodiments, comprises an isolated or purified peptide consisting of or consisting essentially of an amino acid sequence of PKPPSKKR As will be appreciated by one of skill in the art, as used herein, '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
In a yet further embodiment, 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
In some embodiments, the apoptin-deπved peptide is a recombinant peptide and comprises an amino acid sequence of at least one of SEQ ID NO 2-8 As will be appreciated by one of skill in the art, as used herein, 'recombinant' is distinct from 'isolated or purified1 and refers to the amino acid sequence in question being presented in a non- native context That is, 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 ) That is 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 In some embodiments, the apoptin-deπved peptide consists of or consists essentially of a peptide of approximately 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 65 70, 75, 80 85, 90, 95, 100 or more amino acids comprising SEQ ID NO 2 or SEQ ID NO 3 as discussed herein As discussed herein, in some embodiments, there is provided the proviso that the apoptin-deπved peptide is not native apoptin (SEQ ID NO 1 ) In some embodiments, the apoptin-deπved peptide consists of or consists essentially of a peptide of approximately 6-100, 7-100, 8-100, 9-100, 10-100, 15-100, 20- 100, 25-100, 30-100, 35-100, 40-100, 45-100, 50-100, 55-100, 60-100, 65-100, 70-100, 75-100 80-100, 85-100, 90-100, 95-100, 100 or more ammo acids comprising SEQ ID NO 2 or SEQ ID NO 3, as discussed herein As discussed herein, in some embodiments, there is provided the proviso that the apoptin-deπved peptide is not native apoptin (SEQ ID NO 1 )
In a further embodiment of the invention, an apoptin-deπved 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 As will be appreciated by one of skill in the art, these methods involve administering an effective amount of an apoptin-deπved peptide as described above to an individual in need of such treatment As will be appreciated by one of skill in the art, 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 In some embodiments 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
In a further embodiment of the invention, there is provided a method for manufacturing a pharmaceutical composition comprising mixing a purified or isolated apoptin-deπved peptide as described above with a suitable excipient As discussed herein, 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
We have shown that Apoptin interacts with the SH3 domain of the p85 regulatory subunit of PI3-Kιnase which is normally a survival molecule and directs the PI3-kιnase pathway towards cell death away from the cell survival signaling The interaction of Apoptin with the SH3 domain of PI3-Kιnase occurs via the amino acids 80-90 in apoptin, which we later identified as a proline rich motif Further based on the knowledge of this Apoptin and PI3-kιnase interaction, we designed the shorter version of the Apoptin peptides which has the intact proline rich motif and thus can kills the cancer cells specifically via interacting with the PI3-Kιnase and possibly other SH3 domain containing proteins The novelty in the project lies in the fact that the Apoptin derived peptides, which are significantly smaller (as small as 6-10 amino acids) compared to full length Apoptin (121 amino acids) effectively induces cell death in a cancer specific manner and the mechanism utilized by both full Apoptin and its peptide deπvates is based on this novel pathway where the apoptosis inducing molecules redirect the survival/proliferation pathways towards cell death pathways Apoptin induced redirection of PI3-K pathway is based on the translocation of Akt, a downstream molecule of PI3-kιnase, to the nucleus where it phosphorylates different substrates like CDK2 and p27, which might be different from the survival signaling that occurs normally in the cytoplasm
In this study, we have investigated for the first time the role of several signaling molecules involved in cancer-selective cell death triggered by apoptin Interestingly, our results show that the PI3-K/Akt signaling pathway, universally accepted to promote cell survival and proliferation, can also have a critical role in promoting cell death in response to certain stimuli Moreover, we demonstrate that the inhibition of PI3-K activation by either pharmacological inhibitors or by genetic methods can abrogate cell death induced by the viral protein apoptin It is of note that the treatment of cancers using PI3-kιnase inhibitors has been largely unsuccessful Our strategy is novel because it causes the nuclear transfer of Akt and subsequently accesses a new set of substrates, and the possibility to use the same pro-survival pathways towards killing the cancer cells, as described herein Although PI3-K is known to be involved in cell survival, several publications have hinted at the fact that active PI3-K may contribute to apoptosis under certain conditions [43-45] lntriguingly in this context, Akt-inhibitors have proved to be only moderately successful in experimental cancer therapy [46, 47] Here, we provide, for the first time, the molecular basis for the role of the PI3-K/Akt pathway in the induction of apoptosis and its propagation (summarized in Fig 7) The PI3-K/Akt pathway, under normal conditions, targets several substrates either in the cytosol or in the nucleus, and promotes cell survival and proliferation This is achieved by various mechanisms, including the control of the abundance, activity and stability of certain regulators of the cell cycle (reviewed in [16, 30]) We document here for the first time that activated Akt, if translocated to the nucleus, can stimulate rather than protect against apoptosis induced by certain cytotoxic stimuli, including several anticancer drugs We hypothesize that in the presence of apoptin, activated Akt targets substrates and/or pathways that lead to the aberrant activation of CDK2, resulting in perturbation of cell cycle progression, and ultimately cell death Thus, the net-outcome of CDK2-actιvatιon could vary according to the "signaling context ' the type of stimuli and the temporal characteristic of signals that they trigger (e g transient versus constitutive signaling) There are well-established examples of such context- dependent, dramatic changes in the final outcome of activation of certain signaling pathways For example, the proto-oncogene c-myc stimulates cell proliferation in the presence of the appropriate survival stimuli (including an activated PI3-K/Akt-pathway) and triggers apoptosis in their absence [48] This dual capacity ensures that cell growth is restricted to the correct paracrine environment, co-activation of a pro-survival signaling pathway, and/or co-expression of anti-apoptotic molecules, and is thereby strictly controlled by multiple mechanisms [49-51]
We have demonstrated that the interaction of apoptin with the p85 regulatory subunit constitutively activates PI3-K The interaction of different molecules with the SH3 domain of p85 is known to change the conformation and activate its p1 10 catalytic subunit, thus leading to the constitutive PI3-K activation Interestingly, a detailed analysis of the sequence of apoptin revealed a proline-rich motif between ammo acids 80 and 90 The PKPPSK (SEQ ID NO 3) sequence (amino acids 81-86) in fact constitutes a perfect consensus motif (PxxPxR/K)(SEQ ID NO 2) recognized by SH3 domains Derivatives of apoptin lacking these residues are severely impaired in inducing apoptosis, implying that apoptin by interacting with the SH3 domain of the p85 regulatory subunit, activates PI3-K constitutively
In addition to apoptin's interaction with PI3-K, we observed transient interaction with Akt Furthermore, interaction of Akt with apoptin appears to facilitate Akt nuclear localization Recent studies have indicated that under certain conditions, activated Akt can be translocated to the nucleus with the help of certain cytosolic proteins, since Akt itself does not possess an intrinsic nuclear localization signal sequence [52, 53] Here, we show that Akt nuclear translocation occurs early during apoptin-induced apoptosis Furthermore, interaction of activated Akt with apoptin is crucial for its nuclear translocation, as demonstrated by the fact that Akt is translocated to the nucleus only in the presence of apoptin mutants which have the ability to activate PI3-K, and that Akt is observed in the nucleus only in the presence of apoptin derivatives with the ability to accumulate in the nucleus This implies that apoptin interaction with Akt contributes to its nuclear import, possibly through a piggy-back mechanism and the nuclear transporter importin beta 1 [54] Apoptin thus effects Akt's nuclear translocation, presumably enabling access to pro- apoptotic phosphorylation targets that do not normally come into contact with cytoplasmic Akt Nuclear access of Akt alone is not sufficient to induce apoptosis, since NLS-Akt alone does not induce cell death, but can potentiate apoptin-induced cell death Thus, NLS-Akt potentiated classical cell death stimuli, such as methotrexate, docetaxel, doxorubicin, and cispratin, but not others such as staurosporine or CD95-trιggeπng In fact, NLS-Akt even demonstrated some protective effects with respect to staurosporine- or CD95-ιnduced cell death, implying that nuclear Akt may have pro-survival and proliferation-promoting function, depending on the experimental conditions For example, Trotman and colleagues reported that the PML-tumor suppressor prevents cancer by dephosphorylating and inactivating Akt inside the nucleus [55] (see also below) Other researchers have reported that phosphorylated-, nuclear-, but not cytoplasmic- Akt interacts with Ebp1 , (an inhibitor of CAD-dependent apoptotic DNA-fragmentation), and enhances its anti-apoptotic action independently of Akt-kinase activity in a cell-free experimental system [56] Thus, nuclear Akt contributes to cell death pathways only in the presence of certain apoptotic stimuli There are several reported nuclear targets of Akt, including FOXO3a [57], Nur77
[58] and p2i cιp/waf [59] We have previously shown that Nur77 translocates from the nucleus to mitochondria during apoptin-induced cell death [33] We still have to define the connection between nuclear Akt and translocation of Nur77 to the mitochondria We have also observed the downregulation of p2icιp/waf 24 hours after cell death induction by apoptin although this occurs independently of Akt activation, since Akt inhibition by wortmannin did not affect p21 levels Downregulation of p21 may be the result of cleavage by caspases, as a secondary event, downstream of mitochondrial death pathway activation Here, we report p27kip1 as a nuclear target of Akt-phosphorylation and demonstrate the importance of this signaling event in apoptin-induced cell death p27k p1 , a cyclin dependent kinase inhibitor and a negative regulator of the cell cycle, plays a role both in proliferation and apoptosis, in a stimulus-dependent manner [60, 61] p27k p1 is down regulated during apoptin-induced cell death dependent on Akt activation Akt phosphorylates p27k p1 at Thr-157 in the nucleus and targets it for proteosomal degradation Previously, it was shown that p27kιp1-phosphorylatιon at ThM 87 by CDKs triggers the ubiquitination and degradation of p27kιp1 by SCF/Skp2 ubiquitin ligase complexes [62] We found no change in the levels of phospho-p27kιp1-Thr-187 during apoptin treatment, although CDK2 was activated during apoptin-induced cell death Also it was reported that p27kιp1 phosphorylation at SeM 0, Thr-157 and ThM 98 results from activation of the PI3-K/Akt pathway The phospohorylation of p27kιp1 at these sites affects its nuclear localization, resulting in the cytoplasmic accumulation with the help of the 14-3- 3 scaffold protein, thus blocking p27kιp1's ability to inhibit CDKs [63 64] However, our data (not shown) indicate that there is no change in the nuclear localization of p27kιp1 during apoptin-induced cell death Akt is also known to control expression of p27kιp1 at the mRNA level via inactivation of a FOXO3a transcription factor [65] Though, we have observed the phosphorylation and inhibition of FOXO3a by Akt during apoptin-induced cell death, we have not observed changes in p27kιp1 mRNA levels Thus, taken together, Akt mediated phosphorylation of p27kιp1 at Thr-157 only affects the stability of the protein through the proteosome pathway, thereby relieving downstream effector molecules from p27kιp1- mediated inhibition
As a consequence of p27kιp1 downregulation, cyclin A-associated CDK2 is constitutively activated by apoptin Though CDK2 is a crucial player in the ce.l cycle during the progression from G1 to S phase [66], it is also known to regulate apoptosis [67, 68] We show here that CDK2 inactivation by either pharmacological inhibitors or by siRNA, severely impairs apoptin-induced cell death Also, we observed that the levels of CDK2 protein did not change with apoptin expression (Fig 5C), whereas the levels of 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] Furthermore, we demonstrated that cyclin A/CDK2 also regulates apoptin's nuclear localization by direct phosphorylation of ThM 08 (Fig 6A-D) Phosphorylation at ThM 08 has previously been reported to be specific for tumor cells, and required for apoptin induced cell death [37, 38] Interestingly, it has been shown previously that, the phosphorylation level of apoptin is severely impaired by the apoptin mutant lacking residues 80-90, even if residues around the phosphorylation site (ThM 08) are intact [38] The results here explain this interesting observation in that we show that apoptin interacts with PI3-K via ammo acids 80-90, and that this interaction is required for downstream CDK2 activation In contrast to previous results, the Ala-108 apoptin mutant is still toxic for cancer cells under our experimental conditions, suggesting that the Thr-108 phosphorylation is of a secondary importance, and a consequence of CDK2 activation during apoptin-induced cell death, rather than a triggering event directly involved in apoptin's toxicity Our results, however, still confirm that the Thr-108 phosphorylation is critical for apoptin nuclear accumulation Nevertheless, aberrant CDK2 activation is clearly crucial and absolutely necessary for apoptin-induced cell death The targets for CDK2 during apoptosis are still unknown, although p53 has been reported to be activated downstream of CDK2 [67], we and others, have ruled out the role of p53 in apoptin- induced cell death [33 39 69] We do show here, however, that upon cytoplasmic translocation CDK2 phosphorylates Bcl2, and targets it for proteosome-dependent degradation This, in turn, affects the balance between anti- and pro-apoptotic Bcl2-famιly members to promote apoptosis Thus, although CDK2 activation downstream of Akt is important it may not be the sole mechanism for the pro-apoptotic role of the PI3-K/Akt pathway in apoptin-induced cell death We predict that some additional Akt-targets, either in the nucleus or in the cytoplasm, exist that are phosphorylated during the course of apoptin-tπggered cell death Thus, Thr108 phosphorylation is important only for keeping the translocated protein in the nucleus but not necessary for nuclear import itself The nuclear import is mediated by the nuclear localization sequence (NLS) located around the ammo acids 80-90 Thus the smaller peptide still can go to the nucleus via its NLS and doesn't require 108-p as it can't be exported back because it lacks the nuclear export sequence (NES) reported in the full length apoptin
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 lead to the development of a novel class of anti-tumor drugs The invention will now be described by way of examples However, the examples do not necessarily limit the invention Apoptin selectively kills cancer cells relaying on the PI3-K pathway
We firstly reassessed the cancer cell-selective toxicity of apoptin, a controversial question in apoptin biology, using peripheral blood lymphocytes (PBLs) from Chronic Lymphocytic Leukemia (CLL) patients compared to the peripheral blood lymphocytes from a normal healthy individual B-cells in CLL and normal PBLs were detected by double staining for the specific surface markers CD5 and CD19 In normal PBLs, there was no significant difference in the B-cell population before (6 1 %) or after (5 2%) 48 hours of treatment with TAT-apoptin In contrast, there was a significant decrease in the CLL PBLs upon TAT-apoptin treatment (11 2%) compared to the control (40 1 %) (Fig 1A) Thus, the data indicates that apoptin effectively kills the malignant B-cells from CLL patients, but not the normal counterparts, therefore confirming apoptin's selective toxicity (Fig 1A) TAT- GFP, used as a negative control for the toxicity, had no significant effect on the survival of both normal and malignant cells As a first step to identify cellular targets of apoptin action in the cell, we generated an expression construct encoding glutathione-S-transferase (GST) fused to the apoptin N- terminus GST-apoptin and control GST were used in a pull-down assay with cell extracts derived from MCF-7 breast cancer cells (Fig 1 B) Mass spectrometric analysis of proteins specifically bound to apoptin identified the two major components of the PI3-K/Akt pathway the p85 regulatory subunit of PI3-K, and the serine/threonine kinase Akt, a kinase downstream of PI3-K We also found that apoptin interacts with proteins such as chaperones, actin and tubulin family members as observed previously [39] We confirmed the mass spectrometric data by detecting p85 and Akt by Western blot ιr. the samples from the GST-apoptin pull-down assay (Fig 1 C), as well as using total cell extracts from PC-3 prostate cancer cells, L929 mouse fibrosarcoma cells, and 293 transformed human embryonic kidney cells The interaction of apoptin with PI3-K and Akt was also examined in vivo, whereby PC-3 cells were transfected with GFP-apoptin, and total cell extracts immuno-precipitated with anti-GFP antibody at different time points post transfection, with the composition of the immune complexes was analyzed for PI3-K and Akt by immunoblotting In a separate series of experiments, PI3-K (p85), and Akt were immuno- precipitated, and apoptin was detected by Western blot Figure 1 D shows that PI3-K and Akt both interact with apoptin confirming the data from Figure 1 B The interaction of apoptin with PI3-K could be detected 4-6 hours after apoptin transfection, thus preceding apoptin induced cell death which is initiated at least 18-24 hours later The above experiment indicates that PI3-K interaction with apoptin is a very early event in apoptin induced cell death We consistently observed strong interaction of PI3-K with apoptin within 6-48 hours post-traπsfection, whereas interaction of apoptin with Akt seems weaker and is seen only at certain time-points upon apoptin treatment
Apoptin interacts with the SH3 domain of the p85 regulatory subunit via its proline- rich sequence
To identify and map the sites on apoptin responsible for interaction with PI3-K PC- 3 cells were transfected to express either full-length GFP-apoptin, or various deletion mutant derivatives of apoptin tagged with an N-terminal GFP as listed in Figure 1 E Apoptin was immuno-precipitated with anti-GFP antibodies 24 hours post transfection and the immune complexes were analyzed for the interaction with PI3-K by immunoblotting using antι-p85 antibodies PI3-K was found in the immuno-precipitates of full-length apoptin and apoptin derivatives that harbored amino acids from 74-100 (a proline-rich region, see above), implying that this region of apoptin is important for interaction with PI3- K (Fig 1 E) We then tested if interaction of PI3-K with apoptin is essential for apoptin- induced cell death by assessing the percentage of apoptosis induced by the different mutants In agreement with the interaction data, only those mutants that had the intact interaction site for PI3-K were able to induce apoptosis (Fig 1 F) Interestingly, in our model system the mutant Ala-108, which is a non-phosphorylable apoptin stated to be non-toxic to the cells in previous reports [38], was still able to induce significant apoptosis as long as its interaction site with the PI3-K was intact
Next, we investigated the apoptin interaction site on PI3-K by co-transfecting with a vector encoding full-length apoptin, together with full length PI3-K or various deletion derivatives thereof Some 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 ιSH2 domain were immuno-precipitated by antι-p85 antibody, while other mutants were immuno-precipitated using antι-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
To determine the functional significance of apoptin's interaction with the p85 regulatory subunit, we measured PI3-K activity using a non-radioactive ELISA-based method Surprisingly, MCF-7 and PC-3 cells transfected to express GFP-Apoptin revealed constitutive activation of PI3-K in apoptin-transfected cells (Fig 2A) 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 In a control experiment, various PI3-K inhibitors prevented apoptin-tπggered generation of PIP3 (Fig 2B), whilst co-transfection of apoptin with a dominant negative PI3-K vector reduced apoptin-induced PI3-K activation to basal levels (Fig 2B) We then tested if the activation of PI3-K is a direct consequence of apoptin's interaction by monitoring PI3-K activity in cells transfected to express different apoptin deletion derivatives As expected, the full-length apoptin induced six-fold PI3-K activation, while the apoptin 1-89 derivative that harbors only the partial PI3-K interaction site, was much less active The apoptin derivatives 1 -1 11 , 74-121 , Ala-108 and Glu-108 were all able to activate PI3-K to an extent comparable to full-length apoptin, whereas the 1-73 and 104-121 derivatives were unable to activate PI3-K This observation implies that the intact p85 interaction site on apoptin is crucial for the activation of PI3-K (Fig 2C) On the other hand, PI3-K activity in cells co-transfected to express full-length apoptin and p85 deletion mutants confirmed that the interaction of apoptin with the SH3-domaιn of p85 is essential for its activity The p85 derivatives that retain both SH3 and ιSH2 domains have constitutive PI3-K activity in the presence of apoptin, whereas others lacking these domains are severely impaired in their activity (Fig 1 D) Interestingly, the mutant N+l+C SH2 having only the SH2 domains but lacking the SH3 domain have relatively higher PI3- K activity compared to other deletion mutants and to the control, indicating that the intact SH3 domain in the wild-type p85 protein might have an inhibitory action on its activity Thus the interaction of apoptin with the SH3 domain appears to elicit a conformational change in the p85 protein, converting it from an inhibitory- to an active state
In order to investigate the direct effect of PI3-K inhibition on apoptin induced cell death, we pre-treated the cells with the PI3-K inhibitors, wortmannin and LY294002, 30 minutes before transfection to express apoptin and cell death assayed 24 and 48 hours later To our surprise, both inhibitors afforded significant protection against apoptin- mduced cell death, despite generally being known to enhance the cell death process through inhibition of PI3-K activity This effect was further confirmed by co-transfection of cells to express apoptin together with a dominant-negative derivative of PI3-K, which significantly protected cells from apoptin-induced cell death (Fig 2E)
To support the data from the inhibitor and transfection experiments, we used siRNA to test whether knocking down endogenous PI3-K would affect resistance to apoptin-induced cell death In PC-3 cells, the expression of p85 was completely abrogated by p85-specιfιc siRNA but not by the control siRNA (Fig 2F), as assayed approximately 30 hours post-transfection The assessment of cell death revealed that cells that lack p85 expression were strongly resistant towards apoptin induced cell death compared to control siRNA-expressing cells (Fig 2F) Similar observations were also made in the MCF-7 and 293 cell types, consistent with the idea that the effect is not cell type specific
Apart from the negative effect of PI3-K inhibition on apoptin induced cell death, we also tested if 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 residue in apoptin is required for the differential localization of the protein PI3-K is mainly a lipid kinase, though some protein kinase activity has been reported, the possibility that PI3-K might directly phosphorylate apoptin was firstly excluded in in vitro kinase assays (data not shown) It thus seems likely that one of the downstream effectors of the PI3-K pathway phosphorylates apoptin and controls its localization, but in a PI3-K dependent manner (see below)
Akt translocates to the nucleus during apoptin induced cell death
We next investigated the effect of apoptin on downstream targets of the PI3-K pathway by transfecting the PC-3 cells to express apoptin, and assessing Akt activation by Western blotting at different time-points post-transfection In agreement with the results for PI3-K activation, increased levels of activated Akt were seen around 6-12 hours post- transfection, with pronounced levels of phosphorylated Akt detectable even 24 hours post- transfection (Fig 3A) The activation of Akt is downstream of-, and dependent on PI3-K activation pretreatment of cells with the inhibitors wortmannin or LY294002, or co- transfection of apoptin-expressing cells with constructs encoding PI3-K- or PDK1- dominant-negative mutants (PI3-K-DN, PDK1 -DN) impaired Akt activation Also, over- expression of wild type PTEN, a negative regulator of Akt activation, severely reduced apoptin-induced Akt activation, whereas over-expression of a phophatase deficient PTEN mutant had no effect (Fig 3B) To further confirm the role of PI3-K in apoptin-induced cell death and to validate the role of Akt in this process, we transiently over-expressed a PDK1 -DN, and/or the dominant negative Akt (Akt-DN) using adenoviral vectors Both dominant-negative kinase mutants as well as over-expression of wild type PTEN significantly protected against apoptin-induced cell death, thus confirming the key role of the PI3-K/Akt pathway in this process (Fig 3C) Akt is generally regarded as a survival-, or proliferation-promoting kinase and not at all a pro-apoptotic molecule However, in the presence of apoptin, Akt is clearly acting as a pro-cell death molecule as its inhibition severely inhibits the cell death pathways triggered by apoptin Next, we asked if apoptin redirects Akt to different cellular targets by modulating its sub-cellular localization As shown in Figure 3D Akt is mainly localized in the cytoplasm of both PC-3 and MCF-7 cells in the absence of apoptin expression, but is clearly nuclear in its presence Analysis of the kinetics of Akt nuclear translocation revealed that most of the cytoplasmic Akt translocates to the nucleus within 12 hours of transfection to express apoptin (data not shown) Furthermore, to determine the effect of different apoptin mutants on Akt translocation and test if Akt nuclear translocation depends on apoptιn/PI3-K interaction, we investigated the localization of Akt in cells transfected to express different apoptin deletion mutants As shown in figure 3E, Akt translocates to the nucleus only in cells expressing apoptin mutants 1 -121 , 1 -89, 1-1 1 1 74-121 and the Glu-108 mutant, all which are nuclear localizing, and have an intact PI3-K interaction site Cells expressing the apoptin mutants 1-73 and 104-121 did not exhibit Akt nuclear translocation This is in perfect accordance with their ability both to interact with PI3-K and induce apoptosis (Fig 2C) In the presence of the Ala-108 mutant, Akt translocates to the nucleus together with apoptin around 18 hours post transfection, but later at 24 hours traces of Akt were observed in the cytoplasm along with apoptin This is in accordance with previously-published data [37] Apoptin mutant T108A is still able to enter the nucleus, and facilitate the nuclear translocation of Akt but it is deficient in nuclear accumulation This clearly implies that apoptin might be acting as a carrier molecule for Akt, directly mediating its nuclear transport via a "piggy back" interaction
To determine if nuclear Akt alone is sufficient to induce cell death even in the absence of an apoptotic stimulus, we infected PC-3 cells with an NLS-Akt adenoviral vector in the absence and presence of apoptin expression As shown in figure 3F, nuclear Akt alone was unable to induce apoptosis whereas in the presence of apoptin it significantly enhanced the cell death Quantitative analysis, using flow cytometry to determine the extent of cell death (Fig 3G) indicated that NLS-Akt exhibits some residual toxicity (13%) compared to the negative (wild type) control (7%) Co-expression of NLS- Akt together with apoptin sensitized the cells towards apoptin-induced cell death Importantly, NLS-Akt also sensitized the cells towards several other clinically relevant apoptotic stimuli, such as methotrexate, docetaxel, cisplatin, and doxorubicin, but not staurosporine or CD95-trιggeπng (Fig 3H) Together, the data indicates that nuclear Akt alone is not sufficient for cell death induction, but can clearly facilitate cell death in the presence of certain apoptotic stimuli
Akt phosphorylates and downregulates p27kιp1 in the nucleus As most of the Akt is localized to the nucleus during apoptin induced cell death, we next investigated the role of p27k p1 , a cell cycle inhibitor, as a potential nuclear target of Akt activity p27kιp1 was immuno-precipitated from the lysates of untransfected cells and cells transfected to express apoptin and phosphorylation was monitored using anti- phospho-seπne or anti-phospho-threonine antibodies p27kιp1 threonine phosphorylation levels increased in the presence of apoptin, but apoptin had no effect on serine phosphorylation (Fig 4A) We confirmed the phosphorylation of p27kιp1 at threonine residues by using an antibody against phosphorylated ThM 57 (a potential Akt consensus site) Analysis of the kinetics of phosphorylation of p27kιp1 at Thr-157 revealed that it occurs soon after Akt nuclear translocation A significant increase in the phophorylation at Thr-157 of p27kip1 is seen 12 hours after transfection to express apoptin and diminishes after 24-30 hours (Fig 4B) The increase in threonine phosphorylation is dependent on Akt activation, as treatment of cells with wortmannin but not with the MAP kinase inhibitor, PD98059, abolished p27k p1 phosphorylation even in the presence of apoptin (Fig 4C) The decreased p27kιp1 phosphorylation levels after 24 hours may be due to the decrease in the total p27kip1 protein levels (see below) Phosphorylation of p27kιp1 at Thr-157 was revealed to be strongly dependent on Akt activation, which is further dependent on PI3-K and PDK1 upstream activation The co-transfection of cells to express apoptin together with dominant negative PI3-K, PDK1 , or Akt mutant derivatives significantly decreased the phosphorylation at Thr-157 of p27kιp1 In contrast, phosphorylation of the Thr-157 residue was not affected by co-transfection to express a dominant negative form of the unrelated kinase PKC (Fig 4C) Furthermore, we determined that phosphorylation of p27kιp1 at Thr- 157 is directly mediated by Akt, by examining p27kιp1/Akt interaction Co-ιmmuno- precipitation experiments revealed that significant levels of p27kιp1 could be found in the immune complexes, together with Akt, during apoptin-induced cell death (Fig 4D) p27k p1 phosphorylation targets it into the proteosome-dependent degradation pathway To determine the functional significance of p27kιp1 phosphorylation by Akt, we checked the protein levels of p27kιp1 before and after transfection to express apoptin Strikingly, we have found a dramatic decrease in the levels of p27kιp1 in apoptin-expressing cells compared to the GFP control expressing cells (Fig 4E) The kinetics of p27kιp1 downregulation revealed that about 24 hours post-transfection, p27kιp1 is completely downregulated in PC-3 cells While the kinetics were observed to vary somewhat in different cell lines, a similar trend of transient phosphorylation followed by depletion was clearly evident The decrease in the levels of p27k p1 protein is entirely dependent on p27kιp1 phosphorylation by Akt, as the protein levels are restored to the control levels in apoptin expressing cells that have been pre-treated with wortmannin but not with PD98059 (Fig 4F) The restoration of p27kιp1 levels was also seen upon co-expression of PI3-K-DN, PDK1-DN or Akt-DN but not with PKC-DN That the decrease in the protein levels of p27kιp1 was due to proteosome-dependent degradation was indicated by the fact that treatment of cells with the proteosome inhibitor MG-1 15 restored the p27kιp1 levels even in the presence of apoptin expression (Fig 4F) The levels of p27kιp1 mRNA did not change upon apoptin treatment, thus indicating that the decrease in the p27kιp1 protein levels was entirely dependent on Akt-mediated phosphorylation enhancing degradation of the protein via the proteosome pathway
To investigate the role of p27kιp1 downregulation during apoptin-induced cell death, we used a siRNA-based approach p27kιp1 expression was depleted upon transfection to express p27kιp1-specιfιc but not control siRNA (Fig 4G) Furthermore, transfection to express apoptin of the p27kip1-defιcιent cells revealed that p27kιp1 downregulation sensitizes the cells to apoptin-induced cell death, as shown in Figure 4G, the percentage of apoptin induced cell death in PC-3 cells, determined by flow cytometry after propidium iodide staining clearly showed a significant increase upon p27kιp1-ιnhιbιtιon as compared to apoptin alone
CDK2/cyclin A activity is elevated and is required during apoptin-induced cell death
To investigate the role of CDKs downstream of p27k'p1 downregulation during apoptin s pro-apoptotic signaling, we measured the activation of two major cyclin- dependent kinases, CDK1 and CDK2, using an in vitro kinase assay and the substrate histone H 1 PC-3 cells were transfected to express either GFP or GFP-apoptin and the CDK activity was measured 24 hours post transfection by immuno-precipitating using CDK1 or CDK2 specific antibodies In the presence of apoptin expression, CDK2 but not CDK1 activity was elevated as indicated by the H1 phosphorylation activity (Fig 5A) To determine if the increased CDK2 activity during apoptin-induced cell death was associated with cyclin E or A, immuno-precipitation was performed using specific antibodies, either in the presence of over-expressed GFP or GFP-apoptin Figure 5A, shows that only cyclin A- but not cyclin E-associated CDK2 phosphorylated histone H1 , suggesting tnat only cyclin A/CDK2 has a role during apoptin-induced cell death CDK2 activity was detectable at 16 hours and peaked at 24 hours post transfection (Fig 5B) We also tested if this increase in CDK2 activity during apoptin-induced apoptosis is dependent on the upstream PI3-K/Akt activation Figure 5C shows that CDK2 is activated only when the PI3-K/Akt pathway is active, inhibition of Akt activation by wortmannin, Ly294002, PI3-K-DN, PDK1-DN or Akt- DN significantly decreased apoptin-induced CDK2 activation
To test if CDK2 activity is required for apoptin-induced cell death, we used two different approaches The first was to inhibit CDK2 activity using a CDK2-specιfιc inhibitor Roscovitine Figure 5D shows that in the presence-, as compared to the absence of CDK2 inhibitor, PC-3 cells were significantly resistant to apoptin-induced cell death The background levels of cell death seen with the combination of CDK2 inhibitor and apoptin expression may be attributed to the CDK2 inhibitor itself, as the inhibitor itself is slightly toxic The second approach was to knock down the expression of CDK2 by transfection with a plasmid encoding a CDK2 specific siRNA and test its effect on apoptin induced cell death As shown in Figure 5E, CDK2 expression in PC-3 cells was downregulated by CDK2-ιnhιbιtory siRNA 48 hours after transfection, with the CDK2-negatιve cells concomitantly highly resistant to apoptin-induced cell death We have previously shown that apoptin's death signaling converges at the mitochondrial death pathway [33] Here, we further investigated whether CDK2 activation is upstream, or if it is a consequence of mitochondrial death pathway activation As shown in Figure 5F CDK2 activation is unaffected either by Bcl-2 over-expression or by caspase inhibition using the inhibitor zVAD-fmk, but the prevention of CDK2 activation by CDK2 inhibitor completely blocked release of cytochrome c from the mitochondria to the cytosol (Fig 5G), indicating that CDK2 activation is upstream of the mitochondrial death pathway
Sub-cellular localization of CDK2 determines whether it drives either cell proliferation or cell death [40] 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) We tested the functional significance of the cytoplasmic CDK2 by investigating its substrates in the cytoplasm by co-immuno-precipitation, whereby Bcl2 was detected in the CDK2 immune complexes in the presence of apoptin (Fig 5I) Bcl2-phosphorylatιon 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 but not serine residues was observed The increased phosphorylation of Bcl2 appears to occur specifically at Thr-56 (Fig 5J), significant changes in phosphorylation of Bcl2 at Ser-70, Thr-74 and Ser-87 were not observed either in the presence or absence of apoptin expression Bcl2 phosphorylation results in a dramatic decrease of Bcl2 protein (Fig 5J), apparently due to proteosome- mediated degradation, since treatment with the proteosome inhibitor MG-1 15 restored Bcl2 levels (Fig 5K) Both Bcl2 phosphorylation and the decrease in total protein was found to be dependent on CDK2 activity, since downregulation of CDK2-expressιon by siRNA prior to transfection to express apoptin resulted in a significant decrease in Bcl2 phosphorylation and little change to protein levels observed That Bcl2 phosphorylation by CDK2 is dependent on upstream activation of the PI3-K/Akt pathway, was indicated by the fact that depletion of PI3-K-expressιon using a specific siRNA showed an effect on Bcl2 similar to that of the downregulation of CDK2-expressιon Thus, PI3-K/Akt mediated activation of CDK2 leads to the Bcl2-degradatιon, with the low Bcl2 level altering the balance between the pro- and anti-apoptotic Bcl2 family members in favor of pro-apoptotic molecules and ultimately activating the mitochondrial death pathway
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] To test whether CDK2 may directly phosphorylate apoptin, we performed an in vitro kinase assay using recombinant GST-apoptin and TAT-apoptin as substrates As shown in Figure 6A, detection of phosphorylated apoptin using a phospho-threonine-proline specific antibody revealed that apoptin can be phosphorylated by active, recombinant CDK2/cyclιn A Interestingly, active, recombinant CDK2/Cyclιn E was not able to phosphorylate apoptin in vitro, nor was CDK1/Cyclιn B, although both were able to phosphorylate Histone H1 in vitro We further confirmed the CDK2 phosphorylation of apoptin using immuno-precipitated CDK2, CDK1 , cyclin A cyclin E, and cyclin B, with inactive CDK2 (CDK2 T160A) as a neηative control Apoptin was phosphorylated only by the immune complex of active CDK2 and cyclin A, but not by other combinations of CDKs and cyclins (Fig 6B) This data correlates with the activation of cyclin A-associated CDK2, and not other CDKs, in the presence of apoptin (see above)
We next investigated, if activated CDK2 phosphorylates apoptin in vivo at Thr-108 by two different approaches First, we inhibited CDK2 activity in PC-3 cells using a CDK2- specific inhibitor and transfected the cells to express apoptin 24 hours later, apoptin was immuno-precipitated and its phosphorylation-status assessed using the phospho-specific Thr-Pro antibody Figure 6C shows that apoptin is phosphorylated in the presence of active CDK2, with the level of phosphorylation significantly reduced in the presence of the CDK2 inhibitor Roscovitine Secondly, we tested the effect on apoptin phosphorylation of inhibiting the expression of CDK2 using siRNA Figure 6C shows that apoptin phosphorylation was severely abrogated by reduced CDK2 expression in the cells, implying that CDK2 is the apoptin kinase We next investigated the role of CDK2 in controlling apoptin's nuclear localization by inhibiting CDK2 expression in both PC-3 and MCF-7 cells using siRNA Figure 6D shows that in the presence of CDK2, apoptin is exclusively in the nucleus, but knock down of CDK2 by siRNA severely impaired apoptin nuclear accumulation in both cell lines This suggests strongly that apoptin-activated CDK2 is the apoptin kinase controlling apoptin nuclear accumulation in cancer cells Furthermore, upstream activation of PI3-K/Akt is necessary for CDK2 kinase activity towards apoptin, as co-transfection of a PDK1 dominant negative mutant, or Akt-DN, severely reduces apoptin nuclear localization (Fig 6E) Co-expression of apoptin with active PDK1 or Akt facilitated rather than prevented apoptin nuclear accumulation Also phosphorylation of apoptin by CDK2 is reduced in the presence of PDK1-DN or Akt-DN but not upon co-expression of PKC-DN (Fig 6F)
Materials and Methods Cell culture and reagents
MCF-7 PC-3, 293 and L929 cells were grown in RPMI-1640 medium supplemented with 10% FBS (Hyclone), 100μg/ml penicillin and 0 1 μg/ml streptomycin (Gibco BRL) The cells were grown at 37°C with 5% CO2 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 antι-PI3-K (p85), anti-mouse IgG-HRP, anti-rabbit IgG-HRP (all from Upstate Cell Signaling), goat anti-Akt, rabbit antι-p27K p1 , murine anti-tubulin, rabbit anti-GFP, rabbit antι-phospho-p27kιp1-Thr-187, rabbit antι-CDK2, antι-CDK1 , anti-Cyclin E, anti-Cychn A, murine anti-cytochrome c, anti-goat IgG-HRP (all from Santacruz Biotechnologies), murine anti-phospho-Akt Ser-473, Mouse anti-phoshpho-Thr antibody, murine anti-phospho-Thr- Pro antibody (all from Cell Signaling), anti-human CD5-FITC, CD19-PerCP (BD biosciences), murine antι-HA, murine anti-phospho-Ser antibody anti-goat Cy3, anti-rabbit Cy3, anti-muπne Cy3 (all from Sigma), anti-Myc antibody (Invitrogen), and anti-phospho- p27kιp1-Thr-157 (R&D systems) The following inhibitors were used wortmannin (IC50 5nM), LY294002 (IC50 1 4μM), PD98059 (IC50 2μM), CDK2 specific inhibitor, Roscovitine (Kd 70OnM) (all from Calbiochem), and zVAD-fmk Enzyme System Products (Aurora, OH, USA) B-cell staining and FACS analysis
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 5μg per 106 cells) for 30 minutes at 40C in dark The cells were then washed twice with cold PBS and resuspended in 300μl 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
Protein purification, GST-pull down assay and protein identification
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
Plasmids, transfections and adenoviral infections
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 (K Walsh, [79]) Transfection was performed using Lipofectamine (Invitrogen) according to the manufacturer's recommendations Adenoviral transfections with Akt-dominant-negative mutant vector and nuclear targeted Akt were performed as previously [80]
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 antι-p85 antibodies overnight at 40C 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 MgCI2, 20 mM Tris, pH 7 4, 10 mM NaCI, and 25 μM ATP) for 2 h at room temperature The mixtures were then incubated with phosphatidylmositol (3,4,5) triphosphate (PI(3,4,5)P3) detector for 1 h at room temperature in the dark, and subsequently added to PI(3,4,5)P3-coated microplate wells, and incubated for 30 mm at room temperature in the dark After thorough washing, peroxidase-linked secondary detection reagent was added, and PI(3,4,5)P3 detector protein binding to the plate was assessed by measuring absorbance at 450nm The data for the kinase activity are expressed as fold induction in transfected cells compared with the activity in untreated cells
In-vitro kinase assays
The in-vitro kinase assays using Histone H1 (Upstate), GST-apoptin and TAT- apoptin as substrates was performed as described earlier [57], but in a non-radioactive assay Briefly, 5 ng of the recombinant CDK2/Cyclιn A (New England Biolabs), CDK2/Cyclιn E, CDK1/Cyclιn B (Upstate) or the immuno-precipitated CDK2, CDK1 , Cyclin A, Cyclin B, Cyclin E were used in a kinase reaction with 5μg of Histone H1 , GST-Apoptin or TAT-Apoptin as substrates in the presence of 200μM ATP in a kinase assay buffer (25mM Tris pH 7 5, 5mM β-Glycerophosphate, 2mM DTT, 0 1 mM Na3VO4 and 1 OmM MgCI2) The kinase reaction was performed at 3O0C for 30 minutes and the end-products were resolved by SDS-PAGE (12-15%) and detected by immunoblotting using their respective phospho-specific antibodies
Immuno-precipitation and immunoblotting Cells were washed twice with cold PBS, lysed with ice-cold lysis buffer (50 mM
Tris, pH 7 5, 1 % Nonidet P-40, 150 mM NaCI, 1 mM Na3VO4, 2 mM EGTA, Protease inhibitor cocktail), incubated for 10 mm on ice, and centrifuged for 10 mm at 40C lmmunoprecipitations were performed with the indicated antibodies (Figs 1 , 2, 4 and 5) and the immuno-complexes captured with protein A-agarose beads (Amersham Pharmacia Biotech) After three washing steps with cell lysis buffer, bead-bound proteins were subjected to Western blot analysis as described previously [33]
RNA interference The plasmids coding for PI3-K siRNA (pKD-PI3 Kinase, p85-V3), CDK2 siRNA
(pKD-CDK2-v6) and the negative control siRNA were purchased from Upstate cell signaling The p27kιp1 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
Immunolocalization studies
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 antι-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
Cell fractionation
The cytoplasmic and nuclear fractions were separated by using differential centrifugation exactly as described previously [33]
Apoptotic assays The measurement of apoptosis was performed by the Nicoletti method followed by flow cytometry as previously [33]
While the preferred embodiments of the invention have been described above, it will be recognized and understood that various modifications may be made therein, and the appended claims are intended to cover all such modifications which may fall within the spirit and scope of the invention REFERENCES
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Claims

I 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) 2 The method according to claim 1 wherein the peptide comprises
PKPPSKKR (SEQ ID NO 4)
3 The method according to claim 1 wherein the peptide comprises amino acids 1-11 1 of SEQ ID NO 5
4 The method according to claim 1 wherein the peptide comprises amino acids 1 to 48 of SEQ ID NO 6
5 The method according to claim 1 wherein the peptide comprises amino acids 1 to 38 of SEQ ID NO 7
6 The method according to claim 1 wherein the peptide comprises amino acids 1 to 31 of SEQ ID NO 8 7 The method according to claim 1 with the proviso that the peptide is not native apoptin (SEQ ID NO 1 )
8 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 9 The method according to claim 8 wherein the peptide comprises
PKPPSKKR (SEQ ID NO 4)
10 The method according to claim 8 wherein the peptide comprises amino acids 1-1 1 1 of SEQ ID NO 5
I I The method according to claim 8 wherein the peptide comprises amino acids 1 to 48 of SEQ ID NO 6
12 The method according to claim 8 wherein the peptide comprises amino acids 1 to 38 of SEQ ID NO 7
13 The method according to claim 8 wherein the peptide comprises amino acids 1 to 31 of SEQ ID NO 8 14 The method according to claim 8 with the proviso that the peptide is not native apoptin (SEQ ID NO 1 )
15 A purified or isolated peptide comprising amino acids PKPPSK (SEQ ID NO 3)
16 The peptide according to claim 15 wherein the peptide comprises PKPPSKKR (SEQ ID NO 4)
17 The peptide according to claim 15 wherein the peptide comprises amino acids 1 -111 of SEQ ID NO 5
18 The peptide according to claim 15 wherein the peptide comprises amino acids 1 to 48 of SEQ ID NO 6
19 The peptide according to claim 15 wherein the peptide comprises amino acids 1 to 38 of SEQ ID NO 7
20 The peptide according to claim 15 wherein the peptide comprises amino acids 1 to 31 of SEQ ID NO 8
21 The peptide according to claim 15 with the proviso that the peptide is not native apoptin (SEQ ID NO 1 )
EP07719608A 2006-04-21 2007-04-23 Methods of inducing apoptosis of cancerous cells using apoptin derivatives Withdrawn EP2013228A1 (en)

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EP1199363A1 (en) * 2000-10-20 2002-04-24 Leadd B.V. Phosphorylation modifications of apoptin
WO2003089467A1 (en) * 2002-04-19 2003-10-30 Leadd B.V. Fragments of apoptin
US7566548B2 (en) * 2004-08-13 2009-07-28 University Of Massachusetts Methods for identifying therapeutic agents and for treating disease

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