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WO2006103666A2 - Polypeptides bid isoles, polynucleotides les codant et anticorps diriges contre ces polypeptides, methodes d'utilisation pour induire l'arret du cycle cellulaire ou l'apoptose - Google Patents

Polypeptides bid isoles, polynucleotides les codant et anticorps diriges contre ces polypeptides, methodes d'utilisation pour induire l'arret du cycle cellulaire ou l'apoptose Download PDF

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WO2006103666A2
WO2006103666A2 PCT/IL2006/000393 IL2006000393W WO2006103666A2 WO 2006103666 A2 WO2006103666 A2 WO 2006103666A2 IL 2006000393 W IL2006000393 W IL 2006000393W WO 2006103666 A2 WO2006103666 A2 WO 2006103666A2
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bid
polypeptide
phosphorylated
cells
pikk
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WO2006103666A3 (fr
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Atan Gross
Rachel Sarig
Iris Kamer
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Yeda Research and Development Co Ltd
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Yeda Research and Development Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans

Definitions

  • the present invention relates to double strand break (DSB) - induced ATM phosphorylation of BID which induces cell cycle arrest. More particularly, the present invention relates to BID polypeptides capable of inducing cell cycle arrest which can be used to treat diseases associated with genomic instability as well as to BID polypeptides capable of preventing the ATM-mediated BID phosphorylation which can be used to induce apoptosis and treat cancer.
  • DSB double strand break
  • the genetic material is constantly subjected to DNA damage. Following induction of DNA damage, the cell may activate a survival system that enables repair and continuation of its normal life cycle, or it may activate its apoptotic machinery in the face of extensive or irreparable damage.
  • Cell cycle arrest can occur following various stimuli such as exposure to DNA damaging agents such as radiation [e.g., IR, which induces double strand breaks (DSB), ultra violet (UV), which induces single strand breaks (SSB) and thymine dimers], various chemicals (e.g., chemotherapeutic agents such as cisplatin) which induce SSB or DSB in the DNA and/or following exposure to agents inducing replication stress such as inhibitors of topoisomerase II (Etoposide), mitomycin C and hydroxyurea. While a DSB is not the major DNA lesion, it is certainly among the most harmful.
  • DNA damaging agents such as radiation [e.g., IR, which induces double strand breaks (DSB), ultra violet (UV), which induces single strand breaks (SSB) and thymine dimers]
  • various chemicals e.g., chemotherapeutic agents such as cisplatin
  • Etoposide topoisomerase II
  • mitomycin C mitomycin
  • One of the major responses associated with the cell survival network is the temporary arrest of cell cycle progression, which reflects the activation of cell cycle checkpoints.
  • the best-documented, damage-induced cell cycle checkpoints operate in the Gl /S boundary, and at the S and G2 phases.
  • the very early events that take place at the site of a DNA DSB and precede activation of the response network involve several proteins that are rapidly recruited to the damaged site, act as DSB sensors and convey a damage signal to transducers, which in turn deliver it to numerous downstream effectors.
  • ATM a nuclear serine- threonine protein kinase, which is absent or inactivated in patients with the genomic instability syndrome Ataxia-Telangiectasis (A-T).
  • A-T genomic instability syndrome Ataxia-Telangiectasis
  • Cells from A-T patients exhibit genomic instability, radiosensitivity and defective activation of the entire DSB response, most notably, the cell cycle checkpoints.
  • ATM is a member of a group of conserved large proteins, several of which are protein kinases involved in mediating DNA damage responses.
  • ATM and RAD3-related protein (ATR) share several motifs, among them a domain containing a PI3 -kinase signature, which gives this group the title, "PI3-kinase-related protein kinases” (PIKKs).
  • PIKKs PI3-kinase-related protein kinases
  • lymphoid malignancy of a B- or T-cell origin
  • non-Hodgkin's lymphoma Hodgkin's lymphoma
  • leukemia several forms of leukemia.
  • defects in cellular responses to DSBs leading to genetic instability may be a frequent initiating event of lymphoid tumors.
  • the molecular mechanisms leading to DSB- induced tumor formation is only partially understood.
  • Apoptosis is regulated by proteins which function to promote or inhibit programmed cell death. Apoptosis can be in response to diverse signals such as stimulation by growth factors (e.g., TNF ⁇ and Fas), limb and neural development, neurodegenerative diseases, radiotherapy and chemotherapy as well as environmental conditions which induce DNA damage. Apoptotic processes are usually characterized by uncoupling of mitochondrial oxidation, decreased levels of nicotinamide adenine dinucleotide phosphate [NAD(P)H], release of cytochrome c, activation of caspases, DNA fragmentation and externalization of phosphatidylserine.
  • growth factors e.g., TNF ⁇ and Fas
  • Apoptotic processes are usually characterized by uncoupling of mitochondrial oxidation, decreased levels of nicotinamide adenine dinucleotide phosphate [NAD(P)H], release of cytochrome c, activation of caspases, DNA fragmentation and externalization of phosphati
  • the BCL-2 family is comprised of both pro-apoptotic proteins, such as BAX, as well as anti- apoptotic proteins, such as BCL-2.
  • Most BCL-2 family members share three conserved domains; BHl, BH2, and BH3, of which BH3 functions as a death domain in the pro-apoptotic members of the family.
  • BH3-only proteins i.e., proteins which contain only the BH3 domain out of the three conserved BH domains
  • apoptosis BID BH3 interacting death agonist
  • BID is believed to be relatively inactive in the cytosol until proteolytically cleaved by caspase-8. Cleavage of cytosolic BID at As ⁇ 59 yields a pi 5 C-terminal truncated fragment (tBID) that translocates to the mitochondria, where it activates BAX and BAK, resulting in the release of cytochrome c. Phosphorylation seems to regulate BID activity, since it was recently demonstrated that the phosphorylation of BID by casein kinase 1 and/or 2 inhibits its cleavage by caspase-8. However, the apoptotic pathways in which BID plays a role are not yet fully characterized.
  • BID 7' MEFs were found to be less susceptible than BID +/+ MEFs to the DNA damage reagent adriamycin, as well as to the nucleotide analogue 5-fluorouracil (Sax et al., 2002), suggesting that BID may contribute to the DNA damage response.
  • the mechanisms by which BID is involved in the response to DNA damage have not been identified.
  • an isolated polypeptide comprising a BID amino acid sequence having a mutation in a PIKK phosphorylation site. According to another aspect of the present invention there is provided an isolated polypeptide comprising a BID amino acid sequence having a PIKK phosphorylation site and being capable of inhibiting PIKK-mediated phosphorylation of endogenous BID.
  • an isolated polypeptide comprising a BID amino acid sequence which comprises a phosphorylated PIKK phosphorylation site.
  • an isolated polypeptide comprising a BID amino acid sequence wherein the isolated polypeptide being incapable of inducing apoptosis.
  • an isolated polypeptide comprising a BID amino acid sequence attached to a nuclear targeting moiety.
  • an isolated polynucleotide encoding the polypeptide there is provided an isolated polynucleotide encoding the polypeptide. According to still an additional aspect of the present invention there is provided a transformed cell expressing the polypeptide.
  • a method of inducing cell cycle arrest comprising introducing into-, or expressing in the cell a polypeptide comprising a BID amino acid sequence incapable of inducing apoptosis, thereby inducing cell cycle arrest.
  • a method of treating a disease associated with a genomic instability comprising introducing into-, or expressing in cells of an individual in need thereof a polypeptide comprising a BID amino acid sequence incapable of inducing apoptosis, thereby inducing cell cycle arrest and treating the disease associated with the genomic instability.
  • a method of inducing apoptosis comprising introducing into-, or expressing in a cell a polypeptide comprising a BID amino acid sequence incapable of inducing cell cycle arrest, thereby inducing apoptosis.
  • a method of treating cancer comprising introducing into-, or expressing in cells of an individual in need thereof a polypeptide comprising a BID amino acid sequence incapable of inducing cell cycle arrest, thereby inducing apoptosis and treating the cancer.
  • an antibody comprising an antigen recognition domain capable of specifically binding a BID polypeptide phosphorylated on a serine residue corresponding to amino acid 78 of human BID as set forth in SEQ ID NO: 15 but does not bind the BID polypeptide when not phosphorylated on this respective position.
  • an antibody comprising an antigen recognition domain capable of specifically binding a BID polypeptide phosphorylated on a serine residue corresponding to amino acid 78 of mouse BID as set forth in SEQ ID NO: 16 but does not bind the BID polypeptide when not phosphorylated on this respective position.
  • an antibody comprising an antigen recognition domain capable of specifically binding a BID polypeptide phosphorylated on a serine residue corresponding to amino acid 61 of mouse BID as set forth in SEQ ID NO: 16 but does not bind the BID polypeptide when not phosphorylated on this respective position.
  • kits for detecting a presence and/or level of a PIKK-mediated phosphorylated BID comprising the antibody.
  • a method of detecting cellular exposure to DNA damaging agents comprising detecting in a biological sample a presence and/or level of a PIKK-mediated phosphorylated BID, thereby detecting the cellular exposure to the DNA damaging agents.
  • a kit for detecting cellular exposure to DNA damaging agents comprising at least one reagent capable of detecting a presence and/or level of a PIKK-mediated phosphorylated BID.
  • a method of diagnosing a disease associated with an abnormal PIKK-mediated phosphorylation of BID comprising detecting in cells of an individual in need thereof a presence and/or level of a PIKK-mediated phosphorylated BID, thereby diagnosing the disease associated with the abnormal PIKK-mediated phosphorylation of BID.
  • kits for diagnosing a disease associated with an abnormal PIKK-mediated phosphorylation of BID comprising at least one reagent capable of detecting a presence and/or level of a PIKK-mediated phosphorylated BID.
  • a method of inducing apoptosis comprising contacting a cell with the antibody, thereby inducing apoptosis in the cell.
  • a pharmaceutical composition comprising as an active ingredient the antibody and a pharmaceutically acceptable carrier.
  • a method of identifying an agent capable of inducing cell cycle arrest comprising: (a) contacting a plurality of cells with a plurality of molecules; (b) identifying at least one molecule from the plurality of molecules capable of increasing a level of a phosphorylated BID polypeptide, the at least one molecule being the agent capable of inducing cell cycle arrest.
  • a multicellular organism comprising a genome which comprises a genetically modified BID gene which comprises a mutation in a PIKK phosphorylation site.
  • the mutation abolishes phosphorylation at the phosphorylation site.
  • the mutation mimics phosphorylation at the phosphorylation site.
  • the BID amino acid sequence does not exceed 20 amino acids in length.
  • the polypeptide is capable of inducing apoptosis.
  • the nuclear targeting moiety is an NLS.
  • the BID amino acid sequence is at least 20 amino acids long. According to still further features in the described preferred embodiments the polypeptide is capable of inducing cell cycle arrest.
  • the PIKK phosphorylation site comprises a residue corresponding to a serine residue at position 78 of a BID polypeptide set forth by SEQ ID NO: 15. According to still further features in the described preferred embodiments the
  • PIKK is an ATM.
  • the PIKK is an ATR.
  • the cell cycle arrest is induced following DSB in a DNA.
  • the cell cycle arrest occurs at an S phase.
  • polypeptide is the isolated polypeptide. According to still further features in the described preferred embodiments the isolated polypeptide is as set forth in SEQ ID NOs:20-22.
  • the isolated polypeptide is as set forth in SEQ ID Nos:32-34.
  • the disease associated with genomic instability is associated with an abnormal S phase checkpoint.
  • Ataxia-Telangiectasia Fanconi anemia, Bloom's syndrome, hereditary breast and ovarian cancer syndromes involving BRCAl and Nijmegen breakage syndrome
  • the cancer is of a lymphoid origin.
  • the disease associated with the abnormal PIKK-mediated phosphorylation of BID is selected from the group consisting of Ataxia-Telangiectasia, Fanconi anemia,
  • Bloom's syndrome hereditary breast and ovarian cancer syndromes involving BRCAl and Nijmegen breakage syndrome (NBS 1).
  • the detecting in the cells the presence and/or level of the PIKK-mediated phosphorylated BID comprises using the antibody.
  • the at least one reagent comprises the antibody.
  • the phosphorylated BID polypeptide is present in a cell nucleus.
  • the phosphorylated BID polypeptide being phosphorylated on a PIKK phosphorylation site.
  • the mutation abolishes phosphorylation at the PIKK phosphorylation site.
  • the mutation comprises a substitution of a serine residue with an alanine residue at a position corresponding to amino acid residue 78 of a BID polypeptide set forth by SEQ ID NO: 16.
  • the mutation comprises a substitution of a serine residue with an alanine residue at a position corresponding to amino acid residue 61 of a BID polypeptide set forth by SEQ ID NO: 16.
  • the present invention successfully addresses the shortcomings of the presently known configurations by providing polypeptides, polynucleotides and expression vectors encoding same and antibodies directed thereagainst which can be used to induce cell cycle arrest or apoptosis.
  • all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control.
  • the materials, methods, and examples are illustrative only and not intended to be limiting.
  • FIGs. la-d are graphs depicting the dose-response/death curves of MEFs following treatment with DNA damaging agents.
  • BID +/+ and BID "7" MEFs were subjected to treatment with various doses of etoposide (Etop; 20, 50 and 100 ⁇ M Etop for 24 hours; Figure Ia), cisplatin (Cis; 10, 20 and 50 ⁇ M Cis for 14 hours; Figure Ib), ultraviolet radiation (UV; 5, 10 and 20 J/m 2 for 14 hours; Figure Ic), and ionizing radiation (IR; 20, 50 and 100 Gy for 24 hours; Figure Id) and the effect on cell death was monitored by FACScan using propidium-iodide (PI) dye exclusion.
  • PI propidium-iodide
  • FIGs. 2a-c are graphs depicting the dose-response/death curves of BID +/+ and BID "7" splenocytes ( Figure 2a) or SV40-transformed BID "7” and BID +/+ MEFs ( Figures 2b-c) in response to treatment with Etop ( Figure 2a), UV ( Figure 2b) or IR ( Figure 2c).
  • BID "7” or BID +7+ splenocytes were treated for 40 hours with 0, 5, 10 or 50 ⁇ M Etop; SV40-transformed BID "7” and BID +/+ MEFs were treated for 18 hours with 0, 2.5, 5 or 10 J/m 2 UV radiation or for 24 hours with 10, 20 and 50 Gy IR radiation.
  • FIGs. 3a-c are bar graphs depicting the effect of BID on colony survival following IR treatment ( Figure 3a) or cell death following either Etop ( Figure 3b) or IR ( Figure 3c) treatments.
  • Figure 3a - 1000 cells from BID +/+ and BID " ' " MEFs were seeded per well and irradiated with 1, 3, 5, or 8 Gy of IR. Cells were then incubated for 10 days and the percent of colony survival was calculated as the ratio between the No. of colonies formed after IR treatment and the No. of colonies formed in untreated cells.
  • Asterisk (*) represents significant differences (p ⁇ 0.05) based on student's t- test.
  • BID "A MEFs have increased clonogenic survival compared to BID +/+ cells following DNA damage.
  • Figures 3b-c - BID +/+ or BID "A MEFs were either left untreated (N/T) or treated with either Etop (100 ⁇ M for 24 hours; Figure 3b) or ionizing radiation (IR; 100 Gy for 24 hours; Figure 3c).
  • BID "7" MEFs were infected with recombinant adenoviruses carrying a tetracycline-inducible BID vector (Sarig et al., 2003 JBC 278:10707-10715).
  • FIGs. 4a-e are Western blot analyses demonstrating the effect of DNA damaging agents on BID expression pattern.
  • Figures 4a-b - BID +/+ MEFs were either left untreated (N/T; lane 1), or treated for 1 hour with one of the indicated cell death stimuli: Etop (100 ⁇ M; lane 2), IR (50 Gy; lane 3), Cis (50 ⁇ M; lane 4), UV (20 J/m 2 ; lane 5), Thaps (2 niM; lane 6), TNF ⁇ (40 ng/ml together with 2 ⁇ g/ml actinomycin D; lane 7), and STS (4 ⁇ M; lane 8).
  • Etop and IR induce a double electrophoretic mobility shift in BID;
  • Figure 4c - BID +/+ MEFs were treated for 30 minutes with 100 ⁇ M Etop, lysed and either left untreated (-) (lane 1), or further treated for 30 minutes at 37 °C with alkaline phosphatase (+) (lane 2), followed by Western blot analysis using anti-BID Abs.
  • BID-P marks the BID double electrophoretic mobility shift. Note that alkaline phosphatase treatment abolishes the Etop-induced double electrophoretic mobility shift in BID.
  • Figures 4d-e - HeLa cells were transiently transfected with pcDNAIH- wt-BID and 18 hours post-transfection cells were metabolically labeled with P- orthophosphate, treated for 30 minutes with 100 ⁇ M Etop (+) ( Figure 4d lane 2) or remained untreated (-) ( Figure 4d lane 1), following which radiolabeled BID was immunoprecipitated with anti-BID Abs and evaluated by either autoradiography ( Figure 4d) or by Western blot, using anti-BID Abs ( Figure 4e). Note that exposure to Etop resulted in a marked increase in 32 P-labeling of BID, which appears as a doublet (BID-P);
  • FIGs. 5a-b are Western blot analyses demonstrating that BID phosphorylation is mediated by the ATM kinase.
  • Atm/Arf double knockout MEFs (Atm-/-) (lanes 1-3) and Atm+/+Arf-/- MEFs (Atm+/+) (lanes 4-6) were either left untreated (N/T) (lanes 1 and 4), or treated for 30 minutes with 100 ⁇ M Etop (lanes 2 and 5) or 50 Gy IR (lanes 3 and 6), collected, lysed and equal amounts of protein (20 ⁇ g per lane) were subjected to SDS-PAGE, followed by Western blot analysis using anti-BID Abs (Figure 5a). The blot was stripped and reprobed with anti- ⁇ -actin Abs to control for loading ( Figure 5b). Note that the slower migrating forms of BID (BID-P) do not appear in ATM-deficient MEFs.
  • FIGs. 6a-b are Western blot analyses depicting the phosphorylation of exogenous BID (Figure 6a) or ⁇ -actin ( Figure 6b) in Etop - treated HeLa cells in which the expression of LacZ or ATM were downregulated.
  • Control HeLa cells (lanes 1-2) or stably transfected HeLa cells with LacZ siRNA (LacZ downregulated) (lane 3) or ATM siRNA (ATM downregulated) (lane 4) were transiently transfected with pcDNAIII- wt-BID and 18 hours post-transfection the cells were treated for 30 minutes with 100 ⁇ M Etop (lanes 2, 3 and 4) or remained untreated (lane 1).
  • BID '7" MEFs which were similarly treated with Etop, served as a positive control (lane 5).
  • Cells were collected, lysed and subjected to Western blot analysis using anti-BID Abs (Figure 6a). The blots were stripped and reprobed with anti- ⁇ -actin Abs to control for loading ( Figure 6b). Note the presence of the Etop-induced phosphorylation of exogenous BID in LacZ downregulated cells ( Figure 6a lane 3, marked by BID-P), but not in ATM downregulated HeLa cells ( Figure 6a lane 4).
  • FIGs. 7a-g are a sequence diagram ( Figure 7a) and Western blot analyses ( Figures 7b-d) demonstrating the phosphorylation of BID on Serine 61 and Serine 78 of mouse BID.
  • Figure 7a Sequence alignment of mouse (BID_mouse GenBank Accession No. NMJ307544), human (BID_human; GenBank Accession No. CR_456389) and rat (BID_rat; GenBank Accession No. AF_136282) BID proteins depicting the PIKK consensus sites (SQ/TQ). Note that mouse BID carries two PIKK consensus sites (S61Q and S78Q) whereas human and rat BID carry only one (S78Q).
  • Figures 7b-c - HeLa cells were transiently transfected with pcDNAIII-wt-BID (lanes 3-4), pcDNAIII-BID-S61A (lanes 7-8), pcDNAIII-BID-S78A (lanes 5-6), or left untransfected (-) (lanes 1-2).
  • Eighteen hours post-transfection cells were either left untreated (-) (lanes 1, 3, 5 and 7), or treated for 30 minutes with 100 ⁇ M Etop (+) (lanes 2, 4, 6 and 8), collected, lysed and subjected to Western blot analysis using anti-BID Abs (Figure 7b). The blot was stripped and reprobed with anti- ⁇ -actin Abs to control for loading ( Figure 7c).
  • the BID +/+ MEFs were further treated for 30 minutes at 37 0 C with potato acid phosphatase (PAP; +) (lane 3), lysed and subjected to Western blot analysis as above. The blots were stripped and reprobed with anti- ⁇ -actin Abs to control for loading ( Figures 7e and g).
  • BID-P marks the phosphorylated form of BID. Asterisk marks a cross-reactive band. Note that the phospho-specific antibodies to serine 61 and serine 78 recognize endogenous BID in MEFs treated with Etop ( Figures 7d and f, lane 2) which is eliminated following treatment with potato acid phosphatase ( Figures 7d and f, lane 3).
  • FIGs. Sa-d are Western blot analyses depicting the specificity of the anti-pS61 and anti-pS78 antibodies to phosphorylated BID.
  • HeLa cells were transiently transfected with pcDN AIII- wt-B ID (lanes 1-2), pcDNAIH-BID-S61A (lanes 3-4 in Figures Sa-b) or pcDNAIII-BID-S78A (lanes 3-4 in Figures 8c-d).
  • the anti-S61 Abs recognize phosphorylated BID (BID-P) only in HeLa cells transfected with WT BID ( Figure 8a, lane 2) but not in HeLa cells transfecetd with the S61A mutant BID ( Figure 8a, lane 4).
  • the anti-S78 Abs recognize phosphorylated species of BID (BID-P) in HeLa cells transfecetd with WT BID ( Figure 8c, lane 2) but not in HeLa cells transfected with S78A mutant BID ( Figure 8c, lane 4).
  • the anti-pS78 Abs also recognize the lower of the three bands in HeLa cells that were not treated with Etop, indicating a basal level of phosphorylation in healthy cells.
  • FIGs. 9a-e are Western blot analyses depicting the in vitro phosphorylation of BID by ATM and ATR.
  • 293T cells were transiently transfected with empty Flag vector ( Figures 9a-c; lane 1), Flag-tagged ATM ( Figures 9a-c - lane 2; Figures 9d-e - lane 1), Flag-tagged ATR ( Figures 9a-c - lane 4; Figures 9d-e - lane 2), Flag-tagged kinase-inactive (KI) ATM ( Figures 9a-c; lane 3), Flag-tagged KI ATR ( Figures 9a-c; lane 5) and the cells overexpressing the Flagged vectors were immunoprecipitated with anti-Flag M2 Abs (Sigma Cat.
  • FIGs. 10a-d are Western blot analyses depicting Etoposide - induced phosphorylation of BID on both S61 and S78 in mouse splenocytes. Splenocytes purified from either BID +/ * (lanes 1 and 2) or BID '; ⁇ (lane 3) mice were either left untreated (-) (lane 1), or treated for 40 minutes with 50 ⁇ M Etop (lanes 2 and 3) and lysed.
  • Lysates from equal amounts of cells were subjected to SDS-PAGE followed by Western blot analysis using anti-BID Abs (Figure 10a), anti-pS61 ( Figure 10b), or anti-pS78 (Figure 10c) Abs.
  • the blots were stripped and reprobed with anti- ⁇ -actin Abs to control for loading ( Figure 1Od). Note the specific S61 ( Figure 10b, lane 2) and S78 (Figure 10c, lane 2) phosphorylated BID species following Etop treatment.
  • FIGs. 1 la-c are Western blot analyses depicting the phosphorylation of human BID on S78 in response to Etop treatment.
  • HEK293 cells were either left untreated (-)
  • FIGs. 12a-c are Western blot analyses depicting the specificity of the anti- pS78 Abs to Human wt-BID, but not human BID-S78A mutant.
  • HeLa cells were transiently transfected with pcDNAIII-wt-BID (lanes 3 and 6), ⁇ cDNAIII-BID-S78A (lanes 2 and 5) or remained untransfected (lanes 1 and 4).
  • FIGs. 13a-d are Western blot analyses depicting the time course of Etop - induced phosphorylation of endogenous mouse BID on S61 and S78.
  • BID +/+ MEFs were either left untreated (N/T) (lane 1), or treated with 100 ⁇ M Etop, collected at the indicated time points: 15 minutes (lane 2), 30 minutes (lane 3), 1 hour (lane 4), 2 hours (lane 5), 3 hours (lane 6), 5 hours (lane 7), 7 hours (lane 8) and 9 hours (lane 9), lysed, and equal amounts of protein (20 ⁇ g per lane) were subjected to SDS-PAGE followed by Western blot analysis using either anti-BID ( Figure 13a), anti-pS61 ( Figure 13b), or anti-pS78 ( Figure 13c) Abs. The blot was stripped and reprobed with anti- ⁇ -actin Abs to control for loading (Figure 13d).
  • FIG. 14 is a bar graph depicting the time course of Etop-induced apoptosis of
  • FIGs. 15a-d are Western blot analyses depicting that endogenous mouse BID is phosphorylated on S61 and on S78 in an ATM-dependent manner.
  • Atm/Arf double knockout MEFs (Atm-/-) (lanes 1-2) and Atm+/+Arf-/- MEFs (Atm+/+) (lanes 3-4) were either left untreated (-) (lanes 1 and 3), or treated for 30 minutes with 100 ⁇ M Etop (lanes 2 and 4), collected, lysed and subjected to Western blot analysis using either anti-pS61 ( Figure 15a) or anti-pS78 ( Figure 15c) Abs. The blots were stripped and reprobed with anti- ⁇ -actin Abs to control for loading ( Figures 15b and d). The asterisk marks a cross-reactive band.
  • FIGs. 16a-b are Western blot analyses depicting the phosphorylation of mouse BID on S61 only in response to reagents that induce double-strand breaks in DNA.
  • BID +/+ MEFs were either left untreated (N/T) (lane 1) or treated for 1 hour with the death stimuli: Etop (100 ⁇ M; lane 2), IR (50 Gy; lane 3), Cis (50 ⁇ M; lane 4), UV (20 J/m 2 ; lane 5), Thaps (2 niM; lane 6), TNF ⁇ (40 ng/ml together with 2 ⁇ g/ml actinomycin D; lane 7), and STS (4 ⁇ M; lane 8).
  • FIGs. 17a-b are Western blot analyses depicting the phosphorylation of endogenous human BID on S78.
  • HEK293 cells stably transfected with the LacZ siRNA (LacZ downregulated) (lanes 1-5) or ATM siRNA (ATM downregulated) (lanes 6-10) were either left untreated (N/T) (lanes 1 and 6), or treated for 1 hour with Etop (100 ⁇ M), IR (50 Gy), UV (20 J/m 2 ) or STS (4 ⁇ M).
  • Cells were collected, lysed, and equal amounts of protein (20 ⁇ g per lane) were subjected to SDS-PAGE followed by Western blot analysis using anti-pS78 Abs (Figure 17a). The blots were stripped and reprobed with anti- ⁇ -actin Abs to control for loading ( Figure 17b).
  • FIGs. 18a-h are immunofluorescence analyses depicting the intracellular expression pattern of BID.
  • BID +/+ MEFs Figures 18a, c, e and g
  • BID "A MEFs Figures 18b, d, f, and h grown on glass cover slips, were either left untreated ( Figures 18a-d), or treated for 3 hours with 100 ⁇ M Etop ( Figures 18e-h), fixed and immunostaiiied with anti-BID Abs (green, shown in Figures 18a, b, e and f) followed by DAPI nuclear staining (blue, shown in Figures 18c, d, g and h). Note the positive staining of BID in the cytoplasm and the nucleus of healthy MEFs.
  • FIGs. 19a-d are Western blot analyses depicting the subcellular localization of BID.
  • BID +/+ MEFs were either left untreated (-) (lanes 1-2), or treated with formaldehyde (+) (lanes 3-4), and subfractionated.
  • Aliquots of the cytosolic (C) (lanes 1 and 3) and nuclear (N) (lanes 2 and 4) fractions were subjected to SDS- PAGE followed by Western blot analysis using anti-BID ( Figure 19a), anti-MEK ( Figure 19b), anti-BAX (Figure 19c), and anti-lamin B ( Figure 19d) Abs.
  • the asterisk marks a cross-reactive band that might represent a modified form of BID.
  • FIGs. 20a-c are Western blot analyses depicting the phosphorylation of BID following IR treatment.
  • BID +/+ MEFs were either left untreated (N/T) (lane 1), or treated with 50 Gy IR (lane 2), collected 5 minutes later, and lysed.
  • Equal amounts of protein (20 ⁇ g per lane) were subjected to SDS-PAGE followed by Western blot analysis using either anti-BID Abs ( Figure 20a) or anti-pS61 Abs ( Figure 20b). The blot was stripped and reprobed with anti- ⁇ -actin Abs to control for loading (Figure 20c).
  • FIGs. 21a-c are Western blot analyses depicting the phosphorylation of BID following IR treatment.
  • BID +/+ (lanes 1-5) or BID "7" (lane 6) MEFs were either left untreated (N/T) (lane 1), or treated for 30 minutes with the indicated doses of IR: 0.2 Gy (lane 2), 0.5 Gy (lane 3), 1 Gy (lane 4) or 5 Gy (lanes 5 and 6), collected, lysed, and analyzed as described in Figures 20a-c. Note that phosphorylation of BID occurs in response to extremely low, non-apoptotic levels of IR.
  • FIGs. 22a-i are raw data of flow cj1:ometry analyses ( Figures 22a-f) and quantification of the percentage of cells in the Gl ( Figure 22g), S ( Figure 22li) or G2/M ( Figure 22i) cell cycle phases of MEFs following Etop treatment.
  • BID +/+ or BID "7" MEFs were either left untreated (N/T) or treated for 2 hours with 20 ⁇ M Etop, rinsed, and then released into drug-free medium. At the indicated time points the DNA content was analyzed by flow cytometry.
  • Figures 22a-f The actual raw data from a representative experiment together with multi-line plots generated by the ModFit LT computer software program.
  • the data represent the means ⁇ SEM of pooled results from three independent experiments. Filled columns - BID +/+ MEFs; Empty columns - BID "7" MEFs. Note that BID "7" MEFs do not accumulate in the S and G2 phases of the cell cycle following etoposide treatment.
  • FIGs. 23a-c are bar graphs depicting the percentage of DNA synthesis in BID +/+ or BID "7" MEFs following Etop treatment.
  • BID +/+ or BID "7" MEFs were either left untreated (N/T) or treated for 2 hours with 20 ⁇ M Etop, rinsed, and then released into drug-free medium. At the indicated time points following release, the cells were pulse labeled for 30 minutes with BrdU to determine DNA synthesis.
  • Figure 23a - The percentages of BrdU positive cells at eight hours after release as determined in BID +/+ (filled bars) or BID '7" (empty bars). The data represent the means ⁇ SEM of pooled results from three independent experiments.
  • BID "7" MEFs fail to decrease DNA synthesis following Etop treatment.
  • Figures 23b-c The percentages of BrdU positive cells was determined in BID +/+ ( Figure 23b) or BID "7" ( Figure 23c) MEFs at early S (empty bars) or the late S/G2 (filled bars) phases of cell cycles at the indicated time points following drug release (i.e., 0. 4. 6 and 24 hours). The data represent the means ⁇ SEM of pooled results from three independent experiments. Note that BID "7" MEFs are not delayed in their progression from S to G2/M following Etop treatment.
  • FIGs. 24a-c are Western blot analyses of BID "7" MEFs stably expressing the - S61A/S78A mutant depicting absence of phosphorylation in response to Etop treatment.
  • BID "7" MEFs stably expressing either wt-BID (lanes 1-2) or the BID- S61A/S78A mutant (lanes 3-4) were treated for 1 hour with 20 ⁇ M Etop (+) (lanes 2 and 4) or remained untreated (-) (lanes 1 and 3), lysed, and equal amounts of protein (20 ⁇ g per lane) were subjected to SDS-PAGE, followed by Western blot analysis using either anti-BID Abs (Figure 24a) or the S61 ( Figure 24b) or S78 ( Figure 24c) phospho-specific Abs.
  • FIGs. 25a-i are the raw data of flow cytometry analyses ( Figures 25a-f) and quantification of the percentages of cells in the Gl ( Figure 25g), S ( Figure 25h) or G2/M ( Figure 25i) phases of cell cycle.
  • BID "7" MEFs stably expressing either wt-BID or BID-S61A/S78A (two clones from each) were treated for 2 hours with 20 ⁇ M Etop, rinsed and then released into drug-free medium. At the indicated time points the DNA content was analyzed by flow cytometry.
  • Figures 25a-f Actual raw data from a representative experiment together with multi-line plots generated by the ModFit LT computer software program.
  • the dark histograms represent the percent of cells in the Gl and G2/M phases and the hatched histograms represent the percent of cells in S phase.
  • FIGs. 26a-c are a bar graph ( Figure 26a) and Western blot analyses ( Figures 26b-c) demonstrating that BID "7" MEFs expressing the BID-S61A/S78A are more susceptible to Etop-induced apoptosis than those expressing WT BID.
  • Figure 26a - BID "7" MEFs stably expressing either wt-BID or BID-S61A/S78A (the four clones shown in Figure 25g-i) were either left untreated (N/T) or treated with Etop (50 ⁇ M; 18 hours), LTV (20 J/m 2 ; 24 hours) or TNF ⁇ .
  • FIGS 26b-c The two wt-BID and two mutant BID clones were treated with either 50 ⁇ M Etop (for 18 hours) or TNF ⁇ /ActD (for 4.5 hours), lysed and equal amounts of protein (20 ⁇ g per lane) were subjected to SDS-PAGE, followed by Western blot analysis using anti-BID Abs (Figure 26b). For the TNF ⁇ treatment, only clones #1 are shown. The blots were stripped and reprobed with anti- ⁇ -actin Abs to control for loading ( Figure 26c). Note that the enhanced death obtained with the BID-S61A/S78A clones is not due to higher expression of mutant BID or to more cleavage to tBID.
  • FIGs. 27a-b are Western blot analyses depicting the effect of whole-animal irradiation on BID phosphorylation.
  • FIGs. 28a-b are Western blot analyses depicting the effect of dose-dependent whole-animal irradiation on BID phosphorylation in the thymus.
  • Seven-week old BID +/+ (lanes 1-10) or BID "7" (lanes 11-12) female mice were either left untreated (-) (lanes 1-2 and 11) or subjected to whole-body ⁇ -radiation [3 Gy (lanes 3-6) or 6 Gy (lanes 7-10, 12)].
  • the mice were sacrificed by cervical dislocation at 1 hour (lanes 3- 4, 7-8) or 4 hours (lanes 5-6, 9-10, 12) after being exposed to the radiation and the thymus was removed, homogenized and analyzed by Western blot as described in Figures 27a-b.
  • BID-P marks the phosphorylated form of BID.
  • the asterisk marks a cross reactive band. Note that phosphorylation of BID in vivo is transient, and it increases in an ionizing radiation dose
  • FIGs. 29a-c are a diagram ( Figure 29a) and Southern blot analyses ( Figures 29b-c) depicting the generation of BIDS61A/S78A knock-in mice by gene targeting.
  • Figure 29a Diagram of the Bid genomic locus, the targeting vector and the homologous recombinant locus. Also indicated are the restriction enzyme cutting sites (X, Xbal; E, EcoRI; H, Hindlll; C, CM; N, Notl; Xh, XJwI) and the position of external probes.
  • Figures 29b-c are Southern blots of one ES clone that was subsequently aggregated with tetraploid embryos using the 5' external probe (probe-L; Figure 29b) and the 3' external probe (probe-S; Figure 29c) demonstrating homologues recombination within the Bid locus.
  • the 5' external probe (probe-L; Figure 29b) recognizes a 10.1-kb EcoRlIXbal fragment for wild-type loci (lane 1) and a 6.8-kb EcoRl/Xbal recombinant fragment for a targeted loci (lane 2).
  • the 3' external probe (probe-S; Figure 29c) recognizes a 9-kb HindlU fragment for wild-type loci (lane 1) and a 6-kb Hind ⁇ l recombinant fragment for a targeted loci (lane 2).
  • FIGs. 30a-b are PCR analyses depicting the excision of the LoxP-Neo-LoxP cassette. Animals depicted are progeny of a BIDS61A/S78A(neo)/+ male crossed to a PGK-Cre transgenic female. EtBr-stained agarose gels of PCR flanking the Neo locus are presented. PCR primers used were: For 5'-
  • FIGs. 31a-b are Western blot analyses depicting initial characterization of BIDS61A/S78A knock-in mice.
  • Heterozygous mutant BIDS61A/S78A/+ animals were mated to obtain offspring homozygous for the mutant BID gene (BIDS61A/S78A).
  • Three-week old BID +/+ , BIDS61A/S78A/+, and BIDS61A/S78A female mice (+/+, +/KI, KI/KI, respectively) were either left untreated (-) (lanes 1-2) or subjected to whole-body ⁇ -radiation (+; 3 Gy; lanes 3-8).
  • mice were sacrificed by cervical dislocation 1 hour after being exposed to the radiation and the indicated organs were removed, immediately homogenized, and equal amounts of protein (50 ⁇ g from each organ) were subjected to SDS-PAGE, followed by Western blot analysis using either anti-pS78 (Figure 31a) or anti-BID ( Figure 31b) Abs. Lanes 1 and 3-5 - thymus; lanes 2 and 6-8 - spleen. BID-P marks the phosphorylated form of BID. * marks a crossreactive band. Note that in the homozygote KI mice BID is expressed but is not phosphorylated following IR treatment.
  • FIGs. 32a-g demonstrate that BID carries a nuclear export signal that is involved in regulating its cellular location.
  • Figure 32a is a sequence alignment of mouse (BID_mouse; GenBank Accession No. NM 007544), human (BIDJtiuman; GenBank Accession No. CR_456389) and rat (BIDjrat; GenBank Accession No. AF_136282) BID proteins depicting that the classical nuclear export signal (NES) is shared between all three BID homologues.
  • the NES consensus motif (shown in the bottom line) contains four closely spaced leucine residues, which can be substituted by other large hydrophobic residues (Isoleucine/Valine).
  • Figures 32b-e are immunofluorescence analyses ( Figures 32b-c) or DAPI nuclear staining ( Figures 32d- e) of HeLa cells transfected with wt-BID ( Figures 32b and d) or NLS-BID-3LA ( Figures 32c and e). HeLa cells were transfected with wt-BID and NLS-BID-3LA and the transfected cells fixed and stained with anti-BID Abs (green) and with DAPI (blue). Note that transfection of HeLa cells with the NLS-BID-3LA resulted in BID staining which is more confined to the nucleus than wt-BID.
  • Figures 32f-g are Western blot analyses depicting BID expression in the cytosolic (C) or nuclear (N) fractions of HeLa cells which were transfected with either wt-BID ( Figure 32f) or NLS-BID-3LA ( Figure 32g). Transfected cells were treated with formaldehyde subfractionated and aliquots of the cytosolic and nuclear fractions were subjected to SDS-PAGE followed by Western blot analysis using anti-BID Abs. Asterisk marks a cross reactive band.
  • FIGs. 33a-c are bar graphs depicting cell cycle analyses of BID ⁇ MEFs which were transfected with either wt-BID or NLS-BID-3LA.
  • BID " ' " MEFs stabfy expressing either wt-BID or NLS-BID-3LA (two clones from each) were either left untreated (N/T), or treated with 10 ⁇ M Etop and following 8 hours the DNA content was analyzed by flow cytometry. The exact percentage of cells in each phase of the cell cycle is shown. The data represent the means ⁇ SEM of pooled results from three independent experiments.
  • FIG. 34 is a bar graph depicting the effect of the "nuclear-trapped" BID mutant (NLS-BID-3LA) on apoptosis.
  • BID "7" MEFs stably expressing either wt-BID or NLS-BID-3LA (the four clones used in Figures 33a-c) were either left untreated (N/T), or treated with Etop (100 ⁇ M; 24 hours). Cell death was monitored by FACScan using PI dye exclusion. The data represent the means ⁇ SEM of pooled results from three independent experiments. Note that the "nuclear-trapped" BID mutants (NLS-BID-3LA) are less susceptible to Etop-induced apoptosis.
  • FIG. 35 is a schematic model depicting the "double agent" role of BID in the
  • BID is an ATM target, which is important for both cell cycle arrest at the S phase and apoptosis
  • BID clearly induces apoptosis at the mitochondria following activation of the TNF/Fas death-receptor pathway, however it remains an open question whether BID plays a similar role at the mitochondria in the DNA damage pathway (dotted arrow).
  • the present invention relates to a PIKK-phosphorylated BID polypeptide which can be used to induce cell cycle arrest.
  • the present invention relates to isolated polypeptides comprising a BID amino acid sequence which can be used to induce cell cycle arrest and treat diseases associated with genomic instability.
  • the present invention discloses isolated polypeptides comprising a BID amino acid sequence capable of inhibiting the PIKK-mediated phosphorylation of endogenous BID which can be used to induce apoptosis and treat cancer.
  • Genomic instability associated diseases such as Ataxia-Telangiectasia (A-T), Fanconi anemia, Bloom's syndrome, hereditary breast and ovarian cancer syndromes involving BRCAl and Nijmegen breakage syndrome (NBSl) are associated with the inability of cells to activate the survival system which enables DNA repair and continuation of normal cell cycle following exposure to DNA damage.
  • A-T Ataxia-Telangiectasia
  • Fanconi anemia Fanconi anemia
  • Bloom's syndrome hereditary breast and ovarian cancer syndromes involving BRCAl and Nijmegen breakage syndrome (NBSl)
  • NBSl Nijmegen breakage syndrome
  • ATM is a member of a group of conserved large proteins, several of which are protein kinases (e.g., ATM and ATR) involved in mediating DNA damage responses and which share a domain containing a PO -kinase signature (PIKK).
  • PIKK protein kinases
  • Activated ATM phosphorylates a wide spectrum of substrates, many of them at the sites of damage.
  • the functional consequences of some of ATM phosphorylation events have been associated with the activation of the cell cycle checkpoints. However, not all of the phenotypic abnormalities in A-T patients can be explained by a lack of these phosphorylation events, implying that additional, yet unidentified, ATM targets exist.
  • Apoptosis is regulated by proteins which function to promote or inhibit programmed cell death such as members of the BCL-2 family (e.g., the pro-apoptotic BAX), as well as anti-apoptotic BCL-2 protein.
  • a subset of the pro-apoptotic molecules comprises the BH3-only proteins including the major regulator of apoptosis BID. Cleavage of cytosolic BID yields a pl5 C-terminal truncated fragment (tBID) that translocates to the mitochondria, where it activates BAX and BAK, resulting in the release of cytochrome c.
  • BID 7 mice have demonstrated that BID is required for Fas-induced apoptosis.
  • BID 7 MEFs were found to be less susceptible than BID + + MEFs to the DNA damage reagent adriamycin and to the nucleotide analogue 5-fluorouracil (Sax et al., 2002), suggesting that BID may contribute to the DNA damage response.
  • BID is a target for phosphorylation by PIKK proteins such as ATM or ATR mediating cell-cycle arrest.
  • PIKK proteins such as ATM or ATR mediating cell-cycle arrest.
  • the inhibition of such phosphorylation induces apoptosis.
  • BID in the cell On one hand, it has a pro-apoptotic role, which is activated by the TNF/Fas death-receptor pathway, and on the other hand, as an ATM target, it is capable of inducing cell cycle arrest following exposure to DNA damaging agents.
  • TNF/Fas death-receptor pathway a pro-apoptotic role
  • ATM target a pro-apoptotic target
  • modulation of one BID activity will affect the level of the other BID activity in the cell (see schematic model in Figure 35).
  • a method of inducing cell cycle arrest is effected by introducing into-, or expressing in the cell a polypeptide comprising a BID amino acid sequence incapable of inducing apoptosis, thereby inducing cell cycle arrest.
  • the phrase "cell cycle arrest” refers to the delay and/or halt of a cell in any stage of the cell cycle resulting in an abnormal accumulation of the cell in that cell cycle stage.
  • the cell cycle includes 4 main stages: the M phase - cell mitosis; the Gl phase - interphase; the S phase - occurs towards the end of interphase and includes DNA synthesis; and the G2 phase - final stage of interphase in which the cell continues to grow and duplicates in preparation for mitosis.
  • the cell cycle arrest according to this aspect of the present invention occurs at the S phase, entrance to S phase, the G2 phase and/or the G2/M phase.
  • inducing cell cycle arrest refers to initiating or upregulating (e.g., increasing) the rate and/or level of cell cycle arrest at a specific phase (e.g., the S phase).
  • Apoptosis refers to a programmed cell death machinery whereby the cell executes a "cell suicide” program. Apoptosis plays a crucial role in ensuring the normal development and maintenance of cells, organs, and tissues and involves in a number of physiological events such as embryogenesis, regulation of the immune system, and homeostasis.
  • a polypeptide comprising a BID amino acid sequence refers to any natural or chemically synthesized polypeptide, peptide or mimetic thereof which comprises at least a portion of a mammalian (e.g., human, mouse) BID protein [e.g., as set forth in GenBank Accession No. P55957 (SEQ ID NO: 15; human BID) or GenBank Accession No. P70444 (SEQ ID NO: 16; mouse BID)].
  • the polypeptide is incapable of inducing apoptosis.
  • Such a polypeptide is selected of an amino acid length and composition (i.e., at least encompassing PIKK phosphorylation site) sufficient to induce cell cycle arrest.
  • BID exhibits a dual activity in the cell. On one hand it exhibits a pro-apoptotic activity ( Figures 1-3), and on the other hand, it is capable of inducing cell cycle arrest following induction of DNA damage ( Figures 22-23).
  • the induction of cell cycle arrest is regulated by phosphorylation of BID on a PIKJv site (e.g., the serine residue at position 78 as set forth by SEQ ID NO: 15; Figures 24-25) and by the export of BID from the nucleus ( Figures 33a-c).
  • PIKJv site e.g., the serine residue at position 78 as set forth by SEQ ID NO: 15; Figures 24-25
  • the polypeptide comprises a phosphorylated PIKK phosphorylation site.
  • PIKK phosphorylation site refers to the conserved motif of serine or threonine residues followed by glutamine residues, a motif commonly named “SQ/TQ” (Kim et al., 1999) and which is found on proteins that are subject to phosphorylation by "PI3-kinase-related protein kinases” (PIKK) proteins.
  • PIKK proteins can be, for example, ATM (GenBank Accession No. NP_000042), ATR (GenBank Accession No. NP_001175) or DNA-PK (GenBank Accession No. NP_008835).
  • a non-limiting example of a phosphorylated polypeptide which comprises the BID amino acid sequence and which can be used according to this aspect of the present invention is the isolated polypeptide set forth by SEQ ID NO: 17.
  • the polypeptide comprises a mutation in the PIKK phosphorylation site, which mimics phosphorylation at that phosphorylation site.
  • Such a mutation can be, for example, a substitution of a serine residue with an aspartic acid residue essentially as described in Germann UA., et al., 1996, The Journal of Biological Chemistry, 271 : 1708-1716.
  • a mutation in the PIKK phosphorylation site can be introduced to the polypeptide using recombinant techniques or solid phase synthesis methods which are well known in the art and further described hereinbelow.
  • a non-limiting example of a polypeptide which comprises a mutation which mimics phosphorylation at the PIKK site and which can be used according to this aspect of the present invention is the isolated polypeptide set forth by SEQ ID NO: 18.
  • a "nuclear trapped" polypeptide e.g., the NLS-BID-3LA polypeptide described in Example 7 of the Examples section which follows
  • the polypeptide of this aspect of the present invention preferably comprises a BID amino acid sequence attached to a nuclear targeting moiety.
  • nuclear targeting moiety refers to a moiety which is capable of directing the polypeptide to the nucleus.
  • the polypeptide can be further subjected to PIKK-mediated phosphorylation (e.g., by endogenous ATM or ATR).
  • PIKK-mediated phosphorylation e.g., by endogenous ATM or ATR.
  • a non-limiting example of such a moiety is a nuclear localization signal (NLS) such as that of the SV40 large-T antigen (Kalderon, D., et al., Nature, 311 : 33-38, 1984) as set forth by SEQ ID NO: 19 (Lys-Lys-Lys-Arg-Lys- Val-Glu).
  • the nuclear targeting moiety can be a protein interaction domain which may mediate signal to an NLS containing protein.
  • the nuclear targeting moiety can be covalently attached to the isolated polypeptide of the present invention using e.g., solid phase synthesis or can be recombinantly expressed along with the isolated polypeptide as is further described hereinbelow.
  • isolated polypeptides which are incapable of inducing apoptosis can be of any length which enables induction of cell cycle arrest and those of skills in the art are capable of determining the length needed for that activity based on, for example, functional assays (e.g., in vitro assays) capable of detecting apoptosis and/or cell cycle arrest essentially as described in the "General Materials and Experimental Methods" and Example 4 of the Examples section which follows.
  • functional assays e.g., in vitro assays
  • the polypeptide which is used by the method of this aspect of the present invention comprises at least 20 amino acids, more preferably, at least 40 amino acids, more preferably, at least 60 amino acids, more preferably, at least 80 amino acids, more preferably, at least 100 amino acids, more preferably, at least 120 amino acids, more preferably, at least 140 amino acids, more preferably, at least 160 amino acids, more preferably, at least 180 amino acids, even more preferably, about 195 amino acids of the BID polypeptide set forth by SEQ ID NO: 15.
  • Non-limiting examples of such polypeptides include for example, polypeptides which comprise a phosphorylated PIKK phosphorylation site and includes 50-195 amino acids of the BID polypeptide set forth by SEQ ID NO: 15 and thus are incapable of inducing apoptosis.
  • polypeptides can be any of the polypeptides set forth by SEQ ID NOs:20-22.
  • polypeptides which comprise a mutation in the PIKK phosphorylation site which mimic phosphorylation at that site and include 50-195 amino acids of the BID polypeptide set forth by SEQ ID NO: 15 and thus are incapable of inducing apoptosis can be also used according to this aspect of the present invention.
  • a polypeptide can be any of the polypeptides set forth by SEQ ID NOs:23-25.
  • unmodified polypeptides including 50-202 amino acids of the BID pol y peptide set forth by SEQ ID NO: 15 and an NLS, and which are incapable of inducing apoptosis can be also used according to this aspect of the present invention.
  • a polypeptide can be any of the polypeptides set forth by SEQ ID NOs:26-28.
  • polypeptide or "peptide” as used herein encompasses native polypeptides (either degradation products, synthetically synthesized polypeptides or recombinant polypeptides), peptidomimetics (typically, synthetically synthesized peptides), as well as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the polypeptides more stable while in a body or more capable of penetrating into cells.
  • Methods of preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, CA.
  • Natural aromatic amino acids, Trp, Tyr and Phe may be substituted for synthetic non-natural acid such as Phenylglycine, TIC, naphthylelanine (NoI), ring- methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr.
  • the polypeptides of the present invention may also include one or more modified amino acids or one or more non-amino acid monomers (e.g. fatty acids, complex carbohydrates etc).
  • amino acid or “amino acids” is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine.
  • amino acid includes both D- and L-amino acids. Tables 1 and 2 below list naturally occurring amino acids (Table 1) and non- conventional or modified amino acids (Table 2) which can be used with the present invention.
  • the polypeptides of the present invention preferably include one or more non- natural or natural polar amino acids, including but not limited to serine and threonine which are capable of increasing polypeptide solubility due to their hydroxyl- containing side chain.
  • polypeptides of the present invention are preferably utilized in a linear form, although it will be appreciated that in cases where cyclicization does not severely interfere with peptide characteristics, cyclic forms of the peptide can also be utilized.
  • the isolated polypeptide of the present invention can be biochemically synthesized using standard solid phase techniques. These methods include exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation and classical solution synthesis. These methods are preferably used when the polypeptide is relatively short such as a small a peptide of a few amino acids (e.g., 3- 20 amino acids) and/or when it cannot be produced by recombinant techniques ⁇ i.e., not encoded by a nucleic acid sequence) and therefore involve different chemistry.
  • standard solid phase techniques include exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation and classical solution synthesis. These methods are preferably used when the polypeptide is relatively short such as a small a peptide of a few amino acids (e.g., 3- 20 amino acids) and/or when it cannot be produced by recombinant techniques ⁇ i.e., not encoded by a nucleic acid sequence) and therefore involve different chemistry.
  • Polypeptide or peptide mimetics include synthetic structures (known and yet unknown), which may or may not contain amino acids and/or peptide bonds, but retain the structural and functional features of a polypeptide ligand. Types of amino acids which can be utilized to generate mimetics are described hereinabove.
  • peptide mimetics also includes peptoids and oligopeptoids, which are peptides or oligomers of N-substituted amino acids [Simon et al. (1972) Proc. Natl. Acad. Sci. USA 89:9367-9371]. Further included as peptide mimetics are peptide libraries, which are collections of peptides designed to be of a given amino acid length and representing all conceivable sequences of amino acids corresponding thereto.
  • peptide mimetics can be effected using various approaches, including, for example, display techniques as well as computational biology techniques.
  • polypeptides of the present invention can be generated using recombinant techniques such as described by Bitter et al., (1987) Methods in Enzymol. 153:516-544, Studier et al. (1990) Methods in Enzymol. 185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsu et al. (1987) EMBO J. 3:17-311,
  • a polynucleotide sequence encoding the polypeptide is preferably ligated into a nucleic acid construct suitable for expression in a host cell.
  • a nucleic acid construct includes a promoter sequence for directing transcription of the polynucleotide sequence in the cell in a constitutive or inducible manner.
  • Constitutive promoters suitable for use with the present invention are promoter sequences which are active under most environmental conditions and most types of cells such as the cytomegalovirus (CMV) and Rous sarcoma virus (RSV).
  • Inducible promoters suitable for use with the present invention include for example the tetracycline-inducible promoter [Zabala M, et al., Cancer Res. 2004, 64(8): 2799- 804].
  • the nucleic acid construct (also referred to herein as an "expression vector") of the present invention includes additional sequences which render this vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors).
  • a typical expression vector may also contain a transcription and translation initiation sequence, enhancers (e.g., SV40 early gene enhancer; see also Enhancers and Eukaryotic Expression, Cold Spring Harbor Press, Cold Spring Harbor, N. Y. 1983), transcription and translation terminator, and a polyadenylation signal which may increase the efficiency of mRNA translation (e.g., the GU or U rich sequences located downstream from the polyadenylation site and a highly conserved sequence of six nucleotides, AAUAAA, located 11-30 nucleotides upstream).
  • enhancers e.g., SV40 early gene enhancer; see also Enhancers and Eukaryotic Expression, Cold Spring Harbor Press, Cold Spring Harbor, N. Y. 1983
  • transcription and translation terminator e.g., the GU or U rich sequences located downstream from the polyadenylation site and a highly conserved sequence of six nucleotides, AAUAAA, located 11-30 nucleotides upstream.
  • the expression vector of the present invention typically includes a signal sequence for secretion.
  • the expression vector of the present invention can further include additional polynucleotide sequences that allow, for example, the translation of several proteins from a single mRNA such as an internal ribosome entry site (IRES) and sequences for genomic integration of the promoter-chimeric polypeptide.
  • IRS internal ribosome entry site
  • the expression construct of the present invention can also include sequences engineered to enhance stability, production, purification, yield or toxicity of the expressed polypeptide.
  • a variety of cells can be used as host-expression systems to express the recombinant polypeptide of the present invention (e.g., the polypeptide set forth by SEQ ID NO: 17 or 18).
  • These include, but are not limited to, microorganisms, such as bacteria transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector containing the polypeptide coding sequence, mammalian expression systems, yeast transformed with recombinant yeast expression vectors containing the coding sequence (see for example, U.S. Pat.
  • Bacterial systems are preferably used to produce recombinant polypeptides since they enable a high production volume at low cost.
  • a number of expression vectors can be advantageously selected depending upon the use intended for the polypeptide expressed. For example, when large quantities of polypeptide are desired, vectors that direct the expression of high levels of the protein product, possibly as a fusion with a hydrophobic signal sequence, which directs the expressed product into the periplasm of the bacteria or the culture medium where the protein product is readily purified may be desired. Certain fusion protein engineered with a specific cleavage site to aid in recovery of the polypeptide may also be desirable. Such vectors adaptable to such manipulation include, but are not limited to, the pET series of E. coli expression vectors [Studier et al., Methods in Enzymol. 185:60-89 (1990)].
  • mammalian expression vectors include, but are not limited to, pcDNA3, pcDN A3.1 (+/-), pGL3, pZeoSV2(+/-), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMTl, pNMT41, pNMTSl, which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which are available from Stratagene, pTRES which is available from Clontech, and their derivatives.
  • Expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses can be also used.
  • SV40 vectors include pSVT7 and pMT2.
  • Vectors derived from bovine papilloma virus include pBV- IMTHA, and vectors derived from Epstein Bar virus include pHEBO, and p2O5.
  • exemplary vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
  • Various methods can be used to introduce the expression vector of the present invention into host cells.
  • nucleic acids by viral infection offers several advantages over other methods such as lipofection and electroporation, since higher transfection efficiency and specificity can be obtained due to the infectious nature of viruses.
  • Transformed cells are cultured under effective conditions, which allow for the expression of high amounts of the recombinant polypeptide.
  • Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production.
  • An effective medium refers to any medium in which a cell is cultured to produce the recombinant polypeptide of the present invention.
  • Such a medium typically includes an aqueous solution having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins.
  • Cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes and petri plates. Culturing can be carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. Such culturing conditions are within the expertise of one of ordinary skill in the art.
  • resultant polypeptides of the present invention may either remain within the cell, secreted into the fermentation medium, secreted into a space between two cellular membranes, such as the periplasmic space in E. coli; or retained on the outer surface of a cell or viral membrane.
  • recovery of the recombinant polypeptide refers to collecting the whole fermentation medium containing the polypeptide and need not imply additional steps of separation or purification.
  • polypeptides of the present invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concaiiavalin A chromatography, cliromatofocusing and differential solubilization.
  • standard protein purification techniques such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concaiiavalin A chromatography, cliromatofocusing and differential solubilization.
  • the expressed coding sequence can be engineered to encode the polypeptide of the present invention and a fused cleavable moiety.
  • a fusion protein can be designed so that the polypeptide can be readily isolated by affinity chromatography; e.g., by immobilization on a column specific for the cleavable moiety.
  • the polypeptide can be released from the chromatographic column by treatment with an appropriate enzyme or agent that specifically cleaves the fusion protein at this site [e.g., see Booth et al., Immunol. Lett. 19:65-70 (1988); and
  • polypeptide of the present invention is preferably retrieved in "substantially pure” form.
  • substantially pure refers to a purity that allows for the effective use of the recombinant polypeptide (i.e., the polypeptide of the present invention) in inducing cell cycle arrest.
  • the polynucleotide encoding the polypeptide of the present invention is administered to the cell-of-interest (e.g., a cell of a subject who suffers from or is predisposed to a genomic instability associated disease) to thereby induce cell cycle arrest.
  • the phrase "ex vivo gene therapy” refers to the process of expressing the polypeptide of the present invention in cell cultures derived from a subject (e.g., autologous or allogeneic cells) followed by administration of such cells (which express the polypeptide of the present invention) back into the subject in need of therapy.
  • the phrase "in vivo gene therapy” refers to the process of expressing the polypeptide of the present invention in cells of the subject in need of therapy.
  • the type of viral vector and the specific promoter used for ex vivo or in vivo gene therapy will depend on the cell type transformed.
  • the ability to select suitable vectors according to the cell type transformed is well within the capabilities of the ordinary skilled artisan and as such no general description of selection consideration is provided herein.
  • Recombinant viral vectors are useful for in vivo expression of recombinant proteins since they offer advantages such as lateral infection and targeting specificity.
  • Lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. The result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. This is in contrast to vertical-type of infection in which the infectious agent spreads only through daughter progeny.
  • Viral vectors can also be produced that are unable to spread laterally. This characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.
  • nucleic acid transfer techniques include transfection with viral or non-viral constructs, such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems.
  • viral or non-viral constructs such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems.
  • Useful lipids for lipid-mediated transfer of the gene are, for example, DOTMA, DOPE, and DC-Choi [Tonkinson et al., Cancer Investigation, 14(1): 54-65 (1996)].
  • the most preferred constructs for use in gene therapy are viruses, most preferably adenoviruses, AAV, Antiviruses, or retroviruses.
  • a viral construct such as a retroviral construct includes at least one transcriptional promoter/enhancer or locus-defining element(s), or other elements that control gene expression by other means such as alternate splicing, nuclear RNA export, or post-translational modification of messenger.
  • Such vector constructs also include a packaging signal, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate to the virus used, unless it is already present in the viral construct.
  • LTRs long terminal repeats
  • such constructs will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof.
  • Other vectors can be used that are non-viral, such as cationic lipids, polylysine, and dendrimers.
  • agents capable of inducing cell cycle arrest can be identified using the teachings of the present invention. This can be effected by: (a) contacting a plurality of cells with a plurality of molecules; (b) identifying at least one molecule from the plurality of molecules capable of increasing a level of a phosphorylated BID polypeptide, the at least one molecule being the agent capable of inducing cell cycle arrest.
  • the "at least one molecule" or the agent described hereinabove which are capable of inducing cell cycle arrest can be for example a peptide, an oligonucleotide, a carbohydrate or any chemical which is capable of inducing BID phosphorylation, preferably in a PIKK site.
  • Presence or level of a phosphorylated BID polypeptide can be detected using immunological detection methods known in the art such as Western blot analysis, immunohistochemistry and the like. It will be appreciated that since BID-induced cell cycle arrest is mediated by BID phosphorylation on a PIKK site, the method according to this aspect of the present invention preferably detects the level of a PIKK-phosphorylated BID polypeptide.
  • PIKK-phosphorylated BID polypeptide can be detected using, for example, the anti-phosphospecific BID antibodies ( ⁇ pS ⁇ l, ⁇ pS78) described in the Examples section which follows.
  • the method according to this aspect of the present invention preferably detects the level of phosphorylated BID in the cell nucleus. Detection of phosphorylated BID in the cell nucleus can be effected using antibodies which bind phosphorylated BID (e.g., ⁇ pS ⁇ l, ⁇ pS78) which are employed in Western blot analyses of sub-cellular fractions (see for example, Figures 19a-d) or in immunofluorescence analyses (see for example, Figures 18a-h).
  • antibodies which bind phosphorylated BID e.g., ⁇ pS ⁇ l, ⁇ pS78
  • the polypeptides as well as other agents generated using the above-teachings can be qualified by functional assays, such as by monitoring the effect of the polypeptides on cell cycle arrest or apoptosis in cells.
  • the induction of cell c) r cle arrest can be determined following treatment of cells with various agents known to induce DNA damage (e.g., etoposide, mitomycin C, hydroxyurea, UV, or IR) and detecting the fraction of cells in each of the cell cycle phases using, for example, FACS analysis, BrDU labeling, essentially as described in the Examples section which follows (see for example, Figures 22a-i, 23a-c, 25a-i and 33a-c).
  • the effect of the polypeptides on apoptosis can be determined following exposure of cells to DNA damaging agents using various methods such as the Ethidium homodimer-1 staining (Invitrogen-Molecular Probes), the Tunnel assay (Roche, Basel, Switzerland), the Live/dead viability/cytotoxicity two-color fluorescence assay (Molecular Probes, Inc., L-3224, Eugene, OR, USA), FACS analysis [using molecules capable of specifically binding cells undergoing apoptosis, such as propidium iodide and Annexin V], and those of skills in the art are capable of assessing such levels in order to determine the standards of normal levels.
  • Ethidium homodimer-1 staining Invitrogen-Molecular Probes
  • the Tunnel assay (Roche, Basel, Switzerland)
  • the Live/dead viability/cytotoxicity two-color fluorescence assay Molecular Probes, Inc., L-3224, Eugene, OR, USA
  • FACS analysis using molecules capable of specifically binding cells undergoing
  • the polypeptides described hereinabove which are incapable of inducing apoptosis and thus are capable of inducing cell cycle arrest can be used to treat diseases associated with a genomic instability.
  • a method of treating a disease associated with a genomic instability is effected by introducing into-, or expressing in cells of an individual in need thereof the BID polypeptide which is incapable of inducing apoptosis, thereby inducing cell cycle arrest and treating the disease associated with the genomic instability.
  • treating refers to inhibiting, preventing, curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of a pathology or a disease and/or causing the reduction, remission, or regression of a pathology or a disease.
  • Those of skill in the art will understand that various methodologies and assays can be used to assess the development of a pathology or a disease, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of the pathology or the disease.
  • the term "preventing” refers to keeping a pathology or a disease from occurring in a subject who may be at risk for having the pathology, but has not yet been diagnosed as having the pathology or the disease.
  • the term "subject” refers to an animal subject e.g., a mammal, e.g., a human being at any age who suffers from or is at risk of developing the pathology.
  • individuals who are at risk of developing the pathology of the present invention include individuals who are genetically predisposed to develop the pathology (e.g., individuals who cany a mutation associated with the pathology, e.g., a mutation in the BRCAl gene), and/or individuals who are at high risk to develop the pathology due to other factors such as environmental hazard or other pathologies
  • pathology refers to any deviation from the normal structure and/or function of a particular cell, cell type, group of cells, tissue or organ leading to a disease, a disorder, a syndrome or an abnormal condition.
  • the disease is associated with a genomic instability.
  • disease associated with a genomic instability refers to any pathology caused from or characterized by instable genome in at least a portion of the cells of the individual suffering from or predisposed to the pathology.
  • the DNA checkpoints can be abnormal, leading to the accumulation of irreparable mutations which may lead to tumorigenic processes.
  • the disease associated with genomic instability is associated with an abnormal S phase checkpoint which may result in severely damaged genome leading to tumorigenic cells.
  • the disease associated with genomic instability can be Ataxia- Telangiectasia, Fanconi anemia, Bloom's syndrome, hereditary breast and ovarian cancer syndromes involving BRCAl and Nijmegen breakage syndrome (NBSl).
  • Polypeptides and other agents which are capable of inducing cell cycle arrest according to the teachings of the present invention can be administered to the subject per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients as is further described hereinbelow.
  • a method of inducing apoptosis is effected by introducing into-, or expressing in a cell a polypeptide comprising a BID amino acid sequence incapable of inducing cell cycle arrest, thereby inducing apoptosis.
  • the polypeptide used by the method according to this aspect of the present invention comprises a mutation in the PIKK phosphorylation site which abolishes phosphorylation at the PIKK phosphorylation site, while still allowing apoptosis.
  • a mutation can be, for example, a serine to alanine substitution such as the BID-S61A/S78A mutant described in Example 4 of the Example section which follows.
  • Over expression of such a polypeptide in the cell can saturate the BID binding domain on the PIKK protein (e.g., ATM), thus, downregulating the level of endogenous PIKK-phosphorylated BID in the cell.
  • a non-limiting example of such a polypeptide is the polypeptide set forth by SEQ ID NO:5.
  • inhibition of PIKK-mediated BID phosphorylation can be also effected using a polypeptide having a PIKK phosphorylation site which is capable of inhibiting PIKK-mediated phosphorylation of endogenous BID.
  • a polypeptide can be a short peptide (e.g., of 5-25 amino acids) having a BID amino acid sequences and includes the PIKK site (e.g., the peptides set forth by SEQ ID NOs:29-31) and thus competes with endogenous BID on the BID binding domain in the PIKK protein (dominant negative).
  • the polypeptide used by the method according to this aspect of the present invention can be a short phosphorylated peptide (e.g., of 5- 30 amino acids in length) comprising a BID amino acid sequence, which includes the PIKK phosphorylation site.
  • a short phosphorylated peptide e.g., of 5- 30 amino acids in length
  • Such a peptide can bind to proteins capable of recognizing phosphorylated BID and thus block the BID-mediated cell cycle arrest and induce apoptosis.
  • such peptides comprise a BID amino acid sequence which does not exceed 20 amino acids in length.
  • Non-limiting examples of such peptides are set forth by SEQ ID NOs:32-34.
  • the polypeptide used by the method according to this aspect of the present invention can be a short peptide (e.g., of 5-30 amino acids in length) comprising a BID amino acid sequence, which includes a mutation which mimics phosphorylation at the PIKK phosphorylation site.
  • a short peptide e.g., of 5-30 amino acids in length
  • Such a peptide can bind to proteins capable of recognizing phosphorylated BID and thus block the BID-mediated cell cycle arrest and induce apoptosis.
  • such peptides comprise a BID amino acid sequence which does not exceed 20 amino acids in length.
  • Non-limiting examples of such peptides are set forth by SEQ ID NOs:35- 37.
  • the polypeptide used by the method of this aspect of the present invention is capable of inducing apoptosis.
  • Qualified polypeptides can be evaluated using apoptosis functional assays as described hereinabove.
  • the present inventors have generated phospho-specific antibodies against BID polypeptides being phosphorylated on serine 61 ( ⁇ pS61) or serine 78 ( ⁇ pS78) by immunizing rabbits with synthetic peptides being phophorylated on a serine residue corresponding to the serine residue at position 61 of mouse BID or at position 78 of mouse BID.
  • the resulting anti phospho-specific serine 61 ( ⁇ pS61) or anti phospho-specific serine 78 ( ⁇ pS78) antibodies were affinity-purified using the phosphopeptide immobilized on solid support. Such antibodies were qualified by Western blot analysis for being specific to their respective phosphorylated mouse BID proteins ( ⁇ pS61 - Figure 7d; ⁇ pS78 - Figure 7f) as well as for phosphorylated human BID ( ⁇ pS78 - Figure 1 Ia).
  • an antibody comprising an antigen recognition domain capable of specifically binding a BID polypeptide phosphorylated on a serine residue corresponding to amino acid 78 of human BID as set forth in SEQ ID NO: 15 but does not bind the BID polypeptide when not phosphorylated on this respective position.
  • an antibody comprising an antigen recognition domain capable of specifically binding a BID polypeptide phosphorylated on a serine residue corresponding to amino acid 78 of mouse BID as set forth in SEQ ID NO: 16 but does not bind the BID polypeptide when not phosphorylated on. this respective position.
  • an antibody comprising an antigen recognition domain capable of specifically binding a BID polypeptide phosphorylated on a serine residue corresponding to amino acid 61 of mouse BID as set forth in SEQ ID NO: 16 but does not bind the BID polypeptide when not phosphorylated on this respective position.
  • antibody as used in this invention includes intact molecules as well as functional fragments thereof, such as Fab, F(ab')2, Fv or single domain molecules such as VH and VL to an epitope of an antigen.
  • functional antibody fragments are defined as follows: (1) Fab, the fragment which contains a monovalent antigen- binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab 1 , the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab 1 fragments are obtained per antibody molecule; (3) (Fab')2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab 1 fragments held together by two disulfide bonds; (4) Fv, defined as a genetically engineered fragment containing the variable region
  • Antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment.
  • Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
  • antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2.
  • This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab 1 monovalent fragments.
  • a thiol reducing agent optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages
  • an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly.
  • cleaving antibodies such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
  • Fv fragments comprise an association of VH and VL chains. TWs association may be noncovalent, as described in Inbar et al. [Proc. Nat'l Acad. Sci. USA 69:2659- 62 (1972O]. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker. These single- chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide.
  • sFv single- chain antigen binding proteins
  • the structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli.
  • the recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
  • Methods for producing sFvs are described, for example, by Whitlow and Filpula, Methods 2: 97-105 (1991); Bird et al., Science 242:423-426 (1988); Pack et al., Bio/Technology 11:1271-77 (1993); and U.S. Pat. No. 4,946,778, which is hereby incorporated by reference in its entirety.
  • Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR).
  • CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)].
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • donor antibody such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].
  • Fc immunoglobulin constant region
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321 :522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. MoI. Biol., 227:381 (1991); Marks et al., J. MoI. Biol., 222:581 (1991)].
  • the techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boemer et al., J. Immunol., 147(l):86-95 (1991)].
  • human antibodies can be made by introduction of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos.
  • the cDNA encoding the antibody light and heavy chains specific for the target protein of interest are isolated, typically from a hybridoma that secretes a monoclonal antibody specific for the marker.
  • Hybridomas secreting anti-marker monoclonal antibodies, or recombinant monoclonal antibodies can be prepared using methods known in the art.
  • a monoclonal antibody specific for the marker protein is identified (e.g., either a hybridoma-derived monoclonal antibody or a recombinant antibody from a combinatorial library)
  • DNAs encoding the light and heavy chains of the monoclonal antibody are isolated by standard molecular biology techniques.
  • light and heavy chain cDNAs can be obtained, for example, by PCR amplification or cDNA library screening.
  • cDNA encoding the light and heavy chains can be recovered from the display package (e.g., phage) isolated during the library screening process and the nucleotide sequences of antibody light and heavy chain genes are determined.
  • display package e.g., phage
  • nucleotide sequences of antibody light and heavy chain genes are determined.
  • many such sequences are disclosed in Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Sendees, NIH Publication No. 91-3242 and in the "Vbase" human germline sequence database.
  • the antibody light and heavy chain sequences are cloned into a recombinant expression vector using standard methods. It will be appreciated that the antibodies of this aspect of the present invention can be used as neutralizing antibodies capable of inhibiting the cell cycle arrest activity of the endogenous BID polypeptide within the cells.
  • a method of treating cancer is effected by introducing into-, or expressing in cells of an individual in need thereof a polypeptide comprising a BID amino acid sequence incapable of inducing cell cycle arrest, thereby inducing apoptosis and treating the cancer.
  • the cancer treated by the BID polypeptides according to this aspect of the present invention is of a lymphoid origin.
  • the isolated polypeptides or polynucleotides encoding same and/or the neutralizing antibodies of the present invention can be administered to the individual as part of a pharmaceutical composition.
  • active ingredient refers to the isolated BID polypeptide, the polynucleotide and/or expression vector encoding the BID polypeptide of the present invention and/or the neutralizing antibody of the present invention which is accountable for the biological effect.
  • pharmaceutically acceptable carrier refers to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
  • An adjuvant is included under these phrases.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
  • excipients examples include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
  • Suitable routes of administration may, for example, include oral, rectal, neurosurgical strategies (e.g., intracerebral injection, intrastriatal infusion or intracerebroventricular infusion, intra spinal cord, epidural), transmucosal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, intravenous, inrtaperitoneal, intranasal, or intraocular injections.
  • neurosurgical strategies e.g., intracerebral injection, intrastriatal infusion or intracerebroventricular infusion, intra spinal cord, epidural
  • transmucosal, intestinal or parenteral delivery including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, intravenous, inrtaperitoneal, intranasal, or intraocular injections.
  • compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art.
  • the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
  • Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl- cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added. such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol
  • cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl- cellulose, sodium
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
  • the compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro- tetrafluoroethane or carbon dioxide.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro- tetrafluoroethane or carbon dioxide.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • compositions described herein may be fo ⁇ nulated for parenteral administration, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative.
  • the compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes.
  • Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen- free water based solution, before use.
  • a suitable vehicle e.g., sterile, pyrogen- free water based solution
  • compositions of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
  • compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose ⁇ i.e., a therapeutically effective amount as described hereinabove). Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays.
  • a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
  • Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals.
  • the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage may vary depending upon the dosage form employed and the route of administration utilized.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l).
  • Dosage amount and interval may be adjusted individually to provide tissue levels of the active ingredient that are sufficient to regulate cell cycle arrest or apoptosis (minimal effective concentration, MEC).
  • MEC minimum effective concentration
  • the MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.
  • dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
  • compositions to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
  • compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDxA. approved kit, which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may, for example, comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions for human or veterinary administration.
  • Such notice for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
  • compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.
  • the level of PIKK- mediated BID phosphorylation was upregulated in response to DNA damaging agents such as IR and Etoposide.
  • DNA damaging agents such as IR and Etoposide.
  • a level can indicative of the response of cells to DNA damaging agents.
  • a method of detecting cellular exposure to DNA damaging agents is effected by detecting in a biological sample a presence and/or level of a PIKK- mediated phosphorylated BID, thereby detecting the cellular exposure to the DNA damaging agents.
  • cellular response refers to the phosphorylation events occurring in a cell following exposure to DNA damaging agents which induce
  • DNA breaks such as IR, UV, replication stress agents (e.g., Etoposide, mitomycin C and hydroxyurea) (for further details see the Examples section which follows and Zinkel SS., et al., 2005, Cell 122:579-591 which is incorporated herein by reference).
  • replication stress agents e.g., Etoposide, mitomycin C and hydroxyurea
  • biological sample refers to a sample of cells isolated from a tissue or fluid of a subject, including but not limited to, for example, blood, tissue biopsy (e.g., from a tumor such as of a lymphoid origin), as well as cells which are derived from the subject and are cultured ex vivo.
  • the level of PIKK-mediated phosphorylated BID can be detected using various techniques using e.g., Western blot analysis, radio immuno assay, itnmunohistochemistry, FACS analysis with specific anti-BID antibodies (see for example, Figures 4a-e, 5a-b and 6a-b).
  • such detection utilizes phospho- specific antibodies such as the anti-pS61 and/or the anti-pS78 antibodies of the present invention (see for example, Figures 7d-g, 13a-d, 16a-b).
  • PIKK-mediated phosphorylation of BID can be used to diagnose disease(s) associated with abnormal
  • Such diseases can be, for example, a disease associated with genomic instability as described hereinabove (e.g., A-T, breast and ovarian cancer syndromes associated with BRCAl).
  • a method of diagnosing a disease associated with an abnormal PIKK- mediated phosphorylation of BID is effected by detecting in cells of an individual in need thereof a presence and/or level of a PIKK-mediated phosphorylated BID, thereby diagnosing the disease associated with the abnormal PIKK-mediated phosphorylation of BID.
  • diagnosis refers to classifying a disease or a symptom, determining a severity of the disease, monitoring disease progression, forecasting an outcome of a disease and/or prospects of recovery.
  • agents capable of detecting the presence of PIKK-mediated phosphorylated BID e.g., the phospho-specific antibodies of the present invention
  • BID e.g., the phospho-specific antibodies of the present invention
  • the agents capable of detecting the presence of PIKK-mediated phosphorylated BID may be included in a diagnostic kit/article of manufacture preferably along with appropriate instructions for use and labels indicating FDA approval for use in detecting cellular exposure to DNA damaging agents and/or diagnosing disease(s) associated with abnormal PIKK-mediated phosphorylation of
  • kit can include, for example, at least one container including at least one of the above described diagnostic agents (e.g., the phospho-specific - ⁇ pS78 antibody) and an imaging reagent packed in another container (e.g., enzymes, secondary antibodies, buffers, chromogenic substrates, fluorogenic material) which is used to visualize the presence of bound antibodies.
  • diagnostic agents e.g., the phospho-specific - ⁇ pS78 antibody
  • imaging reagent packed in another container
  • another container e.g., enzymes, secondary antibodies, buffers, chromogenic substrates, fluorogenic material
  • the kit may also include appropriate buffers and preservatives for improving the shelf-life of the kit.
  • mice 29a-c, 30a-b and 31a-b are described in Example 6 of the Examples section which follows, the present inventors have generated genetically modified mice in which the BID gene is incapable of PIKK- mediated phosphorylation (i.e., knock-in mice).
  • a multicellular organism comprising a genome which comprises a genetically modified BID gene which comprises a mutation in a PIKK phosphorylation site.
  • multicellular organism refers to any organism having more than one cell, preferably, with differentiated cells that perform specialized functions (e.g., neuronal system, digestive system, cardiovascular system and the like).
  • multicellular organism refers to mammals and their fetuses, but human, such as rodents (e.g., mouse, rat, guinea pig), monkeys (e.g., gorilla, chimpanzee, gibbon, rhesus, apes in particular), pigs, sheep, cattle etc.
  • the multicellular organism is a mouse.
  • the phrase "genetically modified” refers to a sequence alteration which results in altered expression as compared with a wild type equivalent sequence.
  • the sequence alteration (e.g., mutation) may be natural or man-made.
  • it is a mutation in a PIKK phosphorylation site (S/Q motif) which abolishes phosphorylation by PIKK proteins such as ATM and ATR.
  • the mutation comprises the substitution of a serine residue with an alanine residue at a position corresponding to amino acid residue 61 and/or 78 of mouse BID (SEQ ID NO: 16).
  • a BID gene being incapable of PIKK-mediated phosphorylation
  • mutations in control sequences e.g., promoter, enhancer
  • the phrase "genetically modified” is intended to include all such mutations.
  • the term "gene” refers to a nucleic acid sequence from which a protein can be expressed or a part thereof suitable, for example, for directing homologous recombination.
  • a gene can include, for example, a complementary DNA sequence, a genomic DNA sequence or a mixed sequence of genomic and cDNA.
  • Additional sequences can be included, such as, polylinkers, positive and negative selection sequences, and genetically modified sequences such as sequence alterations and the like.
  • the introns of the gene can be for example modified, e.g., shortened.
  • the term gene as used herein further relates to the control sequences flanking the nucleic acid sequence from which a protein can be expressed, in particular upstream (5 1 ) control sequences. Since a gene according to the present invention is expected to undergo homologous recombination, the term as used herein refers also to any portion of a gene that following the homologous recombination is capable of combining with endogenous sequences to reconstruct a nucleic acid sequence from which a protein can be expressed.
  • the BID gene which is genetically modified in the multicellular organism of this aspect of the present invention is derived from mouse (nucleic acids 120942137-120917231 of GenBank Accession No. NC_000072.3).
  • the genetically modified BID gene is incapable of PIKK-mediated phosphorylation.
  • the modified BID which comprises the S61A and S78A mutations in the targeting construct exhibits unique RFLF pattern in both Southern blot analysis ( Figures 29b-c) and PCR analysis ( Figures 30a-b).
  • such genetically modified BID organism e.g., mouse
  • the agents of the present invention e.g., the isolated BID polypeptides
  • a BID polypeptide being incapable of inducing apoptosis e.g., the PIKK-phosphorylated BID polypeptide set forth by SEQ ID
  • a genetically modified BID organism can be used to screen for drugs capable of modulating BID activity in cell cycle arrest and/or apoptosis in vivo.
  • mice (originally kept on a mixed C57BL/6 x 129Sv background) had been bred to wild type C57BL/6 mice twelve times in order to obtain animals that are F12 on a C57BL/6 background.
  • BID '1' MEFs were generated from the Fl 2 mice.
  • BID +/+ and BlD ' ' ' primary MEFs were prepared from 11-13 day-old embryos and were maintained in ISCOVE' s medium containing 10% fetal bovine serum (MEF medium).
  • Atm/Arf double knockout (Atm-/-Arf-/-) and Atm + + Arf ⁇ MEFs were obtained from Chuck J. Sherr (St. Jude Children's Research Hospital).
  • hTERT transformation of primary MEFs The primary MEFs utilized by the present study were immortalized with the catalytic subunit of human telomerase (hTERT) (Wood et al., 2001) by viral transformation, as follows.
  • PA317 packaging cells stably producing pBABE-puro hTERT viral particles (a generous gift from Tej Pandita, Washington University) were grown to 80 % confluence, following which the cells were rinsed and the medium was replaced with complete MEF medium.
  • the medium i.e., infecting media
  • infecting media i.e., infecting media
  • Primary BID-/- and BID+/+ MEFs were grown for 3 passages and were then infected at ⁇ 50 % confluence with 3 ml infecting media mixed with 3 ml MEF media and 4 ⁇ g/ml polybrene (Sigma).
  • the infected K-IEFs were incubated for 16 hours, rinsed and incubated in fresh medium for an additional 8 hours.
  • the cells were infected again as described above, rinsed and incubated in fresh medium for additional 48 hours.
  • the cells were then split 1 :3 and grown for 4 days in a selection medium containing 1 ⁇ g/ml puromycin. After selection, the cells were washed once and incubated with MEF medium (without puromycin). Stable clones were collected 14 to 18 days post-infection.
  • hTERT BID ' ' ' stable clones expressing wtBID or the BID- S61A/S78A mutant - ⁇ NX cells were seeded in a 100-mm plate at 60 % confluence and incubated for one day, following which the medium was replaced and the cells were incubated with a transfection cocktail containing 15 ⁇ g retroviral vector (pBABE-wtBID or pBABE- BID-S61A/S78A) prepared using a calcium phosphate kit (Promega).
  • DMEM fetal bovine serum
  • FBS Gibco-Invitrogen
  • the medium was replaced with fresh DMEM (Gibco-Invitrogen) medium containing 10 % FCS and 1 ⁇ g/ml puromycin. Puromycin was replaced every day for three days. On the fourth day, cells were seeded (100 cells per 10-cm dish) and grown until single clones appeared.
  • SV40 transformation of primary MEFs was performed by transfecting cells with the SV40 whole genome using Lipofectamine 2000 (Invitrogen). Stable clones were collected 14-to-18 days post transfection.
  • splenocytes Preparation of splenocytes -
  • the spleens from either BID '1" or BID +/+ mice were pressed through stainless steel mesh to make a cell suspension in PBS.
  • the cells were centrifuged at 1500 rpm for 5 minutes and then transferred to Ficoll-Paque Plus (Amersham) to separate between erythrocytes and splenocytes. After centrifugation at 1800 rpm for 25 minutes, the fraction of splenocytes was isolated and the cells were washed twice with PBS.
  • the cell pellet was resuspended (1 x 10 6 cells/ml) in RPMI 1640 medium supplemented with 50 U/ml penicillin, 50 mg/ml streptomycin, 1 mM Sodium pyruvate, 0.1 mM ⁇ -mercaptoethanol, 2 mM glutamine, 0.1 mM non-essential amino acids, and recombinant human IL-2 (40 U/ml).
  • BID recombinant adenoviruses and infection of MEFs -
  • Viruses were grown using 293 T cells. Virus preparations were made from freeze/thaw lysis of the cells, and virus titers were done on 293T cells.
  • cells were generally seeded at 70-80 % confluence.
  • Cells were infected with an MOI (multiplicity of infection) of 100 with either the BID or GFP containing viruses and the rtTA containing virus.
  • MOI multiple of infection
  • 1 ⁇ g/ml doxycycline (a synthetic analog of tetracycline; Sigma) was added to the medium 12-to-15 hours post infection to activate gene expression from the tet-inducible promoter.
  • Efficiency of infection was determined using the recombinant adenovirus carrying the inducible expression vector of GFP and was in the range of 70-to-90 %.
  • Cloitogenic survival assays Cells were seeded at a density of 1000 cells per well (6 well plate). The next day, cells were treated with the indicated DNA damage reagent, the medium was replaced with fresh medium, and cells were incubated for 10 days. Once colonies were formed, cells were fixed in 70 % methanol and stained with 0.5 % crystal Violet. The percent of colony survival was calculated as the ratio of the # of colonies after DNA damage to the # of colonies in untreated cells.
  • DNA damage/death reagents and cell viability assays - TNF ⁇ , actinomycin D, and staurosporine were purchased from Sigma. Etoposide, cisplatin and thapsigargin were purchased from Alexis. Cell viability was determined by propidium iodide (PI) dye exclusion. PI (25 ⁇ g/ml) was added to the cells immediately prior to analysis by FACScan (Beckton Dickinson).
  • Transient transfections were performed by the calcium phosphate method (Graham and van der Eb, 1973) or with lipofectamine 2000 (Gibco BRL).
  • HeLa or 293 cells transfected with an ecotropic receptor were co-infected with 293 packaged viral particles of RNAi's and selected with 10 ⁇ g/ml puromycin and 200 ⁇ g/ml hygromycin to generate the stable lines.
  • Metabolic labeling was carried out by pre-equilibrating cells for 3 hours in phosphate-free DMEM (Gibco) prior to the addition of 0.5 mCi/ml 32 P-orthophosphate (NEN) for 1.5 hours. After labeling, cells were treated for 30 minutes with Etop, or remained untreated, following which the cells were lysed with RIPA buffer (150 mM NaCl, 1 % NP-40, 0.5 % deoxycholic acid, 0.1 % SDS, 50 mM Tris, pH 8.0) in the presence of cocktails of protease and phosphatase inhibitors (1 :100 Cat No. P5726 Sigma). Radiolabeled BID was immunoprecipitated using anti-BID Abs (polyclonal anti-mBID Abs described in PCT/IL2006/000021) at a dilution of 1:1000).
  • anti-BID Abs polyclonal anti-mBID Abs described in PCT/IL2006/000021
  • Alkaline or potato-acid phosphatase treatment - MEFs were treated with Etop for either 30 or 60 min, lysed in phosphatase buffer (150 mM NaCl, 1 % CHAPS, 10 mM HEPES, pH 7.5) and were either remained untreated or incubated for 30 minutes at 37 °C with either alkaline phosphatase (1 U/l ⁇ g protein; Roche) or with potato-acid phosphatase (PAP; 1.5 U/30 ⁇ g protein; Sigma). The reaction with PAP was performed in a phosphatase buffer adjusted to pH 5.5. At the end of the reaction the lysates were analyzed by Western blot using anti-BID Abs (1:1000).
  • In vitro kinase assay - 293T cells were transiently transfected with pcDNAIII- Flag ATM WT or kinase inactive (KI) (Canman C.E., et al., 1998, Science 281: 1677- 1679) or pcDNAIII-Flag ATR WT or KI (K2327R) (Tibbetts R.S., et al., 1999, Genes Dev 13: 152-157.) or pcDNAIII vector alone using the ProFection kit (Promega).
  • TNE buffer 100 mM NaCl, 5 mM EDTA, 0.5 % NP40, 1 x protease inhibitor cocktail set III (Calbiochem), 10 mM ⁇ -glycerophosphate, phosphatase inhibitor cocktail I and II (Sigma), 1 mM sodium orthovanadate, 5 mM sodium fluoride, 0.5 mM EDTA, 50 mM Tris, pH 8.0].
  • Lysates were precleared with protein A Sepharose beads (Amersham) for 30 minutes at 4 0 C mixing and Flag- tagged proteins were immunoprecipitated with anti-Flag M2 Abs coupled to agarose beads (Sigma) for 2 hours at 4 0 C mixing.
  • Immunoprecipitates were washed twice with TNE buffer, once with 100 rnM Tris (pH 8.0) containing 0.5 M LiCl 5 and twice with kinase buffer (10 mM ⁇ -glycerophosphate, 10 niM MgCl 2 , 10 mM MnCl 2 , 10 ⁇ M ATP, 50 mM NaCl 5 1 mM DTT 5 phosphatase inhibitor cocktail I and H 5 10 mM HEPES, pH 7.5).
  • Kinase assays were performed by adding 30 ⁇ l of kinase buffer containing 10 ⁇ Ci [ ⁇ - 32 P] ATP and 3 ⁇ g of recombinant mouse wtBID or BID- S61A/S78A per reaction and incubating for 30 minutes at 30 0 C. Proteins were separated by 12 % SDS-PAGE and transferred to PVDF membrane (Immun-blotTM, Bio-Rad) and phosphorylated proteins were visualized by autoradiography. Flag- tagged proteins were subjected to immunoblotting with anti-Flag M2 Abs (Sigma) and BID levels were monitored by coomassie staining.
  • 6xHis-tagged mouse BID was produced and purified from bacteria as previously described (Oh KJ., et al., 2005, J Biol Chem 280: 753-767). Formaldehyde treatment and subcellular fractionation - Formaldehyde was added directly to the tissue culture media to a final concentration of 1 % and the cells were incubated for 10 minutes at room temperature. The cross-linking reaction was stopped by adding glycine to a final concentration of 0.125 M and incubation at room temperature for 5 minutes. Cells were then subfractionated as previously described (Eldar et al., 1992).
  • wash buffer 125 mM KCl 5 5 mM magnesium acetate, 5 mM EGTA, 1 mM ⁇ -mercaptoethanol, 30 mM Tris-HCl, pH 7.5
  • wash buffer 125 mM KCl 5 5 mM magnesium acetate, 5 mM EGTA, 1 mM ⁇ -mercaptoethanol, 30 mM Tris-HCl, pH 7.5
  • KCl concentration was 10 mM and protease (set III; Calbiochem) and phosphatase (set I and II; Sigma) inhibitor cocktails were added.
  • the cells were then lysed in a 2- ml Wheaton Dounce glass homogenizer using 30 complete up and down cycles of a glass "B"-type pestle.
  • the homogenate obtained was overlaid on an equal volume of swelling buffer containing 25 % glycerol and centrifuged (600 x g at 4 °C for 5 minutes).
  • the upper layer of the supernatant was designated the cytosolic fraction. It should be noted that all organelle membranes (besides the nuclear membrane) and the plasma membrane are contained in this fraction.
  • the nuclear pellet was washed once with swelling buffer containing 25 % glycerol and 0.1 % Triton X-100.
  • Nuclei were resuspended in sonication buffer (100 raM NaCl, 2 niM MgCl 2 , 5 niM EGTA, 1 mM ⁇ -mercaptoethanol, 10 mM Tris, pH 9.0). At this stage both the cytosolic and nuclear samples were incubated at 65 °C for 4-5 hours to reverse formaldehyde cross-links. Nuclei were then disrupted by brief sonication. Aliquots of nuclear and cytosolic fractions were separated by 12 % or 15 % SDS-PAGE and transferred to PVDF membrane (Immun-blotTM, Bio-Rad).
  • Protein A purified anti- murine BID Abs and anti-human BID Abs were used for Western blotting.
  • BID-S61A, BID-S78A, and BW-S61 ⁇ /S78A cDNAs - were obtained using the QuickChange site-directed mutagenesis kit (Stratagene). Wild-type BID (SEQ ID NO:6; GenBank Accession No. NM_007544) and the BID mutants (SEQ ID NOs:7- 9) were cloned into the EcoRl site of pcDNAIII (Invitrogen) and into the BamUl and EcoRl sites of the pBABE retroviral expression vector.
  • Anti-pS78 monoclonal antibodies are generated by immunizing mice with the following synthetic peptides which represent portions of mouse BID protein (SEQ ID NO: 16; GenBank Accession No. P70444) around Serine 78: CGRIEPDSESQEE (SEQ ID NO: 10; underlined Serine being phosphorylated) and CEPDSESQEEIIH (SEQ ID NO:38; underlined Serine being phosphorylated).
  • BrdU labeling and analysis A total of 2 x 10 5 cells were treated for 2 hours with 20 ⁇ M Etop, washed twice with PBS and incubated in fresh medium (10 % FBS) for 8 hours. The cells were then pulsed labeled for 30 minutes with 10 ⁇ M BrdU (Sigma; added to the medium), washed with PBS, fixed with cold 70 % ethanol and incubated overnight at -20 °C. The next day, cells were collected and resuspended in 2N HCl with 0.5 % Triton X-100 for 30 minutes at room temperature followed by neutralization with 0.1 M Na 2 B 4 O 7 .
  • Cell cycle assays - MEFs were treated for 2 hours with 20 ⁇ M Etop, rinsed, and released into drug-free medium. Eight or twenty-four hours after release, cells were collected for fixation in methanol. Following fixation, cells were washed and resuspended in PBS with 25 ⁇ g/ml propidium-iodide (PI) and 50 ⁇ g/ml RNAse a half hour before FACScan analysis. Analysis of the cell cycle results was performed using the ModFit LT program (Tripathi et al., 2003).
  • Immunocytochemistty For immunocytochemistry, MEFs or HeLa cells were grown on glass cover slips. At the designated time points, the cells were fixed for 10 minutes with 4 % paraformaldehyde in PBS and permeabilized for 5 minutes with 0.2 % Triton X-100 in PBS. For blocking, the cells were incubated for 1 hour at room temperature in PBS containing 0.1 % Triton and 3 % bovine serum albumin (BSA). 61
  • cells were incubated overnight at 4 °C with either anti-murine BID Abs (diluted 1 :200) or the anti-pS61/ ⁇ S78 Abs (diluted 1:100) in blocking solution. After three washes with PBS containing 0.1 % Triton, the cells were stained for 1 hour at room temperature with Alexa 488-labeled goat anti-rabbit Abs (dilution 1:120, Molecular Probes), followed by 5 minutes incubation with 4',6-diamidino-2- phenylindole dihydrochloride (DAPI) staining (10 ⁇ g/ml). The coverslips were mounted with elvanol, and the cells were viewed under a Nikon fluorescence microscope at a magnification of 200x/400x. Images were captured using a 1310 digital camera (DVC).
  • DVC digital camera
  • BID is important for DNA damage-induced apoptosis -
  • the DNA damaging agents used included Etoposide (Etop; a specific inhibitor of topoisomerase H), cisplatin (Cis; forms covalent adducts with the DNA), ultraviolet radiation (UV; induces thymine dimers), and ionizing radiation (IR).
  • Etop Etoposide
  • Cis cisplatin
  • UV ultraviolet radiation
  • IR ionizing radiation
  • the BID ' ' MEFs were less susceptible to all four treatments than the BID + + MEFs.
  • These DNA-damaging reagents also induced less cell death in primary BID ' ' ' MEFs than in primary BID +/+ MEFs (data not shown), confirming that this decreased sensitivity is not due to hTERT immortalization.
  • Splenocytes from BID " ' ' mice also display less susceptibility to Etop treatment ( Figure 2a), indicating that the decreased death response to DNA damage
  • DNA-damaging reagents causing double-strand breaks induce double electrophoretic mobility shift in BID -
  • hTERT-immortalized BID +/+ MEFs treated with the DNA- damaging reagents described hereinabove and cell lysates were subjected to Western blot analysis using anti-BID antibodies.
  • Etop and IR which are known to induce double-strand breaks (DSBs) in DNA, unlike Cis or UV, induced a double electrophoretic mobility shift in BID.
  • thapsigargin Thaps; stress signaling from the ER, which inhibits the Ca 2+ adenosine triphosphate pump
  • TNF ⁇ TNF ⁇ together with actinomycin D
  • staurosporine STS; a kinase inhibitor
  • the double electrophoretic mobility shift in BID is due to phosphorylation of BID -
  • BID +/+ MEFs were treated for 30 minutes with Etop, lysed and incubated for 30 minutes at 37 °C with alkaline phosphatase or remained untreated.
  • Western blot analysis using anti-BID antibodies demonstrated that treatment with alkaline phosphatase abolished the electrophoretic mobility shifts in BID ( Figure 4c), indicating that these shifts are most likely due to phosphorylation.
  • human cervical adenocarcinoma (HeLa) cells were transfected with mouse BID, labeled with 32 P-orthophosphate, treated with Etop or remained untreated and were subjected to BID- immunoprecipitation. Exposure to Etop resulted in a marked increase in 32 P-labeling of BID, which appeared as a doublet ( Figure 4d). Western blot analysis of the same samples with anti-BID antibodies indicated that the two 32 P-labeled bands correspond to the two slower-migrating forms of BID ( Figure 4e).
  • the phosphorylation of BJD is mediated by the ATM kinase -
  • the ATM kinase plays a pivotal role in the immediate response of cells to double-strand breaks.
  • mouse embryonic fibroblasts deficient in both ATM and the pi 9ARF tumor suppressor gene were utilized, since loss of ARP has been shown to reverse premature replicative arrest of Atm-null MEFs (Kamijo et al., 1999). Accordingly, Atm/Arf double knockout MEFs, as well as MEFs, were treated with Etop or IR, and were subjected to Western blot analysis using anti-BID antibodies.
  • FIGS 5a-b show that following Etop or IR treatment, the slower migrating bands of BID do not appear in the ATM-deficient cells. Thus, the presence of the ATM kinase appears to play an essential role in the process by which etoposide and ionizing radiation induce phosphorylation of BID.
  • MOUSEAND HUMANBID ARE PHOSPHORYLATED ONPIKK CONSENSUS
  • ATM is a member of the PIKK family.
  • the common phosphorylation sites for PIKKs are serines or threonines followed by glutamine residues, a motif commonly dubbed "SQ/TQ" (Kim et al., 1999).
  • Mouse BID carries two such motifs (S 61 Q and S 78 Q), whereas human and rat BID cany only one (S 78 Q) ( Figure 7a). To determine whether mouse BID is phosphorylated on one or both of these sites, each of the serine residues were mutated to alanine residues and the effect of Etop was evaluated on the mutated BID proteins, as follows.
  • Mouse BID is phosphorylated on both Serine 61 and Serine 78 in response to Etop treatment -
  • phospho-specific antibodies to serine 61 (anti-pS61) and serine 78 (anti-pS78) were generated.
  • BID +/+ MEFs were treated for 30 minutes with Etop or remained untreated, lysed, and incubated for 30 minutes at 37 °C with potato-acid phosphatase or remained untreated.
  • the anti-pS61 and anti-pS78 antibodies recognized BID in cells expressing wt- BID and treated with Etop, but not in cells expressing the BID-S61A or BID-S78A mutant, respectively.
  • in vitro kinase assays were performed in 293T cells using flag-tagged human ATM and ATR and the kinase- inactive forms thereof, and purified recombinant mouse wt-BID and the BID- S61A/S78A double mutant.
  • the results demonstrate that wild type ATM and ATR, but not the kinase-inactive forms, efficiently phosphorylate recombinant wt-BID in vitro ( Figures 9a-c), whereas mutating both S61 and S78 completely abolished BID phosphorylation ( Figures 9d-e).
  • BID is phosphorylated on both S61 and S78 in mouse splenocytes -
  • the phosphorylation status of BID in mouse splenocytes was tested by treating cells with Etop and subjecting the cell lysates to Western blot analyses using anti-BID, anti pS61 or anti-pS78 antibodies.
  • Etop treatment induced the phosphorylation of BID on both S61 and S78 in splenocytes.
  • human BID is phosphorylated on S78 in response to Etop -
  • human BID carries only one PIKK consensus site (S78; Figure 7a).
  • Phosphorylation of serine 61 and serine 78 is transient, ATM-dependent, and only occurs in response to reagents that induce double-strand breaks -
  • BID +/Jr MEFs were treated with Etop and were subjected to Western blot analysis using anti-BID, anti-pS61, or anti-pS78 antibodies.
  • phosphorylation of S61 was detected by 15 minutes (the first time point analyzed), reached a peak at 1 hour, and was reduced by 3 hours post Etop treatment.
  • Phosphorylation of S78 was also transient (peak at 2-3 hours), though was somewhat delayed, compared to phosphorylation of the S61 site.
  • BID +/+ MEFs were treated with 100 ⁇ M Etop and cell death was determined following 2, 4, 8, 12 and 22 hours of treatment. As is shown in Figure 14, the onset of apoptosis occurred between 8 and 12 hours following Etop treatment, which is hours after BID phosphorylation.
  • Endogenous mouse BID is phosphorylated on S61 and S78 only in Atm +/+ Arf A MEFs treated with Etop -
  • the phospho- specific antibodies were utilized in Western blot analysis of Atm/Arf double knockout and Atm +M ' Ar/ ' mouse embryonic fibroblasts following Etop treatment.
  • both anti-pS61 and anti-pS78 recognized phosphorylated BID only in Atm +/+ Ar/ ' MEFs which were treated with Etop.
  • Mouse BID is phosphorylated on S61 only in response to reagents that induce double-strand breaks -
  • mouse embryonic fibroblasts were treated with several DNA-damaging and other apoptotic reagents (previously described with respect to Figures 4a-b). Post-treatment, cells were lysed, and the phosphorylation of endogenous mouse BID was examined by Western blot analysis using anti-pS61 antibodies. These results demonstrated that mouse BID is phosphorylated on S61 only in response to Etop and IR treatments which are known to induce double-strand breaks ( Figures 16a-b).
  • Human BID is phosphorylated in an ATM-dependent manner in response to reagents which induce double-strand DNA breaks -
  • stable HEK293 cell line in which ATM was knocked down by siRNA [these cells were generated like the HeLa ATM knocked down cells (Elkon et al., 2005)] or control cells, which carried a siRNA against LacZ, were exposed to Etop, IR, UV, or STS, and were subjected to Western blot analysis using anti-pS78 antibodies. Exposure of LacZ blocked down cells to Etop or IR, but not to UV or STS, induced phosphorylation of endogenous human BID on serine 78, which did not occur in the ATM knocked down cells ( Figures 17a-b).
  • BID + + and BID ' ' MEFs were treated for 3 hours with Etop prior to fixation. These studies have indicated that Etop did not change the staining pattern of BID ( Figures 18e and f).
  • BID partially localizes to the nuclear fraction -
  • MEFs of BID+/+ were subjected to subcellular fractionations followed by Western blot analysis using anti-BID antibodies.
  • cellular BID was detected only in the soluble/cytoplasmic fraction ( Figure 19a, lanes 1 and 2).
  • MEK and BAX cytosolic proteins
  • lamin B a nuclear protein
  • Etop treatment does not change the level of BID associated with the nuclear fraction -
  • cells were treated for 1 and 3 hours with Etop, and then treated with formaldehyde followed by subcellular fractionation. The results demonstrate that Etop treatment did not change the levels of BID associated with the nuclear fraction (data not shown).
  • BID is phosphotylated in the nucleus -
  • immunofluoresence studies were performed with the phospho-specific antibodies using BID ⁇ + and BID '1' MEFs following 30 minutes of Etop treatment. These antibodies detected an increase in nuclear fluorescence in BID +/+ MEFs treated with Etop, however a similar increase was detected in BID ' ' " MEFs (data not shown). Thus, these antibodies cross-react with other phospho-S/T-Q proteins. Nonetheless, the fact that BID partially localizes to the nucleus, and that all currently identified substrates of ATM are nuclear proteins, suggests that BID is phosphorylated in the nucleus.
  • BID is phosphorylated was determined in mouse embryonic fibroblasts. Phosphorylation of serine 61 was detectable immediately after exposing cells to 50
  • BID +/+ and BlD ' ' ' mouse embryonic fibroblasts performed cell cycle analyses on BID +/+ and BlD ' ' ' mouse embryonic fibroblasts following etoposide treatment, as follows.
  • BID ' ' ' mouse embryonic fibroblasts fail to accumulate in the S and G ⁇ phases of the cell cycle following etoposide treatment - Etop induced accumulation of BID +/+ MEFs in the S and G 2 phases of the cell cycle (as measured 8 hours after release into drug-free medium), whereas such an accumulation was not observed in the BID '1' MEFs ( Figures 22a-i).
  • BID is important for the accumulation of cells in the S and G 2 phases of the cell cycle following double-strand break DNA damage.
  • BID 'A mouse embryonic fibroblasts expressing a non-phosphorylatable BID mutant (S61A/S78 ⁇ ) do not accumulate in the S phase following etoposide treatment -
  • BID "1' single stable clones expressing either wt-BID or a non- phosphorylatable BID mutant (S61A/S78A) were generated.
  • Initial analysis showed that the wt-BID (in the wt-BID clones) was phosphorylated on S61 and S78 in response to Etop, and that BID-S61A/S78A (in the mutant BID clones) was not phosphorylated ( Figures 24a-f).
  • the levels of apoptosis was assessed in the two wt-BID and in the two BID-S61A/S78A clones following Etop treatment.
  • the results indicate that Etop induced a significantly higher rate of apoptosis in BID ' ' ' clones expressing BID- S61A/S78A than in the BID 1' clones expressing wt-BID ( Figure 26a).
  • BID is phosphorylated in vivo in response to ionizing radiation - Seven week old BID +/+ or BID " ⁇ mice were subjected to a sub-lethal dose of IR (3 Gy) and one hour after exposure the phosphorylation status of BID and its expression level were studied in a variety of tissues using the anti-pS78 antibodies and antibodies to unmodified BID. As is shown in Figures 27a-b, BID was mainly phosphorylated in the thymus, spleen, bone marrow, and kidney, and these were also the organs in wliich BID was mainly expressed.
  • Phosphorylation of BID in the thymus is transient and increases in an ionizing radiation dose-dependent manner -
  • the present inventors have uncovered that phosphorylation of BID is transient and is increased in an ionizing radiation dose- dependent manner in MEFs (See Figures 21a-b and Example 3 hereinabove).
  • mice were exposed to two doses of IR (3 or 6 Gy), sacrificed 1 or 4 hours later, and the phosphorylation status of BID was analyzed in the thymus.
  • Figures 28a-b show that the phosphorylation of BID in the thymus is also transient (high at 1 hour, and reduced by 4 hours post IR), and it also increases in an ionizing radiation dose-dependent manner.
  • mice in which the endogenous BID gene has been replaced (“knock- in") with a gene that drives the expression of a BID protein carrying mutations in the ATM phosphorylation sites [i.e., BID S61A/S78A, in which both of the serine residues phosphorylation sites (S61 and S78) are converted to alanine residues], as follows.
  • FIG. 29a A targeting vector in wliich mutations in the S61 and S78 serine residues are introduced into the genomic BID sequence was constructed ( Figure 29a). The mutations were linked to silent restriction fragment length polymorphisms (RFLPs) in order to facilitate genotyping.
  • This targeting vector contains a positive selection marker flanked by recognition sites for the Cre recombinase (LoxP-Neo-LoxP), which is used for selection of 129 embryonic stem (ES) cells. Transient expression of the Cre recombinase in ES cells allows excision of the neomycin gene flanked by the two LoxP sites.
  • the targeting vector was transfected into Rl embryonic stem (ES) cells (derived from 129/ola mice), and neomycin resistant clones were selected. Southern-blot analysis was performed using two probes (probe- L and probe-S; Figures 29b-c). Two homologous recombinant Rl clones were identified and aggregated with tetraploid embryos and implanted into white-coated ICR foster mothers. The first generation of black-coated mice, were bora, bred again to white ICR mice, and the second generation of BID S61A/S78A/+ animals were obtained.
  • ES Rl embryonic stem
  • the heterozygous mutant BID S61A/S78A/+ animals are crossed to a general deleter strain, PGK-Cre to excise the neo cassette, which is flanked by two LoxP sites (illustrated by 2 rectangles in Figure 29a). Intercross of BID S61A/S78A/+ animals results in offspring homozygous for the mutant BID gene (BID S61A/S78A) ( Figures 30a-b).
  • BID S61A/S78A mice exhibit endogenous mutated BID which is incapable of being phosphorylated at the ATM regulatory sites -
  • BID +/+ , BID S61A/S78A/+, and BID S61A/S78A animals were exposed to a sub-lethal dose of IR and the phosphorylation status of BID and its level of expression in tissues were examined.
  • Western blot analysis using antibodies to unmodified BID revealed that the amounts of BID protein expressed in spleen and thymi from all three types of animals were similar (Figure 31b).
  • BID carries a nuclear export signal (NES) -
  • NES nuclear export signal
  • NLS nuclear localization signal
  • Figure 32a Examination of the human, rat and mouse BID sequences revealed that they do not carry a classic NLS but do carry a classic nuclear export signal (NES) ( Figure 32a).
  • the Rev type NES sequence is comprised of an 8 to 12 amino acid peptide in which mutations of critical leucines abolish the export activity of the protein (Pemberton, L.F., et al., Curr. Opin. Cell Biol, 10: 392-399, 1998).
  • BID NES regulates the "shuttling" of BID from the nucleus to the cytosol -
  • the present inventors have mutated all three of the conserved leucine residues in the BID NES to alanine residues (L35A/L38A/L42A) by PCR. Wild-type BID and the triple mutant (BID-3LA) were transiently transfected into HeLa cells and the localization of BID was assessed by inimunofloursence. These studies have shown that mutating the BID NES did not seem to increase the nuclear levels of BID (data not shown).
  • LMB leptomycin B
  • CMRl Rev-type proteins
  • NLS-B ID-3 LA variant is expected to be "trapped" in the nucleus due to increased import and decreased export.
  • Figures 32b-e show that NLS-BID-3LA is more confined to the nucleus than wt-BID. It is important to note that fusing the same NLS to wt-BID did not increase the nuclear levels of wtBID (data not shown).
  • the formaldehyde/subcellular fractionation assay was employed. As is shown in Figures 32f-g most of the NLS-BID-3LA was localized to the nuclear fraction.
  • BID export from the nucleus is required for the accumulation of cells in the S phase following Etop treatment -
  • stable BID "7" MEFs clones expressing the wt-BID or NLS-BID-3LA were generated. No difference was found in the phosphorylation of wt-BID and NLS-BID-3LA in the stable clones in terms of level and kinetics of phosphorylation following Etop treatment (data not shown).
  • BID export from the nucleus is needed for Etop - induced apoptosis - Moreover, the levels of apoptosis in the two wt-BID and in the two NLS-BID-3LA clones were assessed following Etop treatment. As is shown in Figure 34, Etop induced a significantly lower rate of apoptosis in BID '1' clones expressing NLS-BID- 3LA than in the BID '1' clones expressing wt-BID.
  • paraformaldehyde/cellular fractionation experiments present a tool that can be used to test whether BID's activities in the DSB DNA damage pathway require nuclear localization, and to purify proteins that bind to nuclear BID.
  • BID "7" MEFs are less susceptible than BID +/+ MEFs to reagents that induce different forms of DNA damage.
  • BID phosphorylation occurs only in response to reagents that induce double-strand breaks in DNA.
  • phosphorylation of BID does not seem to be critical for its pro- apoptotic activity.
  • lack of BID phosphorylation does effect the susceptibility of cells to double-strand break DNA damage since BID "7" MEFs expressing the BID-S61A/S78A mutant are more susceptible than BID "7' MEFs expressing wild-type BID to etoposide-induced apoptosis ( Figures 26a-c).
  • ATM-mediated BID phosphorylation might serve as a mechanism to inhibit BID's apoptotic activity or alternatively as a mechanism to activate a pro-survival activity of BID. If phosphorylation was inhibiting the apoptotic activity of BID, then what would likely be the molecular basis of such inhibition?
  • the only known apoptotic function of BID lies in its ability to induce the release of pro-apoptotic factors from the mitochondria (e.g., cytochrome c).
  • tBID Caspase truncated BID
  • tBID Caspase truncated BID
  • ATM-mediated BID phosphorylation inhibits the cleavage of BID, since casein kinase 1 and/or 2-mediated phosphorylation of BID was demonstrated to inhibit its cleavage (Desagher et al., 2001).
  • the findings of the present invention suggest that ATM-mediated phosphorylation of BID is not related to BID cleavage, for the following reasons: Phosphorylation occurs many hours before the activation of caspases and the onset of apoptosis ( Figures 13-14 and data not shown).
  • BID-S61A/S78A mutant A relatively small amount of BID is phosphorylated ( Figures 4a-e), and phosphorylation occurs also in response to extremely low, non-apoptotic levels of DNA damage ( Figures 20-21). Moreover, the BID-S61A/S78A mutant was not found to be more susceptible to cleavage than wt-BID ( Figures 26b-c). Lastly, though TNF ⁇ relies on the generation of tBID to induce/enhance apoptosis (Yin et al., 1999; Zhao et al., 2001), the BID-S61A/S78A mutant clones are not more susceptible to TNF ⁇ -induced apoptosis than the wtBID clones ( Figure 26a).
  • BID phosphorylation does not seem to serve as a mechanism to inhibit BID's apoptotic activity.
  • the results presented in Figures 22-25 suggest that ATM-mediated phosphorylation of BID regulates a novel, pro-survival function of BID related to cell cycle arrest following DNA double-strand breaks. Etoposide treatment induced accumulation of BID+/+ mouse embryonic fibroblasts in the S and G2 phases of the cell cycle, whereas such an accumulation was not observed in the BID-/- MEFs ( Figures 22a-i). Moreover, using BrdU labeling the present inventors demonstrated that BID is specifically required for S phase arrest.
  • the cell may decide to activate a survival system (mainly through the ATM kinase, which induces cell cycle arrest and DNA repair), or in the face of extensive or irreparable damage, the cell may activate the apoptotic machinery.
  • a survival system mainly through the ATM kinase, which induces cell cycle arrest and DNA repair
  • the cell may activate the apoptotic machinery.
  • the results presented in Figures 26a-c demonstrate that BID "7" MEFs expressing the BID-S61 A/S78A mutant are more susceptible than BID " ' " MEFs expressing wild-type BID to etoposide-induced apoptosis, but not to UV- or to TNF ⁇ - induced apoptosis.
  • BID may play both a pro-survival and a pro-apoptotic role in this pathway. Based on the finding that the phosphorylation of BID occurs in response to extremely low levels of ionizing radiation, and increases in an ionizing radiation dose-dependent manner ( Figures 20-21), the present inventors propose that BID acts as a sentinel of DNA double-strand breaks. BID might translate the damage into either cell cycle arrest/DNA repair processes (at low levels of damage) or apoptosis (at high levels of damage).
  • BID-D59A is a potent inducer of apoptosis in primary embryonic fibroblasts. J Biol Chem 278, 10707-10715. Sax, J. K., Fei, P., Murphy, M. E., Bernhard, E., Korsmeyer, S. J., and El-Deiry, W. S.
  • Tripathi A. K., Chaturvedi, R., Ahmad, R., Asim, M., Sawlani, K. K., Singh, M. K.,
  • Tripathi P., and Tekwani, B. L. (2003). Flow cytometric analysis of aneuploidy and S-phase fraction in chronic myeloid leukemia patients: role in early detection of accelerated phase. Leuk Res 27, 899-902. Wood, L. D., Halvorsen, T. L., Dhar, S., Baur, J. A., Pandita, R. K., Wright, W. E.,
  • Proapoptotic BID is required for myeloid homeostasis and tumor suppression. Genes De ⁇ ' 17, 229-239.

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Abstract

L'invention concerne un polypeptide BID phosphorylé par PIKK pouvant être utilisé afin d'induire l'arrêt du cycle cellulaire. Elle concerne, plus particulièrement, des polypeptides isolés comprenant une séquence d'acides aminés BID pouvant être utilisée afin d'induire l'arrêt du cycle cellulaire et de traiter des maladies associées à une instabilité génomique. Dans un autre mode de réalisation, elle concerne des polypeptides isolés comprenant une séquence d'acides aminés BID capable d'inhiber la phosphorylation médiée par PIKK de BID endogène pouvant être utilisée afin d'induire l'apoptose et de traiter le cancer.
PCT/IL2006/000393 2005-03-28 2006-03-29 Polypeptides bid isoles, polynucleotides les codant et anticorps diriges contre ces polypeptides, methodes d'utilisation pour induire l'arret du cycle cellulaire ou l'apoptose Ceased WO2006103666A2 (fr)

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