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Apoptosis continued
Caspases are cysteine-dependent aspartate specific proteases that cleave target proteins after aspartic acid residues. They exist as inactive zymogens called procaspases in cells and initiate a proteolytic cascade when activated. This leads to cleavage of key proteins and characteristic apoptosis morphology. The Bcl-2 family of proteins regulate apoptosis at the mitochondrial membrane, with pro-apoptotic proteins like Bax activating apoptosis and anti-apoptotic proteins like Bcl-2 inhibiting apoptosis. Many cancers acquire mutations in genes encoding proteins that regulate apoptosis, allowing cancer cells to evade cell death signals and continue uncontrolled proliferation. Targeting apoptosis pathways is a promising strategy for cancer treatment.
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Apoptosis continued
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- 6. Caspases Cysteine-dependent aspartate specificproteases Have a cysteine at the active site Cleave target just after aspartic acid residues Substrate specificity is determined by the 4 residues upstream of cleavage site( towards N-terminal) Exist in cytosol as single chain proenzymes (procaspases) which are activated when cleaved by other caspases Once activated, cleave other caspases – results in proteolytic cascade Also cleave key proteins in the cell, causing the characteristic morphology and biochemistry of apoptosis.
- 7. 3 Types ofCaspases • Inflammatory Caspases: -1, -4, and -5 • Initiator Caspases: -2, -8, -9, -10& -12 – Long N-terminal domain – Interact with effector caspases • Effector Caspases: -3, -6, and -7 – Little to no N-terminal domain – Initiate cell death 14 CASPASES identified in humans.
- 11. Antiapoptotic Proapoptotic Bcl-2 family members Avery large family with 30 members identified and belongs to both: Bid Bim Bik Bad Bmf Hrk Noxa Puma Blk BNIP3 Spike BH1, BH2,BH 3,BH4 BH 3 BH1, BH 2,BH3
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- 13. Regulatory interactions betweenBcl-2 family members
- 15. Youle and Strasser(2008) The BCL-2 protein family: opposing activities that mediate cell death. Nature Reviews Molecular Cell Biology, 9, 47-59 BH3 only protein binding specificity for BCL-2 homologues BIM and PUMA bind to all BCL-2 family members tested; by contrast NOXA only binds to A1 and MCL1. These binding specificities recapitulate the ability of these proteins to activate apoptosis e.g. BIM et al can induce apoptosis alone whereas a combination of NOXA and BAD is required.
- 18. IAPs (Inhibitors ofApoptosis) These proteins act inhibiting caspase activity in 2 different ways: • Direct binding inhibiting the proteolytic activity of caspases • Marking caspases for ubiquitination and so degradation Inhibited by SMAC/DIABLO. Salvesen and Duckett Nat rev mol cell biol (2002)
- 27. Kinchen and Ravichandran (2007) Fig1 Stagesof engulfment of apoptotic cells can be divided into 4 stages Binding Recognition Phagocytosis Internalization
- 28. Lauber et al.(2004)Fig 2 Lack of “don’t eat me” signals on the surface of apoptotic cells
- 30. Anterior chamber ofthe eye and testes fail to elicit an immune response because these cells produce lots of FasL, so kill immune cells when they enter these sites. Possible therapy by inducing production of FasL in other tissues – lowering need for immune rejection drugs ORGAN TRANSPLANTS
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- 39. 1. p53 mediatesapoptosis in response to DNA damage, oncogene expression (adenovirus E1A, myc etc.), or withdrawal of growth factors 2. Overexpression wild-type of p53 leads to apoptosis 3. p53 induces apoptosis through transcriptional activation of proapoptotic genes, such as Puma, Noxa, p53AIP1, Bax, Apaf-1 etc. 4. DNA-damage leads to mitochondrial translocation of p53. 4.p53 binds to Bcl-2 family protein Bcl-xL to influence cytochrome c release. p53 in apoptosis
- 40. p53 binds toBcl-xL and releases Bax . Bax is sequestered by Bcl-xL and inactive Bax is released by p53 from Bcl-xL and forms oligomers, leading to apoptosis . p53 can release both proapoptotic multidomain proteins and BH3-only proteins
- 41. p53 and Pumain apoptosis Ref: K. Vousden, Science, 2005
- 42. p53 in DNArepair and apoptosis Ref: Bensaad & Vousden, Nature Med, 2005 ROS: reactive oxygen species G1- S
- 43. Role of P53in DNA damage induced Apoptosis • DNA damage activate ATM & Chk2 protein kinases • Phosphorylation of P53 • Activate Cdk inhibitor P21, which inhibits Cdk2/cyclin E complexes, halting cell cycle progression in G1 NOXA
- 44. The p53-MDM2 feedbackloop 1. MDM2 binds to p53 N-terminal transactivation domain and inhibits p53-dependent transcription. 2. MDM2 is a transcription target of p53. 3. MDM2 is an E3 ubiquitin ligase of p53, thus targeting p53 for proteolytic degradation. 4. MDM2 knockout is lethal for mouse embryonic development, but simultaneous deletion of p53 and MDM2 genes rescues MDM2-KO.
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- 46. Ref: Nature 408,307 - 310 (2000) Viral oncogenes and the p53 network
- 47. Genetic Control of"Genetically Programmed Cell Death" Cellular Location of Protein Product Mitochondrial Membrane Nuclear Envelope Endoplasmic Reticulum Nucleus Nucleus Cell Membrane Effect on Apoptosis Blocks Stimulates Wild-type Stimulates Mutant Blocks Stimulates ApoGenes bcl-2 myc p53 p53* APO-1/ FAS Apoptosis Gene
- 48. Tumor-derived mutations affecting apoptosis ProteinRole in apoptosis Ref ATM Mutated in ataxia-talangiectasia syndrome. Senses DNA double strand breaks and stabilizes p53. Deficiencies increase risk of developing haematological malignancies and breast cancer Khanna and Jackson, 2001 Bax (p53 target gene) Mutated or decreased expression in some tumors. Mediates mitochondrial membrane damage. Sufficient but not necessary for drug-induced apoptosis. Rampino et al., 1997 Bak Mutated or decreased expression in some tumors. Mediates mitochondrial membrane damage. Sufficient but not necessary for drug-induced apoptosis. Kondo et al., 2000 PTEN (p53 target gene) Mutated or altered expression in cancers. Regulates Akt activation and subsequent phosphorylation of Bad. Loss of PTEN results in resistance to many apoptotic stimuli. Di Cristofano and Pandolfi, 2000 Ref: Cell, 2002, 108:153-164
- 49. Tumor-derived mutations affecting apoptosis ProteinRole in apoptosis Ref Apaf-1 (p53 target gene) Mutated and transcriptionally silenced in melanoma and leukemia cell lines. Necessary for activation of caspase-9 following cytochrome c release. Apaf-1-/- cells are chemoresistant. Soengas et al., 2001 CD-95/Fas Mutated and down-regulated in lymphoid and solid tumors. Initiates the extrinsic apoptotic pathway. Loss of function is associated with resistance to drug- induced cell death. Muschen et al., 2000 TRAIL- R1/R2 Mutated in metastatic breast cancers. Initiate the extrinsic apoptotic pathway. Mutations lead to suppression of death receptor-mediated apoptosis. Shin et al., 2001 Caspase-8 Gene silenced in neuroblastomas. Activates both extrinsic and intrinsic apoptotic pathways. Silencing results in resistance to drug-induced apoptosis. Teitz et al., 2000 Ref: Cell, 2002, 108:153-164
- 50. Tumor-derived mutations affecting apoptosis ProteinRole in apoptosis Ref Bcl2 Frequently overexpressed in many tumors. Antagonises Bax and/or Bak and inhibits mitochondrial membrane disruption. Inhibits drug-induced apoptosis. Reed, 1999 MDM2 Overexpressed in some tumors. Negative regulator of p53. Inhibits drug-induced p53 activation. Sherr and Weber, 2000 IAPs Frequently overexpressed in cancer. Down regulation of XIAP induces apoptosis in chemoresistant tumors. Deveraux and Reed, 1999 NF B Deregulated activity in many cancers. Transcriptionally activates expression of anti-apoptotic members of the Bcl-2 and IAP families. Can inhibit both the extrinsic and intrinsic death pathways and induce drug- resistance. Baldwin, 2001 Ref: Cell, 2002, 108:153-164
- 51. Tumor-derived mutations affecting apoptosis ProteinRole in apoptosis Ref p53 Mutated or altered expression in many cancers. Initiates the intrinsic apoptotic pathway. p53-/- cells are resistant to drug induced apoptosis. Vogelstein et al., 2000 p19ARF Mutated or altered expression in many cancers. Blocks MDM2 inhibition of p53. Enhances drug- induced apoptosis by p53. Sherr and Weber, 2000 Rb Mutated in some cancers, and functionally disrupted in many cancers. Inhibits E2F- medidated transcription. Loss of Rb function induces p53-dependent and independent apoptosis. Harbour and Dean, 2000 Chk2 Mutated in Li-Fraumeni syndrome. Senses DNA double strand breaks and phosphorylates and stabilizes p53. Khanna and Jackson, 2001 Ref: Cell, 2002, 108:153-164
- 52. Tumor-derived mutations affecting apoptosis ProteinRole in apoptosis Ref Myc Deregulated expression in many cancers. Induces proliferation in the presence of survival factors, such as Bcl-2, and apoptosis in the absence of survival factors. Can sensitise cells to drug-induced apoptosis. Evan and Vousden, 2001 Akt Frequently amplified in solid tumors. Phosphorylates Bad. Hyperactivation induces resistance to a range of apoptotic stimuli including drugs. Datta et al., 1999 PI3K Overexpressed or deregulated in some cancers. Responsible for activation of Akt and downstream phosphorylation of Bad. Inhibition of PI3K enhances chemotherapeutic drug-induced apoptosis. Roymans and Slegers, 2001 Ras Mutated or deregulated in many cancers. Activates PI3K and downstream pathways. Induces proliferation and inhibits c-myc and drug-induced apoptosis. el-Deiry, 1997 Ref: Cell, 2002, 108:153-164
- 53. Tumor-derived mutations affecting apoptosis ProteinRole in apoptosis Ref FLIP Overexpressed in some cancers. Prevents activation of caspase-8 and apoptosis induced by some chemotherapeutic drugs. Tepper and Seldin, 1999 Ref: Cell, 2002, 108:153-164
- 54. Tumor advantages followingp53 mutations 1. Cell cycle - mutant cells are able to progress through the cell cycle and divide, passing on mutations. 2. Apoptosis - these cells ignore signals to commit cell suicide. 3. Genetic instability - continued division without checkpoints leads to chromosomal aberrations, incorrect rejoining of chromosomes activation of oncogenes and inactivation of tumor suppressor proteins
- 55. Therapeutic applications of regulatingapoptosis Promote apoptosis in cancer cells: Lymphoma/leukemia Oral cancer Brain tumors Prostate Colon etc. Prevent apoptosis in certain disorders and degenerative diseases: AIDS Ischemia Alzheimer's/Parkinson's etc.
- 56. Apoptosis in thetreatment of cancer An important goal of cancer drug development should be to facilitate apoptosis in neoplastic cells. Drugs that restore apoptosis might selectively kill cancer cells that have triggered a death signal and have become dependent on the deregulation of apoptosis pathways. Strategies already used: • Administration of death ligand • Bcl-2 family inhibitors • XIAP inhibitors Fesik Nat Rev Cancer (2005)
- 58. Ribozyme Inhibition ofBcl-2 Expression • Apoptosis removes damaged cells from the body. The bcl-2 gene prevents this. • The role of bcl-2 in oral cancer and glioblastoma is unexplored. • We constructed a hammerhead ribozyme that would digest the bcl-2 mRNA message and delivered it to oral cancer and glioblastoma cells with an adenovirus vector.
- 59. Ref: Nature, 412,865 - 866 (2001) AAV kills cancer cells
- 62. APOPTOSIS: Role inDisease AGING Aging --> both too much and too little apoptosis (evidence for both) Too much (accumulated oxidative damage?) ---> tissue degeneration Too little (defective sensors, signals? ---> dysfunctional cells accumulate hyperplasia (precancerous lesions)
- 65. „Don't think ofdeath as an ending. Think of it as a really effective way of cutting down your expenses.” Woody Allen
Editor's Notes
- #65 Caspases cleave keratin 18 at two sites during apoptosis. Cleavage at Asp396 generates a neo-epitope recognized by the monoclonal antibody M30.[6] This antibody does not recognize uncleaved K18 and is therefore specific for apoptotic epithelial cells. M30 Apoptosense ELISA utilizes a second monoclonal antibody (M5) which recognizes an epitope N-terminal from the M30 epitope.