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Induction of lysosomal membrane permeabilization by compounds that activate p53-independent apoptosis. Hamidye Erdal, Maria Berndtsson, Juan Castro, Ulf Brunk, Maria C. Shoshan, and Stig Linder. 2005. PNAS 102(1): 192-197. Presented by Linda E. Lewis, B.S. For Hallmarks of Cancer Biology 610 The Death of a Cell Different types of cell death Apoptosis Necrosis Parapoptosis (Necrapoptosis) Characteristics of Apoptosis Morphological Changes Plasma Membrane Blebbing Nuclear Compaction Chromatin Condensation Cell Body Shrinkage Formation of membranebound apoptotic bodies Biochemical Changes Appearance of DNA fragments due to nucleosomal cleavage Flipping of phosphatidylserine from the inner leaflet to the outer leaflet of the plasma membrane Cleavage of other cellular proteins. Characteristics of Necrosis Mitochondrial Swelling Dilation of the endoplasmic reticulum Extensive vacuolation of the cytoplasm Chromatin appears coarse and clumpy Karyolysis – disintegration of the nucleus. Characteristics of Parapoptosis Does not involve activation of caspases Morphologic changes more similar to necrosis than apoptosis No chromatin condensation Extensive vacuolation of the cytoplasm Unlike necrosis, requires de novo protein synthesis like apoptosis What is the benefit of apoptosis over necrosis? In apoptosis, membrane bound vesicular apoptotic bodies are formed so cytotoxic material is not released into the intercellular space, preventing an inflammatory response. In necrosis, the cell contents are released into the intercellular space, causing damage to neighboring cells and inducing an inflammatory response. Molecular Anatomy of a DNA injury response Recognition of injured DNA Period of damage assessment (enforced by checkpoints) Checkpoints provide an opportunity to monitor the necessity of apoptosis over repair They connect cellular processes They erect barriers to prevent replication of injured genomes which can be removed when a cell has recovered Implementation of apoptosis or DNA repair Lysosomes Lysosomes are membrane bound organelles located in the cytosol of cells. The membrane is unique: It enables the final digestion products of macromolecules (amino acids, sugars, and nucleotides) to be transported into the cytosol where they are excreted or reused by the cell. It protects the cytosol from acid hydrolases, but if they leak out minimal damage is done because the cytosolic pH is 7.27.4 and these enzymes function optimally at acidic pH. H+ pump uses ATP hydrolysis to pump H+ into the lysosome to maintain the acidic environment. Can degrade monoubiquinated proteins that do now enter the proteasome. There are approximately 40 types of hydrolases contained in lysosomes, all of them are acid hydrolases. Proteases Nucleases Glycosidases Lipases Phospholipases Phosphatases Sulfatases Acid hydrolases require an acidic environment for efficient function. The lysosome maintains a pH close to 5.0. Lysosomal Rupture and Apoptosis Activation of pro-apoptotic tumor suppressor p53 results in early lysosomal rupture. In oxidant-induced apoptosis, lysosomal rupture occurs in 2 phases: Release of Cathepsins and other hydrolytic enzymes Activation of phospholipase A2 (PLA2) and production of free arachidonic acid Overexpression of anti-apoptotic protein Bcl-2 inhibits the second phase of lysosomal rupture (activation of PLA2) and the release of cytochrome C. In apoptosis caused by non-oxidative agents, increased intracellular generation of reactive oxygen species (ROS), probably produced by the mitochondria, like exogenously added oxidants may act through the lysosomal destabilization pathway. Released lysosomal enzymes, both directly and indirectly (through activation of PLA2) may trigger enhanced mitochondrial production of superoxide anion and H2O2 resulting in the release of cyt c. Release of lysosomal enzymes into the cytoplasm may: Attack mitochondria directly by inducing the release of cyt c Directly and/or indirectly cause enhanced formation of mitochondrial ROS and further pro-oxidant-induced lysosomal destabilization. Activate lytic pro-enzymes such as PLA2, which in turn attacks both mitochondria and lysosomes. Activate Bid and/or other pro-apoptotic proteins. Activate pro-caspases. Lysosomal Permeabilization Lysosomal Permeabilization is the selective release of proteases into the cytosol. It is hypothesized that this release can cause permeabilization in other lysosomes as well. Sometimes proteases can leak out into the cytosol, these can be controlled by inhibitors called stefins (aka cystatins). Induction of Lysosomal Permeabilization P53 activation Increased cellular generation of ROS Sphingosine/ceramide apoptotic pathway Exposure to Ciprofloxacin or hydroxychloroquine Cathepsins B and D Cathepsin B is a cysteine protease and is involved in tumor cell apoptosis via TNF-, and cleavage of Bid to tBid. Cathepsin D is an aspartyl protease and is involved in caspase activation. Organelle-Specific Induction of Apoptosis P53 Apoptosis P53 Signaling in Apoptosis DNA damage is sensed by a member of the ATM family (includes ATM (ataxia telangiectasia mutated), DNA-PK, and ATR (ataxia telangectasia Rad 3 related))causing activation of a checkpoint and phosphorylation of p53. P53 levels increase within minutes of DNA damage and first apoptotic events occur within a few hours. Several cell cycle regulators are induced by p53: P21 GADD45 14-3-3 family Other proteins are induced as well: Bax CD95 (Fas) DR5 (receptor for TRAIL) All classical members of the core apoptosis pathways Lysosomal Apoptosis National Cancer Institute Developmental Therapeutics Program The Developmental Therapeutics Program at NCI facilitates the discovery of chemotherapeutic agents for cancer and AIDS. There are nine branches involved in this process: Grants and Contract Operations Branch Drug Synthesis and Chemistry Branch Natural Products Branch Pharmaceutical Resources Branch Biological Testing Branch Toxicology and Pharmacology Branch Information Technology Branch Biological Resources Branch Screening Technology Branch Biological Testing Branch The mission of the Biological Testing Branch has 5 components: To plan, direct and implement a contract supported program to screen compounds for indications of clinical efficacy in vivo. To develop new screening models. To produce, provide quality control for, and distribute to NCI, NIH, and grantee community genetically and biologically defined rodents. To maintain a repository of experimental and human tumor cell lines for use in research performed by the program and other qualified investigators. To define and publish biological testing screening protocols. Toxicology and Pharmacology Branch The primary function of this branch is to obtain the toxicology and pharmacology data necessary for NCI to file an Investigational New Drug application with the FDA in order to conduct Phase I clinical trials of new oncolytic agents in humans. This is accomplished by working with other agencies to: Determine or develop analytical methods for quantifying drug levels in biological fluids and tissues. Plasma drug distribution and elimination kinetics in animal models. Plasma protein binding and stability. Metabolic potential. Maximum tolerated doses and dose-limiting toxicity. Biological Resources Branch Conducts studies to assess effects of novel biological agents and their relationships with anti-tumor activity. NCI Repository distributes selected agents for peer review preclinical studies. Production and in vivo evaluation of monoclonal antibodies, immunoconjugates, and other biologicals. National Cancer Institute Mechanistic Set Consists of 879 compounds. These compounds were screened for growth inhibitory activity on 60 different human tumor cell lines. Growth inhibitory effect is measured by GI50 which is the dose needed to cause 50% growth inhibition. Experimental Procedures Materials NCI Mechanistic Set Reagents Pepstatin A – strong inhibitor of acidic proteases Cisplatin – alkylating agent that inhibits cell growth Doxorubicin – anthracycline glycoside that impairs DNA synthesis Thapsigargin – weak tumor promoter with structural properties similar to TPA. Ciprofloxacin – fluoroquinolone antibiotic that inhibits tumor cell growth and induces apoptosis Procedures Assessment of Cell Viability and Apoptosis M30 ELISA and M65 ELISA Caspase-Glo 3/7 Preparation of Cytoplasts (enucleated cells) Western Blot Analysis SDS/PAGE gel electrophoresis Anti-p53 antibodies Anti-mouse pro-caspase-12 antibodies Anti-GRP78 Anti-GRP94 Anti-tubulin as standard Flow Cytometric Analysis FITC-conjugated DAKO A/S antibody Antibody staining for -H2AX Immunofluorescence Staining Anti-p53 antibodies Anti-Cathepsin B antibodies Anti-Cathepsin D antibodies Acridine Orange Staining Lysosomotropic metachromatic fluorophore Red fluorescence in the lysosome, green fluorescence when released from lysosome. M30-Apoptosense ELISA Cytokeratin 18 (CK18) containing M30 neo-epitope is generated in epithelial cells or tissues as a result of caspase activation (cleavage). M30 is a monoclonal antibody that specifically recognizes apoptotic (not necrotic or viable) epithelial cells. In this assay, the neo-epitope is determined by horseradish peroxidase labeled M30 (neo-epitope at the C-terminal domain of CK18). The M65 ELISA measures soluble CK18 released extracellularly from dying cells. It is used to assess overall cell death (apoptosis and necrosis) to determine the relative contribution of apoptosis to the total degree of cell death. M30-Apoptosense ELISA Figure from Peviva Erdal,Hamdiye et al. (2005) Proc. Natl. Acad. Sci. USA 102, 192-197 Fig. 1. Graphic representation of apoptosis induction by the compounds in the NCI mechanistic set Erdal,Hamdiye et al. (2005) Proc. Natl. Acad. Sci. USA 102, 192-197 Copyright ©2005 by the National Academy of Sciences Fig. 2. Dose-response curves of CK18 cleavage of 20 compounds selected from the mechanistic set Erdal, Hamdiye et al. (2005) Proc. Natl. Acad. Sci. USA 102, 192-197 Copyright ©2005 by the National Academy of Sciences Caspase-GLO 3/7 Assay This assay measures activity of executioner caspases 3 and 7. When reagent (aminoluciferin) is cleaved by caspase it reacts with the enzyme luciferase to produce a glo (firefly). Fig. 4. Induction of caspase-3 activation in enucleated cells Erdal, Hamdiye et al. (2005) Proc. Natl. Acad. Sci. USA 102, 192-197 Copyright ©2005 by the National Academy of Sciences What Does Caspase-3 Do? Activated Caspase-3 cleaves DFF (DNA fragmentation factor complex) which results in chromatin condensation and nuclear DNA fragmentation. Activates Acinus which causes chromatin condensation without DNA fragmentation. Cleaves Gelsolin (an actin protein) causing actin reorganization and ultimately membrane blebbing. Interacts with -fodrin and FAK (focal adhesion kinase) resulting in cell body shrinkage, detachment from neighboring cells, and detachment from the basement membrane. Interacts with PAK2 resulting in formation of apoptotic bodies containing condensed cytoplasmic material from fragmented apoptotic cells. Inactivates Cdc27 ubiquitin ligase complex, which mediates degradation of mitotic cyclins Inactivates Weel kinase, inhibiting phosphorylation on Cdks Interacts with p21Cipl and p27 Kip1 to decrease their association with Cdks, allowing CDK activity to accumulate during apoptosis Cleavage of cell cycle regulators occurs late in apoptosis via caspase-3-like activities that coincide with dismantling of transcription and translation machinery Caspase activated CDK activity cannot activate normal mitotic program, spindles do not form in apoptotic cells leading to mitotic catastrophe Mitotic catastrophe – cell division without completion of S phase of c3ell cycles, results in cell body shrinkage, DNA condensation, nuclear envelope breakdown and cell death. Inactivates PARP enzyme activity, turning off energetically expensive repair pathway following commitment to apoptosis. Fig. 3. Induction of p53 expression and DNA damage Erdal, Hamdiye et al. (2005) Proc. Natl. Acad. Sci. USA 102, 192-197 Copyright ©2005 by the National Academy of Sciences Fig. 5. Induction of LMP Erdal, Hamdiye et al. (2005) Proc. Natl. Acad. Sci. USA 102, 192-197 Copyright ©2005 by the National Academy of Sciences Fig. 6. NCS267461 induces necrosis at higher concentrations Erdal, Hamdiye et al. (2005) Proc. Natl. Acad. Sci. USA 102, 192-197 Copyright ©2005 by the National Academy of Sciences Key Points Compounds that induce p53 expression do not always induce p53 apoptosis. P53-independent apoptosis can be induced as a result of Lysosomal Membrane Permeabilization. Lysosomal Membrane Permeabilization may be an important target in the development of chemotherapeutic agents against p53 resistant tumors. References Erdal, Hamdiye; Berndtsson, Maria; Castro, Juan; Brunk, Ulf; Shoshan, Maria C. and Linder, Stig. Induction of lysosomal membrane permeabilization by compounds that activate p53independent apoptosis.PNAS 102(1):192-197 (2005). Cirman, Tina; Oresic, Kristina; Mazovec, Gabriela Droga; Turk, Vito; Reed, John C.; Myers, Richard M.; Salvesen, Guy S. and Turk, Boris. Selective Disruption of Lysosomes in HeLa Cells Triggers Apoptosis Mediated by Cleavage of Bid by Multiple Papain-like Lysosomal Cathepsins. J. Biol. Chem. 279(5):35783587 (2004). Ferri, Karine F. and Kroemer, Guido. Organelle-specific initiation of cell death pathways. Nature Cell Biol. 3:E255-E263 (2001). Hengartner, Michael O. The Biochemistry of Apoptosis. Nature 407:770-775 (2000). National Cancer Institute www.dtp.nci.nih.gov.