Induction of lysosomal membrane permeabilization Induction of lysosomal membrane permeabilization by compounds that activate p53-independent by compounds that activate p53-independent

apoptosis.apoptosis.

Hamidye Erdal, Maria Berndtsson, Juan Castro, Ulf Brunk, Maria C. Hamidye Erdal, Maria Berndtsson, Juan Castro, Ulf Brunk, Maria C. Shoshan, and Stig Linder. 2005. PNAS 102(1): 192-197.Shoshan, and Stig Linder. 2005. PNAS 102(1): 192-197.

Presented by Linda E. Lewis, B.S.Presented by Linda E. Lewis, B.S.ForFor

Hallmarks of CancerHallmarks of CancerBiology 610Biology 610

The Death of a CellThe Death of a Cell Different types of cell deathDifferent types of cell death

ApoptosisApoptosis NecrosisNecrosis Parapoptosis (Necrapoptosis)Parapoptosis (Necrapoptosis)

Characteristics of Characteristics of ApoptosisApoptosis

Morphological Morphological ChangesChanges Plasma Membrane Plasma Membrane

BlebbingBlebbing Nuclear CompactionNuclear Compaction Chromatin Chromatin

CondensationCondensation Cell Body ShrinkageCell Body Shrinkage Formation of Formation of

membrane-bound membrane-bound apoptotic bodies apoptotic bodies

Biochemical ChangesBiochemical Changes Appearance of DNA Appearance of DNA

fragments due to fragments due to nucleosomal cleavagenucleosomal cleavage

Flipping of Flipping of phosphatidylserine phosphatidylserine from the inner leaflet from the inner leaflet to the outer leaflet of to the outer leaflet of the plasma the plasma membranemembrane

Cleavage of other Cleavage of other cellular proteins.cellular proteins.

Characteristics of NecrosisCharacteristics of Necrosis Mitochondrial SwellingMitochondrial Swelling Dilation of the endoplasmic reticulumDilation of the endoplasmic reticulum Extensive vacuolation of the Extensive vacuolation of the

cytoplasmcytoplasm Chromatin appears coarse and clumpyChromatin appears coarse and clumpy Karyolysis – disintegration of the Karyolysis – disintegration of the

nucleus.nucleus.

Characteristics of Characteristics of ParapoptosisParapoptosis

Does not involve activation of Does not involve activation of caspasescaspases

Morphologic changes more similar to Morphologic changes more similar to necrosis than apoptosisnecrosis than apoptosis No chromatin condensationNo chromatin condensation Extensive vacuolation of the cytoplasmExtensive vacuolation of the cytoplasm

Unlike necrosis, requires Unlike necrosis, requires de novode novo protein synthesis like apoptosisprotein synthesis like apoptosis

What is the benefit of What is the benefit of apoptosis over necrosis?apoptosis over necrosis?

In apoptosis, membrane bound vesicular In apoptosis, membrane bound vesicular apoptotic bodies are formed so cytotoxic apoptotic bodies are formed so cytotoxic material is not released into the intercellular material is not released into the intercellular space, preventing an inflammatory response.space, preventing an inflammatory response.

In necrosis, the cell contents are released In necrosis, the cell contents are released into the intercellular space, causing damage into the intercellular space, causing damage to neighboring cells and inducing an to neighboring cells and inducing an inflammatory response.inflammatory response.

Molecular Anatomy of a Molecular Anatomy of a DNA injury responseDNA injury response

Recognition of injured DNARecognition of injured DNA Period of damage assessment (enforced Period of damage assessment (enforced

by checkpoints)by checkpoints) Checkpoints provide an opportunity to monitor Checkpoints provide an opportunity to monitor

the necessity of apoptosis over repairthe necessity of apoptosis over repair They connect cellular processes They connect cellular processes They erect barriers to prevent replication of They erect barriers to prevent replication of

injured genomes which can be removed when injured genomes which can be removed when a cell has recovereda cell has recovered

Implementation of apoptosis or DNA repairImplementation of apoptosis or DNA repair

LysosomesLysosomes Lysosomes are membrane bound organelles located in the Lysosomes are membrane bound organelles located in the

cytosol of cells.cytosol of cells. The membrane is unique:The membrane is unique:

It enables the final digestion products of macromolecules It enables the final digestion products of macromolecules (amino acids, sugars, and nucleotides) to be transported (amino acids, sugars, and nucleotides) to be transported into the cytosol where they are excreted or reused by the into the cytosol where they are excreted or reused by the cell.cell.

It protects the cytosol from acid hydrolases, but if they leak It protects the cytosol from acid hydrolases, but if they leak out minimal damage is done because the cytosolic pH is out minimal damage is done because the cytosolic pH is 7.2-7.4 and these enzymes function optimally at acidic pH.7.2-7.4 and these enzymes function optimally at acidic pH.

HH++ pump uses ATP hydrolysis to pump H pump uses ATP hydrolysis to pump H++ into the lysosome into the lysosome to maintain the acidic environment.to maintain the acidic environment.

Can degrade monoubiquinated proteins that do now enter the Can degrade monoubiquinated proteins that do now enter the proteasome.proteasome.

ProteasesProteases NucleasesNucleases GlycosidasesGlycosidases LipasesLipases PhospholipasesPhospholipases PhosphatasesPhosphatases SulfatasesSulfatases

Acid hydrolases Acid hydrolases require an acidic require an acidic environment for environment for efficient function.efficient function.

The lysosome The lysosome maintains a pH maintains a pH close to 5.0.close to 5.0.

There are approximately 40 types of hydrolases There are approximately 40 types of hydrolases contained in lysosomes, all of them are acid contained in lysosomes, all of them are acid hydrolases.hydrolases.

Lysosomal Rupture and ApoptosisLysosomal Rupture and Apoptosis

Activation of pro-apoptotic tumor suppressor p53 results in early Activation of pro-apoptotic tumor suppressor p53 results in early lysosomal rupture. lysosomal rupture.

In oxidant-induced apoptosis, lysosomal rupture occurs in 2 In oxidant-induced apoptosis, lysosomal rupture occurs in 2 phases:phases: Release of Cathepsins and other hydrolytic enzymesRelease of Cathepsins and other hydrolytic enzymes Activation of phospholipase A2 (PLA2) and production of free Activation of phospholipase A2 (PLA2) and production of free

arachidonic acidarachidonic acid Overexpression of anti-apoptotic protein Bcl-2 inhibits the second Overexpression of anti-apoptotic protein Bcl-2 inhibits the second

phase of lysosomal rupture (activation of PLA2) and the release phase of lysosomal rupture (activation of PLA2) and the release of cytochrome C.of cytochrome C.

In apoptosis caused by non-oxidative agents, increased In apoptosis caused by non-oxidative agents, increased intracellular generation of reactive oxygen species (ROS), intracellular generation of reactive oxygen species (ROS), probably produced by the mitochondria, like exogenously added probably produced by the mitochondria, like exogenously added oxidants may act through the lysosomal destabilization pathway.oxidants may act through the lysosomal destabilization pathway.

Released lysosomal enzymes, both directly and indirectly Released lysosomal enzymes, both directly and indirectly (through activation of PLA2) may trigger enhanced (through activation of PLA2) may trigger enhanced mitochondrial production of superoxide anion and Hmitochondrial production of superoxide anion and H22OO22 resulting in the release of cyt c.resulting in the release of cyt c.Release of lysosomal enzymes into the cytoplasm may:Release of lysosomal enzymes into the cytoplasm may:

Attack mitochondria directly by inducing the release of Attack mitochondria directly by inducing the release of cyt ccyt cDirectly and/or indirectly cause enhanced formation of Directly and/or indirectly cause enhanced formation of mitochondrial ROS and further pro-oxidant-induced mitochondrial ROS and further pro-oxidant-induced lysosomal destabilization.lysosomal destabilization.Activate lytic pro-enzymes such as PLA2, which in turn Activate lytic pro-enzymes such as PLA2, which in turn attacks both mitochondria and lysosomes.attacks both mitochondria and lysosomes.Activate Bid and/or other pro-apoptotic proteins.Activate Bid and/or other pro-apoptotic proteins.Activate pro-caspases.Activate pro-caspases.

Lysosomal Lysosomal PermeabilizationPermeabilization

Lysosomal Permeabilization is the Lysosomal Permeabilization is the selective release of proteases into the selective release of proteases into the cytosol.cytosol.

It is hypothesized that this release can It is hypothesized that this release can cause permeabilization in other cause permeabilization in other lysosomes as well.lysosomes as well.

Sometimes proteases can leak out into Sometimes proteases can leak out into the cytosol, these can be controlled by the cytosol, these can be controlled by inhibitors called stefins (aka cystatins).inhibitors called stefins (aka cystatins).

Induction of LysosomalInduction of Lysosomal PermeabilizationPermeabilization

P53 activationP53 activation Increased cellular generation of Increased cellular generation of

ROSROS Sphingosine/ceramide apoptotic Sphingosine/ceramide apoptotic

pathwaypathway Exposure to Ciprofloxacin or Exposure to Ciprofloxacin or

hydroxychloroquinehydroxychloroquine

Cathepsins B and DCathepsins B and D Cathepsin B is a cysteine protease Cathepsin B is a cysteine protease

and is involved in tumor cell and is involved in tumor cell apoptosis via TNF-apoptosis via TNF-, and cleavage , and cleavage of Bid to tBid.of Bid to tBid.

Cathepsin D is an aspartyl protease Cathepsin D is an aspartyl protease and is involved in caspase and is involved in caspase activation.activation.

Organelle-Specific Induction of Organelle-Specific Induction of ApoptosisApoptosis

P53 ApoptosisP53 Apoptosis

P53 Signaling in ApoptosisP53 Signaling in Apoptosis DNA damage is sensed by a member of the ATM DNA damage is sensed by a member of the ATM

family (includes ATM (ataxia telangiectasia family (includes ATM (ataxia telangiectasia mutated), DNA-PK, and ATR (ataxia mutated), DNA-PK, and ATR (ataxia telangectasia Rad 3 related))causing activation telangectasia Rad 3 related))causing activation of a checkpoint and phosphorylation of p53.of a checkpoint and phosphorylation of p53.

P53 levels increase within minutes of DNA P53 levels increase within minutes of DNA damage and first apoptotic events occur within damage and first apoptotic events occur within a few hours.a few hours.

Several cell cycle Several cell cycle regulators are induced regulators are induced by p53:by p53: P21P21 GADD45GADD45 14-3-3 family14-3-3 family

Other proteins are Other proteins are induced as well:induced as well: BaxBax CD95 (Fas)CD95 (Fas) DR5 (receptor for DR5 (receptor for

TRAIL)TRAIL) All classical members All classical members

of the core apoptosis of the core apoptosis pathwayspathways

Lysosomal ApoptosisLysosomal Apoptosis

National Cancer InstituteNational Cancer InstituteDevelopmental Therapeutics ProgramDevelopmental Therapeutics Program

The Developmental Therapeutics Program at NCI The Developmental Therapeutics Program at NCI facilitates the discovery of chemotherapeutic agents facilitates the discovery of chemotherapeutic agents for cancer and AIDS.for cancer and AIDS.

There are nine branches involved in this process:There are nine branches involved in this process: Grants and Contract Operations BranchGrants and Contract Operations Branch Drug Synthesis and Chemistry BranchDrug Synthesis and Chemistry Branch Natural Products BranchNatural Products Branch Pharmaceutical Resources BranchPharmaceutical Resources Branch Biological Testing BranchBiological Testing Branch Toxicology and Pharmacology BranchToxicology and Pharmacology Branch Information Technology BranchInformation Technology Branch Biological Resources BranchBiological Resources Branch Screening Technology BranchScreening Technology Branch

Biological Testing BranchBiological Testing Branch The mission of the Biological Testing Branch has 5 The mission of the Biological Testing Branch has 5

components:components: To plan, direct and implement a contract supported To plan, direct and implement a contract supported

program to screen compounds for indications of clinical program to screen compounds for indications of clinical efficacy efficacy in vivoin vivo..

To develop new screening models.To develop new screening models. To produce, provide quality control for, and distribute to To produce, provide quality control for, and distribute to

NCI, NIH, and grantee community genetically and NCI, NIH, and grantee community genetically and biologically defined rodents.biologically defined rodents.

To maintain a repository of experimental and human tumor To maintain a repository of experimental and human tumor cell lines for use in research performed by the program and cell lines for use in research performed by the program and other qualified investigators.other qualified investigators.

To define and publish biological testing screening protocols.To define and publish biological testing screening protocols.

Toxicology and Toxicology and Pharmacology BranchPharmacology Branch

The primary function of this branch is to obtain the The primary function of this branch is to obtain the toxicology and pharmacology data necessary for NCI toxicology and pharmacology data necessary for NCI to file an Investigational New Drug application with to file an Investigational New Drug application with the FDA in order to conduct Phase I clinical trials of the FDA in order to conduct Phase I clinical trials of new oncolytic agents in humans.new oncolytic agents in humans.

This is accomplished by working with other agencies This is accomplished by working with other agencies to:to: Determine or develop analytical methods for Determine or develop analytical methods for

quantifying drug levels in biological fluids and tissues.quantifying drug levels in biological fluids and tissues. Plasma drug distribution and elimination kinetics in Plasma drug distribution and elimination kinetics in

animal models.animal models. Plasma protein binding and stability.Plasma protein binding and stability. Metabolic potential.Metabolic potential. Maximum tolerated doses and dose-limiting toxicity.Maximum tolerated doses and dose-limiting toxicity.

Biological Resources Biological Resources BranchBranch Conducts studies to assess effects of Conducts studies to assess effects of

novel biological agents and their novel biological agents and their relationships with anti-tumor activity.relationships with anti-tumor activity.

NCI Repository distributes selected NCI Repository distributes selected agents for peer review preclinical agents for peer review preclinical studies.studies.

Production and Production and in vivoin vivo evaluation of evaluation of monoclonal antibodies, monoclonal antibodies, immunoconjugates, and otherimmunoconjugates, and other biologicals.biologicals.

National Cancer National Cancer InstituteInstituteMechanistic SetMechanistic Set

Consists of 879 compounds.Consists of 879 compounds. These compounds were screened These compounds were screened

for growth inhibitory activity on 60 for growth inhibitory activity on 60 different human tumor cell lines.different human tumor cell lines.

Growth inhibitory effect is Growth inhibitory effect is measured by GImeasured by GI5050 which is the dose which is the dose needed to cause 50% growth needed to cause 50% growth inhibition.inhibition.

Experimental Experimental ProceduresProcedures

MaterialsMaterials NCI Mechanistic SetNCI Mechanistic Set ReagentsReagents

Pepstatin A – strong inhibitor of acidic proteasesPepstatin A – strong inhibitor of acidic proteases Cisplatin – alkylating agent that inhibits cell Cisplatin – alkylating agent that inhibits cell

growthgrowth Doxorubicin – anthracycline glycoside that Doxorubicin – anthracycline glycoside that

impairs DNA synthesisimpairs DNA synthesis Thapsigargin – weak tumor promoter with Thapsigargin – weak tumor promoter with

structural properties similar to TPA.structural properties similar to TPA. Ciprofloxacin – fluoroquinolone antibiotic that Ciprofloxacin – fluoroquinolone antibiotic that

inhibits tumor cell growth and induces apoptosisinhibits tumor cell growth and induces apoptosis

ProceduresProcedures Assessment of Cell Viability and ApoptosisAssessment of Cell Viability and Apoptosis

M30 ELISA and M65 ELISAM30 ELISA and M65 ELISA Caspase-Glo 3/7Caspase-Glo 3/7

Preparation of Cytoplasts (enucleated cells)Preparation of Cytoplasts (enucleated cells) Western Blot AnalysisWestern Blot Analysis

SDS/PAGE gel electrophoresisSDS/PAGE gel electrophoresis Anti-p53 antibodiesAnti-p53 antibodies Anti-mouse pro-caspase-12 antibodiesAnti-mouse pro-caspase-12 antibodies Anti-GRP78Anti-GRP78 Anti-GRP94Anti-GRP94 Anti-tubulin as standardAnti-tubulin as standard

Flow Cytometric AnalysisFlow Cytometric Analysis FITC-conjugated DAKO A/S antibodyFITC-conjugated DAKO A/S antibody Antibody staining for Antibody staining for -H2AX-H2AX

Immunofluorescence StainingImmunofluorescence Staining Anti-p53 antibodiesAnti-p53 antibodies Anti-Cathepsin B antibodiesAnti-Cathepsin B antibodies Anti-Cathepsin D antibodiesAnti-Cathepsin D antibodies

Acridine Orange StainingAcridine Orange Staining Lysosomotropic metachromatic Lysosomotropic metachromatic

fluorophorefluorophore Red fluorescence in the lysosome, Red fluorescence in the lysosome,

green fluorescence when released green fluorescence when released from lysosome.from lysosome.

M30-Apoptosense ELISAM30-Apoptosense ELISA Cytokeratin 18 (CK18) containing M30 neo-epitope is Cytokeratin 18 (CK18) containing M30 neo-epitope is

generated in epithelial cells or tissues as a result of generated in epithelial cells or tissues as a result of caspase activation (cleavage).caspase activation (cleavage).

M30 is a monoclonal antibody that specifically recognizes M30 is a monoclonal antibody that specifically recognizes apoptotic (not necrotic or viable) epithelial cells.apoptotic (not necrotic or viable) epithelial cells.

In this assay, the neo-epitope is determined by horseradish In this assay, the neo-epitope is determined by horseradish peroxidase labeled M30 (neo-epitope at the C-terminal peroxidase labeled M30 (neo-epitope at the C-terminal domain of CK18).domain of CK18).

The M65 ELISA measures soluble CK18 released The M65 ELISA measures soluble CK18 released extracellularly from dying cells. It is used to assess overall extracellularly from dying cells. It is used to assess overall cell death (apoptosis and necrosis) to determine the cell death (apoptosis and necrosis) to determine the relative contribution of apoptosis to the total degree of cell relative contribution of apoptosis to the total degree of cell death.death.

Figure from Peviva

M30-Apoptosense ELISA

Table 1. Summary of the biological effects of selected compounds

1 69187 9-methoxy-ellipticine

+ + ++ – – – –

2 622627 + + + + + – –3 102811 Formycin A + + + + – – –4 72961 8-azaadenosine + + + + – – –5 93419 Steffimycin + + ++ + – – –6 651079 – + ++ + + + –7 687852 – + + + + + –8 106408 Anthramycin

meth.-ether– + + + – – –

9 267461 Nanaomycin – + + – + + –10 13973 Acridine yellow – + ++ – + – –11 166381 – + – – – – –12 24819 Peltatin A – + ++ – – – –13 10010 – + + – + – –14 647889 – + + – + + –15 285116 Siomycin A – + + – + + +16 313981 Gold Cl-3-

ethylphosphine– + + + + + –

17 140377 Arnebin – + + – + – –18 157389 – + – – + – –19 85236 Helenalin – + – – + + –20 24818 Podophyllotoxin – + + – – – –

* p53-supported apoptosis was defined as 2-fold higher CK18 cleavage in HCT116 WT cells (area under curve in Fig. 2) Drugs that showed Bax-supported apoptosis induced <10% caspase activation in HT116 Bax (null) cells compared with HCT116 control cells See Fig. 5 for details ER, endoplasmic reticulum. Induction of GRP78 in HCT116 p53 WT cells; cleavage of caspase-12 in mouse CT51 cells

LMP

Apoptosis supported by

ER stressNo. Compound NSC no.

Compound name

p53* BAX Induction of p53

DNA damage

Cytoplasmic target

Erdal,Hamdiye et al. (2005) Proc. Natl. Acad. Sci. USA 102, 192-197

Copyright ©2005 by the National Academy of Sciences

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

Copyright ©2005 by the National Academy of Sciences

Erdal, Hamdiye et al. (2005) Proc. Natl. Acad. Sci. USA 102, 192-197

Fig. 2. Dose-response curves of CK18 cleavage of 20 compounds selected from the mechanistic set

Caspase-GLO 3/7 AssayCaspase-GLO 3/7 Assay This assay measures activity of This assay measures activity of

executioner caspases 3 and 7.executioner caspases 3 and 7. When reagent (aminoluciferin) is When reagent (aminoluciferin) is

cleaved by caspase it reacts with cleaved by caspase it reacts with the enzyme luciferase to produce a the enzyme luciferase to produce a glo (firefly). glo (firefly).

Copyright ©2005 by the National Academy of Sciences

Erdal, Hamdiye et al. (2005) Proc. Natl. Acad. Sci. USA 102, 192-197

Fig. 4. Induction of caspase-3 activation in enucleated cells

What Does Caspase-3 Do?What Does Caspase-3 Do? Activated Caspase-3 cleaves DFF (DNA fragmentation Activated Caspase-3 cleaves DFF (DNA fragmentation

factor complex) which results in chromatin condensation factor complex) which results in chromatin condensation and nuclear DNA fragmentation.and nuclear DNA fragmentation.

Activates Acinus which causes chromatin condensation Activates Acinus which causes chromatin condensation without DNA fragmentation.without DNA fragmentation.

Cleaves Gelsolin (an actin protein) causing actin Cleaves Gelsolin (an actin protein) causing actin reorganization and ultimately membrane blebbing.reorganization and ultimately membrane blebbing.

Interacts with Interacts with -fodrin and FAK (focal adhesion kinase) -fodrin and FAK (focal adhesion kinase) resulting in cell body shrinkage, detachment from resulting in cell body shrinkage, detachment from neighboring cells, and detachment from the basement neighboring cells, and detachment from the basement membrane.membrane.

Interacts with PAK2 resulting in formation of apoptotic Interacts with PAK2 resulting in formation of apoptotic bodies containing condensed cytoplasmic material from bodies containing condensed cytoplasmic material from fragmented apoptotic cells.fragmented apoptotic cells.

Inactivates Cdc27 ubiquitin ligase complex, Inactivates Cdc27 ubiquitin ligase complex, which mediates degradation of mitotic cyclinswhich mediates degradation of mitotic cyclins

Inactivates Weel kinase, inhibiting Inactivates Weel kinase, inhibiting phosphorylation on Cdksphosphorylation on Cdks

Interacts with p21Cipl and p27 Kip1 to decrease their Interacts with p21Cipl and p27 Kip1 to decrease their association with Cdks, allowing CDK activity to accumulate association with Cdks, allowing CDK activity to accumulate during apoptosisduring apoptosis Cleavage of cell cycle regulators occurs late in apoptosis Cleavage of cell cycle regulators occurs late in apoptosis

via caspase-3-like activities that coincide with dismantling via caspase-3-like activities that coincide with dismantling of transcription and translation machineryof transcription and translation machinery

Caspase activated CDK activity cannot activate normal Caspase activated CDK activity cannot activate normal mitotic program, spindles do not form in apoptotic cells mitotic program, spindles do not form in apoptotic cells leading to mitotic catastropheleading to mitotic catastrophe

Mitotic catastrophe – cell division without completion of S Mitotic catastrophe – cell division without completion of S phase of c3ell cycles, results in cell body shrinkage, DNA phase of c3ell cycles, results in cell body shrinkage, DNA condensation, nuclear envelope breakdown and cell death.condensation, nuclear envelope breakdown and cell death.

Inactivates PARP enzyme activity, turning off energetically Inactivates PARP enzyme activity, turning off energetically expensive repair pathway following commitment to apoptosis.expensive repair pathway following commitment to apoptosis.

Copyright ©2005 by the National Academy of Sciences

Erdal, Hamdiye et al. (2005) Proc. Natl. Acad. Sci. USA 102, 192-197

Fig. 3. Induction of p53 expression and DNA damage

Copyright ©2005 by the National Academy of Sciences

Erdal, Hamdiye et al. (2005) Proc. Natl. Acad. Sci. USA 102, 192-197

Fig. 5. Induction of LMP

Copyright ©2005 by the National Academy of Sciences

Erdal, Hamdiye et al. (2005) Proc. Natl. Acad. Sci. USA 102, 192-197

Fig. 6. NCS267461 induces necrosis at higher concentrations

Key PointsKey Points Compounds that induce p53 expression Compounds that induce p53 expression

do not always induce p53 apoptosis.do not always induce p53 apoptosis. P53-independent apoptosis can be P53-independent apoptosis can be

induced as a result of Lysosomal induced as a result of Lysosomal Membrane Permeabilization.Membrane Permeabilization.

Lysosomal Membrane Permeabilization Lysosomal Membrane Permeabilization may be an important target in the may be an important target in the development of chemotherapeutic development of chemotherapeutic agents against p53 resistant tumors.agents against p53 resistant tumors.

ReferencesReferences Erdal, Hamdiye; Berndtsson, Maria; Castro, Juan; Brunk, Ulf; Erdal, Hamdiye; Berndtsson, Maria; Castro, Juan; Brunk, Ulf;

Shoshan, Maria C. and Linder, Stig. Induction of lysosomal Shoshan, Maria C. and Linder, Stig. Induction of lysosomal membrane permeabilization by compounds that activate p53-membrane permeabilization by compounds that activate p53-independent apoptosis.independent apoptosis.PNASPNAS 102102(1):192-197 (2005).(1):192-197 (2005).

Cirman, Tina; Oresic, Kristina; Mazovec, Gabriela Droga; Turk, Cirman, Tina; Oresic, Kristina; Mazovec, Gabriela Droga; Turk, Vito; Reed, John C.; Myers, Richard M.; Salvesen, Guy S. and Vito; Reed, John C.; Myers, Richard M.; Salvesen, Guy S. and Turk, Boris. Selective Disruption of Lysosomes in HeLa Cells Turk, Boris. Selective Disruption of Lysosomes in HeLa Cells Triggers Apoptosis Mediated by Cleavage of Bid by Multiple Triggers Apoptosis Mediated by Cleavage of Bid by Multiple Papain-like Lysosomal Cathepsins. Papain-like Lysosomal Cathepsins. J. Biol. Chem.J. Biol. Chem. 279279(5):3578-(5):3578-3587 (2004).3587 (2004).

Ferri, Karine F. and Kroemer, Guido. Organelle-specific Ferri, Karine F. and Kroemer, Guido. Organelle-specific initiation of cell death pathways. initiation of cell death pathways. Nature Cell Biol. Nature Cell Biol. 33:E255-:E255-E263 (2001).E263 (2001).

Hengartner, Michael O. The Biochemistry of Apoptosis. Hengartner, Michael O. The Biochemistry of Apoptosis. NatureNature 407407:770-775 (2000). :770-775 (2000).

National Cancer Institute National Cancer Institute www.dtp.nci.nih.gov.www.dtp.nci.nih.gov.