Transcript Document

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
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Different types of cell death
Apoptosis
 Necrosis
 Parapoptosis (Necrapoptosis)
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Characteristics of Apoptosis
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Morphological Changes
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Plasma Membrane
Blebbing
Nuclear Compaction
Chromatin Condensation
Cell Body Shrinkage
Formation of membranebound apoptotic bodies
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Biochemical Changes
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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.
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Characteristics of Parapoptosis
Does not involve activation of caspases
 Morphologic changes more similar to
necrosis than apoptosis
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No chromatin condensation
 Extensive vacuolation of the cytoplasm
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Unlike necrosis, requires de novo
protein synthesis like apoptosis
What is the benefit of
apoptosis over necrosis?
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In apoptosis, membrane bound vesicular
apoptotic bodies are formed so cytotoxic
material is not released into the intercellular
space, preventing an inflammatory response.
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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)
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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
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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.
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Proteases
Nucleases
Glycosidases
Lipases
Phospholipases
Phosphatases
Sulfatases
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Acid hydrolases
require an acidic
environment for
efficient function.
The lysosome
maintains a pH close
to 5.0.
Lysosomal Rupture and Apoptosis
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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).
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Induction of Lysosomal Permeabilization
P53 activation
 Increased cellular generation of ROS
 Sphingosine/ceramide apoptotic
pathway
 Exposure to Ciprofloxacin or
hydroxychloroquine
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Cathepsins B and D
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Cathepsin B is a cysteine protease and
is involved in tumor cell apoptosis via
TNF-, and cleavage of Bid to tBid.
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Cathepsin D is an aspartyl protease and
is involved in caspase activation.
Organelle-Specific Induction of Apoptosis
P53 Apoptosis
P53 Signaling in Apoptosis
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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.
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P53 levels increase within minutes of DNA damage
and first apoptotic events occur within a few hours.
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Several cell cycle regulators
are induced by p53:
 P21
 GADD45
 14-3-3 family
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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
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The Developmental Therapeutics Program at NCI
facilitates the discovery of chemotherapeutic agents for
cancer and AIDS.
There are nine branches involved in this process:
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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
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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
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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:
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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.
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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.
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Experimental Procedures
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Materials
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NCI Mechanistic Set
Reagents
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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
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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
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Flow Cytometric Analysis
FITC-conjugated DAKO A/S antibody
 Antibody staining for -H2AX
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Immunofluorescence Staining
Anti-p53 antibodies
 Anti-Cathepsin B antibodies
 Anti-Cathepsin D antibodies
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Acridine Orange Staining
Lysosomotropic metachromatic fluorophore
 Red fluorescence in the lysosome, green
fluorescence when released from
lysosome.
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M30-Apoptosense ELISA
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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).
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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?
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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.
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Inactivates Cdc27 ubiquitin ligase complex, which
mediates degradation of mitotic cyclins
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Inactivates Weel kinase, inhibiting phosphorylation on
Cdks
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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.
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References
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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.