LCCC Brown Bag Seminar 2009 Apoptosis, Cell Survival, and Cancer Rebecca Riggins 202.687.7451...
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Transcript of LCCC Brown Bag Seminar 2009 Apoptosis, Cell Survival, and Cancer Rebecca Riggins 202.687.7451...
LCCC Brown Bag Seminar 2009
Apoptosis, Cell Survival, and Cancer
Rebecca Riggins202.687.7451
Overview
• What is apoptosis, and why is it important?
• What genes/pathways are involved?• How do we measure it?• How do cancer cells combat it?• How do we manipulate apoptosis and
cell survival in the treatment of cancer?• What are some alternatives to
apoptosis?
Definition of Apoptosis
• Also called “programmed cell death”• A specific program of events leading to
the death of a cell• Program is active in organisms from
yeast to humans• Important in removing cells that are
infected, growing inappropriately or otherwise damaged
• Essential in development
Apoptosis in Development
• Can be visible at the macroscopic and microscopic levels
http://transdeath.uni-graz.at/
PlantsFilamentousFungi
Dictyostelium Caenorhabditiselegans
Apoptosis in the Worm
• C. elegans is the best-studied model of developmental apoptosis
Lettre et al. Nature Reviews Molecular Cell Biology 7, 97–108 (February 2006) | doi:10.1038/nrm1836
Apoptosis in the Mammary Gland
• Cycle of cell growth and apoptosis during pregnancy and lactation
Ductal/Lobulo-Alveolar differentiation during four mammary gland stages
http://mammary.nih.gov/
Cellular Events in Apoptosis• Historically, apoptosis is identified by
morphology (appearance)• Chromatin condensation (pyknosis)• Nuclear fragmentation due to endonuclease
cleavage of DNA (karyorhexis)• Cell shrinkage• Ruffling of the plasma membrane• Key feature: cell membranes do not rupture…
they remain intact
←pyknosis
←karyorhexis
Wojcik, C. et al, Apoptosis. 1997;2(5):455-462.
Molecular Events in Apoptosis
• There are 2 major signal transduction pathways that control apoptosis
• Extrinsic pathway• Intrinsic pathway
Extrinsic = Outside the Cell
• Death Receptor Pathway
• Signals are received by receptors on the cell surface
• Other proteins are recruited to the cytoplasmic tails of these receptors
• These proteins activate the caspase enzyme family
Ashkenazi and Dixit, Science. 1998 Aug 28;281(5381):1305-1308.
PlasmaMembrane
Intrinsic = Inside the Cell
• Mitochondrial or Cell Stress Pathway
• Responds to DNA damage, other internal signals
• Mitochondrial membrane becomes permeable
• Cytochrome c is released, leading to activation of caspases
http://www.scq.ubc.ca/wp-content/uploads/2006/07/apoptosispathways.gif
Cross-Talk between the Pathways
Shajahan AN, Riggins RB, and Clarke R., 2008
Caspases
• A family of cysteine-dependent, aspartate-specific proteases
• Caspases hydrolyze peptide bonds on the carboxyl side of an aspartate residue – Eg.) DEVD↓….
Alnemri et al, Cell. 1996 Oct 18;87(2):171
Caspase Cooperation
Initiator Caspases
Effector CaspasesShajahan AN, Riggins RB, and Clarke R., 2008
What are the substrates of Effector Caspases?
• Cytoplasmic, structural proteins (actin)• Nuclear, structural proteins (lamin)• DNA repair proteins (PARP)• Kinases, other signaling molecules• Cell cycle proteins
Apoptotic Control of Cell Cycle Progression
http://www.cancerline.com/gUserFiles/stages_of_cell_cycle.gif
Apoptosis
Apoptosis in the Lab
• Visualization of DNA fragments by gel electrophoresis
Induce apoptosis
Isolate DNA and load onto agarose gel
Wojcik, C. et al, Apoptosis. 1997;2(5):455-462.
DNA Fragmentation, cont’d.
• Propidium iodide (P.I.) staining and fluorescence activated cell sorting (FACS)
• PI intercalates between DNA molecules, 1 dye per 4-5 base pairs
Induce apoptosis
Remove cells fromdish, stain with PI
MeasureFluorescence
Ex. 535 nm
Em. 617 nm
DNA Fragmentation Results
S
M
G1G2
control
0.95%
100 nM paclitaxel
7.5%
G1S
G2/M
- where a cell is in the cell cycle determines how much DNA is present
Cel
l C
ou
nt
subG1 peak
More Apoptosis in the Lab
Induce apoptosis
Isolate protein andload onto poly-acrylamide gel
• Western blot analysis…use of specific antibodies to detect full-length and cleaved caspase substrates
MW - +
Apoptosis
Apoptosis in Cancer: What goes wrong?
• Cancer cells live because they “forget” how to die
• This can happen:– When key genes that induce apoptosis get
deleted, or turned off– When genes and pathways that induce cell
growth become amplified, or turned on
• Cell survival signaling directly counter-acts apoptotic signaling
Cancer’s “Drive to Survive”o growth factors and their receptors o all receptors are transmembrane proteinso 3 major features: o extracellular domain (ectodomain)o transmembrane regiono intracellular domaino where, when, and how they are expressed determines their biological function
Figure 5.10 The Biology of Cancer (© Garland Science 2007)
ErbB/HER Family
Epidermal Growth Factor Receptor (EGFR)
Seminars in Cancer Biology, Volume 14, Issue 4, August 2004, Pages 262-270
Growth Factor Receptor Action
Figure 5.15 The Biology of Cancer (© Garland Science 2007)
Cell Cycle Progression SURVIVAL
RASP
RTK
PGrb2SOS
PAkt
PI3K
p85
p110
RAS
Raf
MEK
P
P
ERK
GROWTH FACTOR
PIP3 PIP3
PDK1
PROLIFERATION
MDM2BAD
P
NF-ĸB
P
FKHR
P
CELL SURVIVAL
p70S6K
P
GSK3
P P
PROTEIN SYNTHESIS
Aaronson, Growth factor and receptor tyrosine kinases. Sci. STKE 2005, tr6 (2005).
Growth Factor Signaling is Complex!
Incidence of RAS mutations in human cancer
Lung (30% ki-ras)Breast
Brain
Colon (50% Ki-ras)
OvaryProstate
Pancreas (90% Ki-ras)
Bladder (10% Ha-ras)
Thyroid (50% ras)
Liver (30% N-ras)
Skin (14% N-ras)
Head & Neck
Leukemia (30% N-ras)
Kidney (10% Ha-ras)
A. Chan, Ras-MAPK Pathways. Sci. STKE 2005, tr5 (2005).
A. Chan, Ras-MAPK Pathways. Sci. STKE 2005, tr5 (2005).
Lung carcinoma K-Ras codon 12 (GGTgly) >AGTser
Pancreatic Carcinoma K-Ras codon 12 (GGTgly) >GTTval
Bladder Carcinoma H-Ras codon 12 (GGCgly) >GTCval
Melanoma N-Ras codon 61 (CAAgln)>CGAarg
Ras Mutations display Tumor Specificity
Apoptosis and Cell Survival in Cancer Therapy
• How do we stop inappropriate cell growth and survival, or re-start the process of apoptosis?
Drug Primary Target Mechanism(s) of Cell Death
Anthracyclines
Doxorubicin, Epirubicin DNA intercalation, topoisomerase II
BCL2 family regulation, NFκB inhibition, p53 activation
Alkylating Agents
Cisplatin, Cyclophosphamide
DNA crosslinking caspase activation, p53 activation, cytochrome c release
Antimetabolites
5-fluorouracil, Capecitabine
thymidylate synthase p53 activation, thymineless death
Microtubule Inhibitors
Docetaxel, Paclitaxel microtubule stabilization caspase activation, phosphorylation of BCL2 and BCL-X, JNK activation, CD95/FAS expression, autophagy
Vinblastine, Vincristine microtubule dissolution p53 activation, post-translational modification of BCL2 family members
Apoptosis and Cell Survival in Cancer Therapy
Shajahan AN, Riggins RB, and Clarke R., 2008
Drug Primary Target Mechanism(s) of Cell Death
Signal Transduction Inhibitors
Gefitinib EGFR kinase activity phosphorylation of BAD, downregulation of BCL2
Trastuzumab, CH401 HER2 extracellular domain inhibition of PI3K/Akt, phosphorylation of BAD, JNK activation
TRAIL TRAIL death receptor Death receptor/caspase 8 activation
Genasense BCL2 downregulation of BCL2
ABT-737, others BCL2 and BCL-X prevention of BCL2 and BCL-X interaction with pro-apoptotic BAX and BAK
Apoptosis and Cell Survival in Cancer Therapy
Shajahan AN, Riggins RB, and Clarke R., 2008
Some Final Thoughts• Is there more than apoptosis?
– Senescence– Mitotic Catastrophe– Necrosis– Autophagy
SENESCENCE• As cells divide, telomeres atchromosome ends become shorter• At some point, cells are unableto re-enter the cell cycle
MITOTIC CATASTROPHE• Cells die during mitosis (M
phase)• Caused by improper
chromosome segregation
NECROSIS• Chaotic death process in which
plasma membrane ruptures• Release of intracellular
contents leads to inflammation• Mechanism(s) still poorly
understood
Autophagy
• Autophagy, autophagocytosis = “self eating”
• Degradation of cellular components in a sequestering vacuole (autophagosome)
• This merges with the lysosome, where acidic hydrolases degrade its contents
Rubinsztein, Nat Rev Drug Disc. 2007 Apr; 6:304-312.
Autophagy vs. Apoptosis
Shajahan AN, Riggins RB, and Clarke R., 2008
Rubinsztein, Nat Rev Drug Disc. 2007 Apr; 6:304-312.
Mood stabilizer
Anti-malarial
Anti-estrogen
Regulating Autophagy Pathways
• If you would like to download this Powerpoint file for your reference, please visit:
http://openwetware.org/wiki/Riggins_Lab
• Under Resources, click Lectures then Summer Brown Bag
Thank You!