Transcript Caspase Activity in Apoptotic Cells

Caspase Activity in Apoptotic
Cells
Cell Bio 220
Tenzin Dolkar
Stephanie Kim
Victoire Ndong
Patricia Wong
Caspases
• Are families of proteases which are involved
in initiating apoptotic events
• Synthesized in the cell as precursorsprocaspase
• Cleave proteins at aspartic acid residue sites
• There are initiator caspase (procaspase-8)
And executioner/effector caspase
(procaspase-3)
Caspase Structure
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NH2-terminal domain
Large subunit
(~20kD) – (lavender)
Small subunit (~10kD)
– (gray)
Red represents an
inhibitor
Thornberry and Lazebnik. Caspases:
Enemies Within. 1998. p 1313
Caspase Role in Apoptosis
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Cut off contact with surrounding cells
Reorganize cytoskeleton
Shut down DNA replication and repair
Interrupt splicing
Destroy DNA
Disrupt nuclear structure
Induce cell to display signals marking it for
phagocytosis
• Disintegrate cells into apoptotic bodies
Initiator Caspase Activation
• Initiator Caspases are activated by either
internal (DNA damage) or external signals
(ligand binding)
• External: caspase-8, an initiator caspase is
activated by binding to it’s specific cofactor,
FADD (Fas-associated protein with death
domain)
Executioner/Effector Caspase
Activation
• Effector caspase are activated generally by
other caspases.
• Caspase activation results in proteolytic
cascade which is self-amplifying and
irreversible.
• Caspase-3 is an effector caspase
Our Experiment
• MDCK cells treated with camptothecin
(interferes with DNA replication and
transcription)
• Added “D2-R110,” a bis-L-aspartic acid
amide of Rhodamine 110
• When the compound is cleaved by
caspase, such as caspase-3, it fluoresces
green
Hypothesis/Goal
• Cells treated with camptothecin will have a
greater number of fluorescing cells,
indicating a higher rate of caspase activity
and the occurrence of apoptosis
• To compare caspase activity in MDCK
cells treated with camptothecin with
control cells in order to determine the rate
of caspase activity
Sample A – Camptothecin Treated
Average Percentage of Apoptotic Cells: 73.40%
Sample B - Control
Average Percentage of Apoptotic Cells: 2.95%
Cellular Pathway
Sources of Error and Uncertainty
• Human errors
• Direct causal relationship between
caspase activity and fluorescence not
proven
Further Experimentation
• Disable caspase-3 activation to see the
extent of its importance in apoptosis
• Disable caspase-9 to determine if
apoptosis is a result of internal signaling
• Compare caspase activity in cells
undergoing apoptosis to those undergoing
necrosis
• Examine caspase activity at different time
intervals