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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 precursors-procaspase Cleave proteins at aspartic acid residue sites

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Caspase activity in apoptotic cells

Caspase Activity in Apoptotic Cells

Cell Bio 220

Tenzin Dolkar

Stephanie Kim

Victoire Ndong

Patricia Wong


Caspases
Caspases

  • Are families of proteases which are involved in initiating apoptotic events

  • Synthesized in the cell as precursors-procaspase

  • Cleave proteins at aspartic acid residue sites

  • There are initiator caspase (procaspase-8)

    And executioner/effector caspase

    (procaspase-3)


Caspase structure
Caspase Structure

  • 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
Caspase Role in Apoptosis

  • 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 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
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
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
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
Sample A – Camptothecin Treated

Average Percentage of Apoptotic Cells: 73.40%


Sample b control
Sample B - Control

Average Percentage of Apoptotic Cells: 2.95%



Sources of error and uncertainty
Sources of Error and Uncertainty

  • Human errors

  • Direct causal relationship between caspase activity and fluorescence not proven


Further experimentation
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


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