Suppose you can use siRNA to knockdown cytochrome C expression from cultured HeL

Question

Suppose you can use siRNA to knockdown cytochrome C expression from cultured HeLa cells, and furthermore that these cells can survive just fine using glycolysis as their sole source of ATP production. To test the relationship between cytochrome C and various apoptotic pathways, you will apply the following treatments in cyto C-depleted cells and then measure apoptosis. Based upon what we learned in class, predict the experimental outcomes and briefly rationalize each prediction.

(1) Stimulate apoptosis using UV light-induced DNA damage

(2) Stimulate apoptosis using FAS ligand added to culture media.

(3) Stimulate apoptosis by overexpression of BAX.

(4) Stimulate apoptosis by any of the methods above in the presence of the drug zVAD, a general caspase inhibitor.

Explanation / Answer

1. UV light induced DNA damage will lead to activation of p53 protein which will lead to expression of pro apoptotic genes like PUMA and Bax.Membrane permeability of the mitochondria is changed due to these genes, which leads to release of cytochrome C in the cytoplasm. This leads to activation of Caspases 9, which causes activation of a cascade of caspases resulting in apoptosis.As Cyt C is knocked down by siRNA , therefore no apoptosis will take place.

2. Fas ligand results in apoptosis due to binding with its Fas receptor. It results in apoptosis due to extrinsic pathway.Knocking down Cyt C will no affect on apoptosis if apoptosis is activated by Fas ligand.

3. Bax is a pro apoptotic protein that results in cleavage of Bcl 2 which is a anti apoptotic protein.Function of Bcl 2 is to inhibit the membrane permeabilization of mitochondria.Due to overexpression of Bax ,cleavage of Bcl 2 will take place, which will result in release of Cyt C due to permeability of outer membrane of mitochondria.Cyt C will result in activation of caspases9, which results in actvation of cascade of caspases leading to apoptosis.As Cyt C is knocked down, no apoptosis will take place.

4. No apoptosis will take place if caspases inhibitor zVAD is added because apoptosis occurs due to activation of caspases.

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