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 and various apoptotic pathways, you will apply the following treatments in cyto C-depleted cells and then measure apoptosis. upon what we learned in class, predict the experimental outcomes and briefly rationalize each prediction. A. Stimulate apoptosis using UV light-induced DNA damage B. Stimulate apoptosis using FAS ligand added to culture media. C. Stimulate apoptosis by overexpression of BAX. D. Stimulate apoptosis by any of the methods above in the presence of the drug zVAD, a general caspase inhibitor.

Explanation / Answer

Apoptosis can be initiated in the cells by two pathways-

Intrinsic pathway - In this pathway apoptosis signal is generated inside the cell. It may be DNA damage, cellular stress.

Extrinsic pathway - In this type apoptosis signal is generated outside the cell. Signalling molecules bind to the receptor as a result of it a cascade of caspases is activated which is responsible for apotpsois.

A - UV-induced DNA damage- It activate the p53 protein which in turn start the expression of pro-apoptotic genes like Bax, PUMA. These proteins change the membrane permeability of the mitochondria. Due to change in membrane permeability Cyt-C is released in the cytoplasm where is activates the Casapses-9. caspases-9 activates a cascade of caspases which leads to apoptosis.

Since the Cyt-C is Knocked down so the cell will not undergo apoptosis.

B- Fas ligand induce apoptosis by binding to its receptor Fas. It activates the apoptosis by extrinsic pathway. So if the apoptosis is activated by Fas ligand then in that case knocking down Cyt-C will not affect the apoptosis process.

C - Bax is a proapoptotic protein which cleaves the Bcl-2 which is anti-apoptotic protein. Bcl-2 prevents the membrane permeabilization of the mitochondria. Overexpress of Bax leads to cleavage of Bcl-2 and as a result of it, the outer membrane of the mitochondria become permeable which results in the release of the Cyt-C. Cyt-C in the cytoplasm activates caspases-9. caspases-9 activates a cascade of caspases which leads to apoptosis.

Since the Cyt-C is Knocked down so the cell will not undergo apoptosis.

D- As we have seen that UV-induced apoptosis, overexpression of Bax-induced apoptosis and Fas ligand-induced apoptosis all of them requires the activation of caspases to undergo apoptosis. If we add caspases inhibitor zVAD then in that case cell will not undergo apoptosis.

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