Apoptosis, Necrosis or Autophagy?

[porty1]

Apoptosis, Necrosis or Autophagy?

that is a good question.

How can one differentiate between the three and what are the methods.

I know some:

DNA laddering
Electron Microscopy

Are there are faster or easier (more accurate) methods that differentiate between them?

[amicia]

Really good question. It's difficult to asnwer,I suggest you the lecture of these papers that can help you.

Szabo C. Mechanisms of cell necrosis. Crit Care Med. 2005 Dec;33(12 Suppl):S530-4.

Malhi H, Gores GJ, Lemasters JJ. Apoptosis and necrosis in the liver: a tale of two deaths? Hepatology. 2006 Feb;43(2 Suppl 1):S31-44.

In my opinion is really hard to distinguish between late apoptosis and necrosis, but it's depend either from the stimuli and type of cells that you're studing. the new tendency is to consider the fall of ATP levels, but you need a technique to detect the level of ATP.
If you need some specifications about same technique to detect apoptosis/necrosis don't hesitate to poste it.

[admin]

Hello Amicia and porty1! welcome to the forums.

Tha is a very good paper, and a great response. I think technological advances will provide easier detection methods (and faster! - who wants to do Electron Microscopy ) in the future...

[rodicaolteanu]

Are apoptosis(late) and necrosis really different?????Will seee!!!

[kiki06]

Hmm.. good point Rodicaolteanu.

I think they still can be separated, as apoptosis leads to blebbing and budding which is eventually cleaned up easily.

Necrosis leads to leakage of cell contents, and causes death to nearby cells.
Apoptosis does not.

This seems clear-cut, but maybe in vivo it is not.

Any more ideas to post?

thanks
kiki

[stefulj]

What about fragmentation of the genomic DNA? I suppose agarose gel electrophoresis of genomic DNA might distinguish between these two processes.

[admin]

Yes that is an excellent point.

Both Apoptosis and Autophagy do not get fragmented DNA I believe.

Necrosis you get fragmentation of nuclear genomic DNA...

[stefulj]

Hi Admin!

Apoptosis seem to be accompanied by DNA fragmentation,
please see
Mol Biotechnol. 1998 Jun;9(3):247-51 Electrophoretic DNA analysis for the detection of apoptosis
(and similar)

Í thought necrosis does not get DNA fragmentation...

stef

[molecule2005]

Hey Stefulj,

Apoptosis is accompanied by specific DNA fragmentation.


Necrosis may get DNA damage/fragmentation but is not specific....

Apoptosis you get laddering and specifc fragments.

[jpap]

Hi there

I agree that at the late stage of the processes it is difficult to distinguish them, but there are tools and reagents available to help you determine if the type of death is apoptotic or not: caspase activation, cytochrome c release from the mitochondria, chromatin laddering etc etc... Again, whatever your marker of choice it is important to do a detailed time course making sure you include both early and late time points.

To my knowledge there are no reliable molecular of biochemical markers for necrotic death. A quick assay we use in vitro in populations undergoing death by novel/uncharacterised types of stress, is double staining with propidium iodide and Hoechst, the PI will stain the damaged cells with compromised membrane integrity and the DNA staining by Hoechst will distinguish the intact nuclei from the apoptotic fragmanted ones.

Measurements of cellular ATP levels can also be done relatively easily, there are kits available.

As for the contribution of autophagy to cell death, I again agree that the best experimental approach would be EM, but for a quick test in vitro we usually do staining of acidic vacuoles by acridine orange and quantification by FACS, and western blotting for LC3 protein processing. I believe the anti-LC3 antibody is now commercially available. If anyone is interested I could post some references and our protocols.

And on the level of function of course, the big question about autophagy is whether it is a form of death or actually protects from it!

The contribution and detection of these processes in vivo is another story, as people have already said...