If I have more number of foci, does it represent nuclear damage or nuclear repair? Some cells show pan nuclear staining, does it mean the cell underwent apoptosis?
I am new to this area. Any help would be appreciated.
Hi Adity Pore ,
H2AX is a variant of the H2A histone. When DNA undergoes double-strand break damage, H2AX is phosphorylated by ATM (or ATR). This phosphorylation of H2AX is named gamma-H2AX (y-H2AX). y-H2AX is not a marker for apoptosis; it is the starting point to recruit the DSB repair machinery. Hence, the y-H2AX foci will form in DSB areas.
If you need more, check this article, it will basically explain everything you need to know about y-H2AX: [https://www.ncbi.nlm.nih.gov/pubmed/18610740].
Gamma H2AX foci are always formed in case of DNA double strand breaks as a first step in its repair. As there is no repair without a previous damage I would not say that these foci represent neither damage nor repair. It´s rather both. The more foci you detect the higher the amount of damage and the more repair is necessary. In case of successful repair the number of foci (and amount of damage) should decrease over time. Pan-nuclear signals are supposedly induced differently and have been shown to increase over time after damage. In that case additional staining to check for apoptotic cells might be useful as described here: https://www.nature.com/articles/cddiscovery201611
Hi Adity,
H2AX phosphorylation is part of the signaling cascade mostly in response to DNA damage (double strand breaks) through activation of PI3KK (ATM, ATR or DNA-PK). In this regard gamma-H2AX foci should be regarded as a marker for DSBs rather than for repair, although H2AX phosphorylation may promote DSBR.
Gamma-H2AX dynamics (i.e. its decay) may even be used as a readout for DSBR.
Pan-nuclear staining may indeed result from DNA fragmentation during apoptosis, but it could also occur in the absence of (massive) DSBs like during hypoxia, replication stress or alteration of osmolarity.
Hope this helps.
This paper is an excellent summary of DNA damage foci by Kai Rothkamm https://www.ncbi.nlm.nih.gov/pubmed/?term=foci%2C+meaning+and+significance%2C+rothkamm
Steve
Both of Steve (Stephen G R Barnard) and Kai Rothkamm were the expert on gamma-h2AX foci analysis. You could ask them in detail about gamma-h2AX foci analysis.
Regards,
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