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## Re: C26 Concerning survival determinations and not viability From: D.E. Godar Date: 12/8/97 Time: 9:32:32 PM Remote Name: 150.148.47.70 ### Comments Thank you for your focused answers and very interesting paper. Concerning survival, I think it is important to distinguish between viability, which changes over time, and survival (clonogenic), which measures reproductive capacity and does not change. Especially when using transformed cell lines that grow and divide and consequently obscure death at later time points, if not all together. Granted, PI determines viability, and Annexin V early apoptosis, but neither enddpoint (or combined) measures survival! Only plating (liquid or soft agar) can determine survival (based on the colonies that form). Viability and survival are mixed up by many investigators (I even saw this mistake published in a paper in Science!) and the difference really needs to be straightened out if we are to make expedient progress in this field. The reason I make such a BIG point about this is because viability sometimes does not change very much for many hours or days, and may not change at all, while survival data can show that, say, as much as 90% of the cells actually died over time (cannot reproduce). This is important when analyzing data that distinguishes between programmed cell death (PCD) and preprogrammed cell death (prePCD) apoptotic mechanisms. For example, if doses of UVB (PUVA, UVC, or any DNA damaging agent, such as diaminotoluene) are used that reduce the 'survival' between 1-99 % then the mechanism will clearly be by PCD, but if doses beyond that point are used ("overkill") then the prePCD mechanism is usually initiated (probably from an oxidative mechanism), and if too much is used then necrosis will occur. So, anyone can get any cell death mechanism using any insulting agent available, but the most likely mechanism in vivo will only be evident when either using physiologically relevant doses (if known) or doses that allow some survival (say around 1%). Many insulting agents have more than one route of action and they will only act specifically in a narrow dose range. Beyond that range they can act on other cell constituents (such as the membranes) and intitiate immediate apoptosis, or prePCD. On another but similar note, I noticed that your cells are p53 -/-, so they should not be able to undergo delayed apoptosis or PCD. They only undergo apoptosis at higher doses (as I would have predicted), and they do so quickly (immediate apoptosis). It would be very nice if your group transfected your cell line with p53, or used the a close relative that was p53 +/- at least, to show that both mechanisms can be induced in those cells as opposed to the p53 -/- cells, which are only capable of undergoing prePCD. I predict that the p53 +/- or +/+ cells will undergo delayed apoptosis (> 12 h, probably 24 or 48 h) at much lower dose levels than the mutated cell line your group used in this paper. Have you tried a p53 +/- cell type? We used a p53 +/- cell line and found both mechanisms were inducible. UVA1-induced immediate prePCD apoptosis, while UVB and UVC induced delayed PCD apoptosis using doses that reduced the survival, not viability, to 10% (90% reproductive death). It would be interesting to see what UVA would do, I think both mechanisms would be induced, which one would win is another question. Probably some cells would incur enough DNA damage (but not enough membrane damage) to die by PCD and some enough membrane damage (and in this case the DNA damage wouldn't matter) to die by prePCD. In which case a biphasic time course may be more noticable and could be asscociated with each of the two mechanisms. If nothing else, it would mean another publication for your group! (But get ready for some contradictory looking data, one is p53 dependent the other is not, same for Ca2+ and PKC and almost everything else you can think of) Thank you for making this a discussion, and an interesting one at that! Back to the list of comments