I am assessing the effects of a hypoxia selective compound in FaDu cells (a HNSCC cell line). When I treated the cells with the compound for 24 hours (at increasing concentrations) under hypoxia, and replate (reoxygenation), the cells become round, and stop dividing (for up to 7 days). I stained these cells with Calcein AM/PI 48 hours after replating, the round cells stain positive for Calcein AM (alive). I did a flow analysis with Annexin V/PI, and I only see a modest increase (from 6% in control to 11% in drug treated) in apoptotic population; most cells stain negative for Annexin V/PI. I am wondering what this phenotype might be? I have not done a beta-gal staining, but I am assuming senescent cells will become flat, not round.
Hi Faisal - in brief yes it can. Calcein AM fluorescence depends upon the activity of intra-cellular esterases not the cycling status of the cell. Hypoxia does not generally increase/induce senescence in cells - and the morphology you mention is not one typically associated with senescence ("fried egg phenotype"). Furthermore senescence is mainly associated with primary cells in culture (not cancer lines). As I don't know the drug used it is hard to give a firm conclusion but (if I had to guess) I would say that the cells have probably been frozen at a particular stage of the cell cycle. I would perform a cell cycle profile of the treated cells (versus untreated controls) to confirm which part they have halted in i.e. G0/G1, S or M. These cells can also "round up" as they prepare for cell division - so could be blocked in M-phase (cell cycle analysis should reveal what is going on). There is an interesting paper here looking at MMPs (not sure what compound you are looking at but the FaDu behave differently to the other lines examined - see bit about stromelysin-1).
Article Stromelysin-1 Activation Correlates with Invasiveness in Squ...
Hope this helps Gary
Should just say - if you are loking at hypoxia/invasiveness this also may be an interesting referrence
http://www.nature.com/articles/onc20178
Thanks Gary for your comments! Arrest in cell cycle seems like a plausible explanation.
I am assessing a nitroimidazole compound for its hypoxic cytotoxicity and radiosensitization properties.
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