Hi,
Iam working on a oral cancer cell line and Iam facing a problem. The DNA damage shows apoptosis but the caspase 3 and 9 levels are low. Is this possible or what Iam seeing is an artifact. Confused and not able to conclude. Suggestions are welcome.
Jaya
Hello,
For this matter, I have need to know something more. What types of experiments you did to confirm DNA damage? Usually, in our lab we use TUNEL assay to detect DNA damage. That pictures also very nice and clear. If you didn't do this assay then I would like to suggest you for this experiment. But if you already did it then I think in your picture, there may be lots of artifacts.
I could not agree more with the comment of Dr. Md Moniruzzaman.
Cell death is classified into three categories; apoptosis, necrosis, and autophagic cell death.
Depending on the cause to induce cell death, we cannot determine whether or not caspase-dependent cell death, I mean apoptosis, was induced or not.
Could you tell me what kind of stimuli did you give the cells of interest?
i did a knock down of some genes and saw the apoptotic effect. My concern is I see the DNA fragmentation but the levels of caspase were decreased. I would certainly do a tunnel effect to confirm it.
Following article will help you to understand the phenomena.
http://www.ncbi.nlm.nih.gov/pubmed/22244258
http://www.ncbi.nlm.nih.gov/pubmed/19273205
It is important to use different assays to measure apoptosis. DNA fragmentation cannot clearly say whether it is apoptosis or necrosis. Tunel assay as suggested before or 7AAD / Annexin V assay are good assays. The kinectics of caspase activity is also important to keep in mind and different cell lines can have different kinetics (http://www.nature.com/cdd/journal/v8/n1/full/4400800a.html).
I agree with Dr Breuning, it is neccesary to use different assays to confirm apoptosis, I used 3. In the caspases case I had the same results that you with a technique based in chemiluminiscense to mesure caspase activity but I changed by fluorescense method complementing with WB.
It's likely that DNA fragmentation observed may be an artifact. So, this needs to be confirmed and whether apoptosis was caused or not?
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