Is it possible for a cell to express CASP3 (I have seen this by Western blotting assays) without cell death (I have seen no cell death by time laspe assays under the same conditions).
In my cell-based model I use A549 (KRAS-active lung cancer cell) and treat them with Aurora Inhibitor II (a dual inhibitor which blocks both mitotic Aurora kinases A and B) for 72h. There is an incresing in CASP3 expression. However, during time-lapse assays I noticed that my cells enter in mitosis and readhere to the plate without completing cell divison. So they became polyploid, without dying...
How could I explain that?
Dear Edmilson Ozorio
1- You might wanna check the related lncRNA (long non-coding RNA) for your Casp3.
Because Casp3 expressions may not be enough for the protein activation. Some specific lncRNAs also needed for the protein activation.
Is there any ncRNA which required to co-activate your expressed Casp3 protein? Because some proteins need specific lncRNAs to activate.
2- Another possibility is there can be second activator corresponding to your Casp3 functioning. I mean, proteins mostly activeted by upstream molecules.
If your subjected protein is activated by up or downstream signalling, this tricky result may emerged by abrogated upstream/downstream signal pathway of Casp3. In such case, after its expression Casp3 protein requires some other proteins to functioning.
In both case you should check for the any co-activators or pathway function concordance.
Hi Edmilson;
Are you detecting an increase in Caspase 3, or in cleaved caspase 3? If caspase 3 is not claved that means that it is not active, And that could explain the fact that you have not observed apoptosis.
As mentioned above cleaved form and not full length Casp3 expression corresponds to enhanced apoptosis. I think in the case of initiator caspases, increasing expression levels could lead to activation,but the effector caspases like CASP3 would require cleavage and activation. It is still possible that cells with higher expression are more sensitive to secondary pro-apoptotic stimuli.
Caspase 3 requires Caspase 8 and Caspase 9 uptream signalling to be activated. Following the cleaved Caspase 3 alone is not the clear answer. Showing why/how this protein activated/cleaved allows more healthy results.
Hi all, thank you! All of you are right! But I have detected a higher expression of Cleaved CASP3 by WB. So I need to explain why I see it without cell death.
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