My compound increased PARylation (PAR) protein levels in time and-concentration depedent manner in HCT116 and HT-29 cells. Usually PAR activation induced AIF mediated cell death in neuronal cells (called Parthanatos cell death). In my case PAR was increased, but I could not absorb cell death. then what doest it mean? there are two other enzyme which is responsible for PAR degradation such as PARG and Iduna. PARG is an enzyme resposible for the PAR degradation and duna is a PAR-dependent E3 ubiquitin ligase that regulates DNA damage. My compound down regulated the both protein levels (PARG and Iduna), it ma increase the PAR level. Finally there is no cell death why?
Thanks in Advance.
Your results suggest that activated PARP is not enough to induce apoptosis in HCT-116 and HT-29 cells. Why not check the activation of caspase3?
PARP activation is an important component of the DNA damage response, however the outcome of this activation depends on the cell-type as well as the compound and treatment conditions. PARylation may stimulate DNA repair, however if this is not achieved cell death pathways may be activated, including apoptosis - which may ultimately result in PARP cleavage which is frequently tracked as an indicator of end-stage apoptosis. Perhaps you could assay for DNA damage using gamma-H2AX immunofluorescence or a COMET assay and also monitor DNA repair?
Thanks Go J Yoshidaand William H Gmeiner,
Caspase no changes, usually apoptotic cells can be observed by microscope, shrinkage or simple MTT assay or western blot analysis of apoptotic related protein cleavage such as PARP1, Casapase3,8 and 9, in my case i did not observe cleavages. the strange thing the cells are looking good after treament, but the PAR (PARylation protein) was increased and PARP1 was downregulated why?
My observation looks very strange but the fact is true.
Why PARP1, Iduna, and PARG was downregulated, if this protein was downregulated what will happen?
When you mentioned PARylation, do you mean the level of PARylation on PARP1 itself? The top subject of PARP1 is PARP1 itself, however, it still have other subjects, I am not sure what subject are you talking about. Maybe the PAR signal comes from other PARP1 subjects.
Thanks for your comments Mei-Kuang Chen,
We dont know PARP1 itself or other proteins
What we absorb is that PARP1 protein level was dramatically downregulated, like PARP1 inhibitor. its well known PARP1 inhibitor reduced cell growth via apoptosis.
In my case PARP1 completely abrogated after treatment. but there is no cell death.
Then we decide to check PAR level, it was dramatically increased, but still PAR increases induced parthanatos cell death. Again there is no cell death.
Already, I said PAR degrading enzyme PARG level and PAR ubiqutination E3 ligase Iduna was down regulated, utimately PAR accumulation in cell.
In any of this case it should induce cell death, then why not for me? but i am continously aboserved that there is morpholical changes between control and treatment.
In an another aspect, i am thinking about differentiation. Is it possiblie that the cancer cells can become normal cells?
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