To assess whether expression of the gene of our interest is associated with mutant status of p53 we have treated FaDu cells( mut-p53) with CP-31398, a Known p53 stabilizer, that restore a wild-type DNA-binding conformation of mutant p53. we check the expression by TaqMan based q-PCR, and got expected result, but when we treated the Fadu cells with Pifithrin-α( an inhibitor of p53 ) we again got the same trend( in both cases RQ is 0.2 & 0.4 respectively), while we are expecting No change or Induction. According to the publication: Cancer Biol Ther. 2002 Jan-Feb;1(1):47-55, FaDu expressed only 50% of the normal level of p53 mRNA, either because only one allele was present (A431), or because only one of the two alleles was transcribed. What could be the possible reason of same trend of gene expression after the exposure of CP-31398 & Pifithrin-α in Fadu Cells?
May the the molecule of your interest which you are working is simply not involved in cascade. The result can be interpreted as it has no role in particular pathway or may be any other signaling deivn the proces. Thanks
Thanks for the answer Tariq Khan, but wild type p53 is a established negative regulator of our gene of interest. we identified a novel feedback loop downstream of our gene of interest, but we want to establish the effect of mutant p53 status on the feedback mechanism. I have no confusion about the involvement of our target gene with p53, but what causes the same effect of chemicals with opposite function on a cell line that has a unique p53 status.
Based on the information you shared, it is likely that both treatments (CP-31398 or Pifithrin-alpha) of cells resulted in a conformational change in the p53, resulting in alterations in the expression of your gene of interest. To rule out other possibilities, you may want to check for the expression of p53 target genes that are either repressed or stimulated by p53 under your treatment conditions.
Thank you for the answer Prof. Divaker Choubey , I recently learned that Pifithrin alpha also has a p53-independent inhibition mechanism( Article Pifithrin-alpha has a p53-independent cytoprotective effect ...
). I think I have to try more specific methods like RNAi-mediated p53 knockdown to study the association.- Badges
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