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Wnt/b-catenin pathway - LEf/TCF transcription activity (Nov/02/2008 )

Hi

I'm working on the b-catenin pathway and I'm quite new to this. Would appreciate any help in clarifying some doubts that I have.

According to a research article, of the 7 HCC cell lines studied,only two, (i.e. HepG2 and
HuH6)cell lines demonstrate beta-catenin/Tcf-regulated transcriptional activity, thrugh pTOP-FLASH transfection assay. Does
this mean that in the remaining HCC cell lines, b-catenin is not stabilised in the cytoplasm and is degraded

My concern is that if I want to study the effect of an antagonist to the b-catenin patwhay, wich cell line do I choose? Do I chose HepG2, since it demonstrates beta-catenin/Tcf-regulated transcriptional activity? BUt than the transcriptional activity in this cel line is due toa mutation to the b-catenin gene and thus may not be a fair cell line to study my antagonist.

My answer (let me know if wrong approach): I should co-transfect all my HCC cell lines with pTOP-FLASH and wnt 3a (agonist). Which ever cell lines gives me high transcriptional activation, can be selected for studying the antagonists?

Thank you

-Sarwat-

what about the matched HCT116 cells which are Wnt On and Wnt Off? (vogelstein papers)
My guess is that if you are trying to antagonise the pathway, it needs to be on. But in saying that, on because of mutation is not ideal????? I suggest you work in more than 1 cell line and clarify where the Wnt mutations are first (b cat and Apc), so you know what you are dealing with....
Or, you could take a cell line where the pathway is off and artificially turn it on.....Or.....??????

good luck!



QUOTE (Sarwat @ Nov 3 2008, 08:13 AM)
Hi

I'm working on the b-catenin pathway and I'm quite new to this. Would appreciate any help in clarifying some doubts that I have.

According to a research article, of the 7 HCC cell lines studied,only two, (i.e. HepG2 and
HuH6)cell lines demonstrate beta-catenin/Tcf-regulated transcriptional activity, thrugh pTOP-FLASH transfection assay. Does
this mean that in the remaining HCC cell lines, b-catenin is not stabilised in the cytoplasm and is degraded

My concern is that if I want to study the effect of an antagonist to the b-catenin patwhay, wich cell line do I choose? Do I chose HepG2, since it demonstrates beta-catenin/Tcf-regulated transcriptional activity? BUt than the transcriptional activity in this cel line is due toa mutation to the b-catenin gene and thus may not be a fair cell line to study my antagonist.

My answer (let me know if wrong approach): I should co-transfect all my HCC cell lines with pTOP-FLASH and wnt 3a (agonist). Which ever cell lines gives me high transcriptional activation, can be selected for studying the antagonists?

Thank you

-aussieuk-