MCF-7's not responding to estrogen - (Jul/21/2009 )
Hi,
I was hoping someone might lend me some advice to a very frustrating problem. I am working on a study with MCF-7 and some stable transfected MCF-7 cell lines, and I had a recent breakthrough that might potentially involve estrogen. So, I tried to simply do an expeirment to see the rate of increase in proliferation when treating the cells with 17-B-Estradiol (treating the cells with either 1, 10, 100 nM and measuring using Promega AQuone Proliferation Assay), though it didn't work in serum free media (w/o phenol red). So, then I tried it with 10% serum (w/o phenol red) and it still didn't work. Any idea of how to troubleshoot this bc I'm really confused.
At first I thought the lack of an effect in serum free media meant that estradiol might not be able to activate proliferation, but it's clear there's another issue at hand. Something to note is that I have first tried it in regular FBS, but my next step is trying dextran-coated charcoal stripped serum to see if that makes a difference.
Any thoughts?
jshaya on Jul 21 2009, 08:47 PM said:
I was hoping someone might lend me some advice to a very frustrating problem. I am working on a study with MCF-7 and some stable transfected MCF-7 cell lines, and I had a recent breakthrough that might potentially involve estrogen. So, I tried to simply do an expeirment to see the rate of increase in proliferation when treating the cells with 17-B-Estradiol (treating the cells with either 1, 10, 100 nM and measuring using Promega AQuone Proliferation Assay), though it didn't work in serum free media (w/o phenol red). So, then I tried it with 10% serum (w/o phenol red) and it still didn't work. Any idea of how to troubleshoot this bc I'm really confused.
At first I thought the lack of an effect in serum free media meant that estradiol might not be able to activate proliferation, but it's clear there's another issue at hand. Something to note is that I have first tried it in regular FBS, but my next step is trying dextran-coated charcoal stripped serum to see if that makes a difference.
Any thoughts?
Might be the cell line. MCF7 are mutated up to the sky. How long was the line passaged? Check if they have receptors for estriadol, by simple western blotting.
Maybe you want to avoid phenol red ( has estrogen-like activity) and use FBS that has been treated with Charcoal (to remove the endogenous estrogen)?
Well, I have been avoiding phenol red and I have tried different batches of MCF-7's of different passage numbers but I'm not sure why it isn't working. I am looking for estrogen-mediated activation of RTK's, so I usually only carry my experiment out to 72 hours max.
My initial observation was that in serum free media, my stablely transfected MCF-7 cells respond to phenol red (in terms of their proliferation) whereas the MCF-7's do not respond, so I attributed this response to phenol red impurities' estrogenic activity, yet I have been unable to show that estradiol can stimulate proliferation in at 0 or 10% serum.
Also, I have tried a calcium assay and shown that when treated with diff concentrations of estradiol, the cells do have a calcium increase which shows they are still responding to estrogen.
Any thoughts, thanks in advance!
The response of MCF-7 to estradiol is pretty limited, I haven't seen much change in the rate of proliferation with and without estradiol. Personally I would look for an estrogen response by looking for internalization of the ER with immunocytochemistry.
Or you can do a transient transfection with a reporter plasmid bearing an ER resposive element;
RT-PCR for ER responsive genes is another way.