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aza treatment p16 - (May/23/2007 )

Hi,

I am treating cells with 5'aza'2'deoxycytidine to remove methylation from the p16 gene
in breast cancer cells.
In the literature people generally treat cells for 3-4 days with 5-10 micromolar AZA and rarely show a time course.

However, I was wondering if people know how short a period you can treat cells (especially breast cancer)
with AZA to induce p16. For example can you treat for 1-2 days and see a p16 induction?

Also, what do people generally dissolve AZA in? Media or DMSO?

Thanks.

-mikew-

Hi, the time course of AZA treatment depends on the rate of replication of the cells, as AZA only works when DNA is replicated. As cancer cells are normally growing rapidly, you may see p16 induction as early as you mentioned.

-kr├╝melmonster-

Hi Mike,

I dissolve 5-aza-dC in aliquots of PBS and freeze at -80 degrees. In my experiments, this 5-aza-dC still works after at least 2 months in the freezer.
As for how long you need to treat cells to see re-expression, the best way would be to do a few experiments to see what applies to your cells and gene(s).

Dave

ps. My aliquots of 5-aza-dC are frozen at 10,000uM if that makes any difference.

-Davo-

you would probably see some induction at day 2, but if your cells are staying viable, it would be easier to wait another day.

-sneth-

Hey guys,

Thanks for the replies. In case anyone is curious, I did the initial experiments and
saw induction of p16 at 48 hours post aza treatment but not at 24.
Interestingly, in these cells AZA does not seem to decrease viability
to a significant degree.

-mikew-

QUOTE (mikew @ Jun 5 2007, 08:32 AM)
Interestingly, in these cells AZA does not seem to decrease viability
to a significant degree.


In my experience, I have treated cells for three days with rather high 5-aza-dC concentrations. After three days they don't seem too bad but if you let some of the treated cells grow without 5-aza-dC in the media, in the coming days you will then observe a lot of sick cells! This article may be of use to you:

"De novo DNA methyltransferases Dnmt3a and Dnmt3b primarily mediate the cytotoxic effect of 5-aza-2'-deoxycytidine"

Do a pubmed search for 15735669 and it will bring it up.

Dave

-Davo-