positive control for aza treatment - (Jan/31/2006 )
Hi,
I have problems with aza treatment of a lymphoid cell line - looks like there is no demethylation, at least in the region of interest. I used concentration from 1 to 10 uM for 72h. I changed the medium every day adding fresh aza. It looks like aza itself was O.K. because I observed a drop in cell proliferation (above 5uM of aza).
Does anybody know anything about a commonly used gene that could serve as positive control for 5-aza treatment, that is - which expression is upregulated after demethylation in manner that is detectable by RT-PCR?
Help!
Gosia
Hi Gosia,
72 hrs may be not enough. Try 5-6 days. You can treat cell consecutively using a lower dose or non-consecutively at a higher dose. Any gene that is often methylated in your cell may serve as a control. Another possibility is that methylation doesn't play a role in the silencing of the gene in your cell.
Hi pcrman,
the problem is that I know nothing about genes that can be methylated in my cell line (it's a pre-B cell line 18.81) and I didn't find any literature. I was thinking about something more universal.
I suspect that my gene is regulated by methylation because it has a CpG island on its 5' end and the methylation of this island is highly correlated with the gene expression in a tissue specific manner.
My problem with aza treatment is that bisulfite treatment of DNA after aza treatment shows that the promoter is still methylated - at least one of the CpG sequences which I can monitor by restriction enzyme digestion.
So mayby I really should prolong the aza treatment, thanks for that tip.
Gosia
Gosia
I'm not the specialist in this, but I hope that this info will help somehow.
This abstract provides some interesting information about the TIME role too:
http://www.citeulike.org/user/tuck/article/418578 -
This study is the first report on the radiation-induced epigenetic changes in radiation-target murine thymus during the pre-lymphoma period. We have demonstrated that acute and fractionated whole-body irradiation significantly altered DNA methylation pattern in murine thymus leading to a massive loss of global DNA methylation. We have also observed that irradiation led to increased levels of DNA strand breaks 6 h following the initial exposure. The majority of radiation-induced DNA strand breaks were repaired 1 month after exposure. DNA methylation changes, though, were persistent and significant radiation-induced DNA hypomethylation was observed in thymus 1 month after exposure. In sharp contrast to thymus, no significant persistent changes were noted in the non-target muscle tissue
Another problem - the random character of DNA methylation processes (see Susan Clark...)
Kestutis Urba
sorry - now I'm at the wrong computer - it doesn't allow to creat direct link...
I have problems with aza treatment of a lymphoid cell line - looks like there is no demethylation, at least in the region of interest.
Gosia
It is one more way:
Biochemical Society Transactions (2004) Volume 32, part 6
Zebularine: a new drug for epigenetic therapy
C.B. Yoo, J.C. Cheng and P.A. Jones
When cancer cells were transiently treated with 5-Aza-CdR and
then by continuous treatment with zebularine, remethylation
was hindered and gene expression was maintained, suggesting
a combinatorial treatment method that would give a better
response to the inhibition of DNA methylation [26].
ftom this article
Good luck.
Kestutis Urba
Not sure about your cell line. I used 5uM AZA to treat HepG2 cells for 72hrs. There were more than 50% drop in global methylation level (By LC-MS method). The quick way to detect this is by doinging dot-blot.
72 hrs may be not enough. Try 5-6 days. You can treat cell consecutively using a lower dose or non-consecutively at a higher dose. Any gene that is often methylated in your cell may serve as a control. Another possibility is that methylation doesn't play a role in the silencing of the gene in your cell.
I have a similar problem. The gene expression show well corelation with promoter methylation status. After 6 day's 5aza-doxycytidine treatment, the expression of mRNA was restored. However, the sequencing results showed no change in methylation status after the treatment.
[you still see the product for msp because 5 aza 2 deoxycitidine leads to situation when methylase is covalent bond to new synthesised DNA, after repliecation when the dna is hemimethylated but one strand is still methylated and youyr msp is trand specific so you should have in the best situation 2 products