Differences in zinc induction of MT 3 mRNA expression between normal and transformed UROtsa cells following inhibition of histone deacetylase activity As described above, the parental and transformed UROtsa cells were allowed to proliferate to confluency in the presence of MS 275 and then allowed to recover for 24 h in the absence of the drug. After the recovery per iod, the cells were then exposed to 100 uM zinc for 24 h and prepared for the analysis of MT 3 mRNA expression. The parental UROtsa cells previously exposed to MS 275 showed no increase in MT 3 mRNA expression when treated with 100 uM Zn 2 for 24 h. In contrast, MT 3 expression was induced over a 100 fold when the Cd 2 and As 3 transformed cell lines that had been previously treated with MS 275 were exposed to 100 uM Zn 2.
Histone modifications associated with the MT 3 promoter in the UROtsa parent and transformed cell lines Two regions of the MT 3 promoter were analyzed for his tone modifications before and after treatment of the respective cell lines with MS 275. These were chosen to be regions containing sequences of selleck chemical Doxorubicin the known metal response elements. The first region chosen spans the lar gest cluster of MREs and is desig nated as region 1. The second region is immediately upstream from region 1, extends up to and includes MREg and is designated region 2. The level of acetyl H4, trimethyl H3K4, trimethyl H3K9 and trimethyl H3K27 modifications were determined for each of the two regions of the MT 3 promoter using ChIP qPCR.
In the distal region 2, it was shown that the modification of acetyl H4 was increased in the parental UROtsa cells and both transformed cell lines following treatment with MS 275. For all three cell lines, there was only a marginal modification for acetyl H4 selleckchem in cells not treated with MS 275. In addition, the relative increase in acetyl H4 modification following MS 275 treatment was greater in the Cd 2 and As 3 transformed cell line compared to parental cells. There was modification of trimethyl H3K4 in both the normal and transformed UROtsa cell lines under basal conditions and the level of modification increased for the parental UROtsa cells and the Cd 2 transformed cell line following treatment with MS 275. There was no increase in the level of modi fication of H3K4 following MS 275 treatment of the As 3 transformed UROtsa cells.
Modification of trimethyl H3K9 was present in both the parental and transformed UROtsa cells under basal conditions. The basal level of H3K9 modification was increased for both transformed cell lines when compared to parental cells and also when the As 3 transformed cell line was com pared to the Cd 2 transformed cell line. There was a dif ferential response in the level of H3K9 modification when the cells were treated with MS 275. The parental UROtsa cells showed an increase in the modification of H3K9 following MS 275 treatment, whereas, both transformed cell lines showed a decrease in the level of H3K9 modifica tion. The relative magnitude of these differences was large for the parental and As 3 transformed cell lines.
There was a large difference in the level of modification of H3K27 between the parental and the transformed cell lines, with the parent having a very low level and the transformed lines highly elevated in their modification of H3K27. Treatment of both the Cd 2 and As 3 transformed cell lines with MS 275 resulted in a large decrease in the level of H3K27 modification, return ing to a level similar to that found in parental cells. In themore proximal, down stream promoter region 1, the modification pattern of acetyl H4 was similar to that of region 2, with the exception that the basal level of modification was increased in the Cd 2 and As 3 trans formed cell lines.