Investigation of the mechanism by which inhibitors of histone deacetylases (HDI) disrupt assembly of kinetochores in human tumor cell lines was continued. During the previous reporting period we had found that treatment with HDI prevented pre-mitotic pericentromeric phosphorylation of histone H3 at Ser 10. We have now established that: 1. In HDI-treated cells phosphorylation of H3(Ser10) is delayed until prophase, occurring simultaneously with phosphorylation of H3(Ser 28). 2.
AIM -1 kinase, a member of the Aurora B branch of the Aurora/Ipl family of Ser/Thr kinases, shown by others to be responsible for mitotic phosphorylation of H3(Ser10) in C. elegans and D. melanogaster, is present in HDI-treated cells, but is hyperacetylated within three hours of addition of the inhibitors. 3. In vitro kinase activity of AIM-1 immunoprecipitated from control or HDI-treated cells is similar in assays employing non-acetylated amino-terminal peptides of histone H3; however, when assayed with mono-and di-acetylated peptides, enzyme from untreated cells exhibited 5-times greater activity than that from HDI-treated cells. Current efforts include 1. using transient transfection of an inducible, dominant-negative mutant of AIM-1 to ascertain whether that enzyme is responsible for pre-mitotic phosphorylation of H3(Ser 10) in human cells; 2. further examination of the activity of acetylated AIM-1 in vitro.
