Investigation of the mechanism by which inhibitors of histone deacetylases (HDI) block human tumor cell lines in prometaphase was continued. Progression from prometaphase to metaphase requires proper attachment of spindle microtubules to kinetochores. Results of immunofluorescence studies demonstrate that treatment with HDI prevents maturation of prekinetochores into kinetochores, despite normal levels of kinetochore proteins in HDI-treated cells. We hypothesized that the presence of acetylated histones at the centromere, a region in which histones are normally hypoacetylated, in some way interferes with kinetochore assembly. An attractive possibility is that acetylated histone H3 may not undergo pre-mitotic phosphorylation at Ser10. This phosphorylation begins at the centromeres of G2 cells, then spreads along the chromosomes. Using an antibody specific for H3-PhoS10 a 2-fold decrease in cells exhibiting immunostained patches of chromatin was observed after incubation with HDI for one hour, an 8-fold decrease after 3 hours. While total acid extractable protein contained H3 that was both acetylated and phosphorylated, chromatin fragments prepared by brief nuclease digestion revealed a subpopulation of acetylated H3 that was not phosphorylated. Current efforts involve determining 1. whether these chromatin fragments are centromeric, and 2. whether failure to phosphorylate acetylated centromeric H3 results from the modification of H3, or a decrease in the relevant kinase.
