Inhibitors of Histone Deacetylases Alter Kinetochore Assembly by Disrupting Pericentromeric Heterochromatin

Abstract

The kinetochore, a multi-protein complex assembled on centromeric chromatin in mitosis, is essential for sister chromosome segregation. We show here that inhibition of histone deacetylation blocks mitotic progression at prometaphase in two human tumor cell lines by interfering with kinetochore assembly. Decreased amounts of hBUB1, CENP-F and the motor protein CENP-E were present on kinetochores of treated cells. These kinetochores failed to nucleate and inefficiently captured microtubules, resulting in activation of the mitotic checkpoint. Addition of histone deacetylase inhibitors prior to the end of S-phase resulted in decreased HP1-beta on pericentromeric heterochromatin in S-phase and G(2), decreased pericentromeric targeting of Aurora B kinase, resulting in decreased premitotic phosphorylation of pericentromeric histone H3(S10) in G(2), followed by assembly of deficient kinetochores in M-phase. HP1-beta, Aurora B and the affected kinetochore proteins all were present at normal levels in treated cells; thus, effects of the inhibitors on mitotic progression do not seem to reflect changes in gene expression. In vitro kinase activity of Aurora B isolated from treated cells was unaffected. We propose that the increased presence in pericentromeric heterochromatin of histone H3 acetylated at K9 is responsible for the mitotic defects resulting from inhibition of histone deacetylation.

Heterochromatin and its Relationship to Cell Senescence and Cancer Therapy

“…62 In contrast, normal primary human cells tolerated HDIs quite well, despite minor chromosome segregation defects and a delay to progression through mitosis, as described by others previously. [63][64][65][66][67] We also found that in normal primary human cells not expressing HP1bDN, treatment with HDIs caused a rapid relocalization of endogenous HP1 proteins to pericentromeric regions, adjacent to chromosome kinetochores. 62 This relocalization was blocked in the cells expressing HP1bDN.…”

Belinostat and vincristine demonstrate mutually synergistic cytotoxicity associated with mitotic arrest and inhibition of polyploidy in a preclinical model of aggressive diffuse large B cell lymphoma

“…However, at similar submicromolar concentrations belinostat is unable to induce a strong mitotic arrest in the resistant DLBCL cell lines. Studies of the mitotic effects of HDACi in other cell types have established that, in addition to activating the SAC, they cause it to fail prematurely and induce apoptotic signaling 17,19,30,31 ). We therefore hypothesized that forced mitotic arrest in the resistant cells might sensitize them to belinostat-induced cytotoxicity.…”

“…17,19 HDAC inhibition causes destabilized interactions between microtubules (Mt) and kinetochores assembled at centromeres. 17,31 A recent study showed that aberrant acetylation of the Mt endbinding protein, EB1, prevents stable chromosome alignment at the metaphase plate. 47 SAC activation prevents progression or with belinostat (Bel) and vincristine (VCR) either alone or in combination for 48 h prior to harvest as shown below the timeline.…”

“…17,19,31 In addition, HDACs appear to be necessary to maintain the SAC once it has been activated. 17,19,30,31 Based on these studies and our results we predict that vincristine induces prolonged activation of the SAC in the HDACi-resistant cell lines and belinostat causes it to fail. To address this hypothesis we examined the phosphorylation of BubR1, an event that indicates SAC activation.…”

Histone deacetylase inhibitors: molecular mechanisms of action

“…In culture with TSA, histones in newly synthesized chromatin remain acetylated, and this disrupts the structure and function of the centromere and the pericentric heterochromatin, with loss of binding to heterochromatin binding proteins (Taddei et al, 2001;Cimini et al, 2003). Histone acetylation interferes with histone phosphorylation and disrupts the function of mitotic spindle checkpoint proteins, such as BubR1, hBUB1, CENP-F and CENP-E (Dowling et al, 2005;Robbins et al, 2005). As a result, the cells show a transient arrest at prometaphase, followed with aberrant mitosis such as missegregation and loss of chromosomes, resulting in cell death by either apoptosis or, mitotic cell death/ catastrophe (Qiu et al, 2000;Cimini et al, 2003;Xu et al, 2005).…”