Examining the involvement of Slx5 in the apoptotic response to chronic activation of the spindle assembly checkpoint.

Microtubule-targeting agents represent one of the most successful groups of anticancer drugs used in cancer therapy today. These drugs induce a prolonged mitotic arrest through chronic spindle assembly checkpoint (SAC) activation. Apoptosis, an outcome of the prolonged mitotic arrest, is the main mechanism by which these anticancer drugs kill cancer cells. However, not much is known about the mechanism that directs chronic SAC activation to apoptosis among other possible outcomes. The aim of this study is to investigate whether Slx5, a sumo-targeted ubiquitin E3 ligase, is involved in directing chronic SAC activation to apoptosis. We show that chronic SAC activation triggered by a 10-h nocodazole incubation leads to a prolonged mitotic arrest in the slx5Δ strain similar to wild type (WT). However, the proportion of cells displaying apoptotic features such as nuclear fragmentation, DNA fragmentation, and reactive oxygen species (ROS) production were increased more in the WT strain during the chronic SAC activation compared to slx5Δ, indicating that Slx5 may be involved in the chronic SAC-activation-apoptosis relation. We also showed that the possible role of Slx5 in the chronic SAC activation-apoptosis association was not through ubiquitin dependent degradation of 3 apoptosis-related and sumoylated candidate proteins.

Hydrogen peroxide prolongs mitotic arrest in a dose dependent manner and independently of the spindle assembly checkpoint activity in Saccharomyces cerevisiae.

Oxidative stress and chromosome missegregation are important factors that are linked to aneuploidy. A major reason for chromosome missegragation is the inappropriate activity of the spindle assembly checkpoint (SAC), a conserved surveillance mechanism that monitors the state of kinetochore-microtubule attachments to ensure equal chromosome segregation in mitosis. SAC-activation induces a prolonged mitotic arrest. Mitosis is considered the most vulnerable cell cycle phase to several external signals, therefore increasing the time cells spent in this phase via mitotic arrest induction by SAC-activating agents is favorable for cancer therapy. Cancer cells also display elevated oxidative stress due to abnormally high production of reactive oxygen species (ROS). However, the effect of increased oxidative stress on the duration of mitotic arrest remains largely unknown. In this study, we investigated the effect of H2O2-induced oxidative stress on the mitotic arrest induced by a SAC-activating agent (nocodazole) in Saccharomyces cerevisiae. Our data suggest that oxidative stress prolongs SAC-activation induced mitotic arrest in a dose dependent manner. We, in addition, investigated the effect of H2O2 treatment on the mitotic arrest induced independently of SAC-activation by using a conditional mutant (cdc23) and showed that the effect of H2O2-induced oxidative stress on mitotic arrest is independent of the SAC activity.