Dissecting the role of Mad2 in the mitotic checkpoint

Eukaryotic organisms have evolved a mitotic checkpoint to prevent sister chromatid separation until all chromosomes have been captured by mitotic spindle microtubules. The mitotic checkpoint signal originates from kinetochores located on each chromosome. A single unattached kinetochore can inhibit anaphase onset by generating a diffusible inhibitor that binds and inhibits Fizzy, an activator of the anaphase-promoting complex/cyclosome responsible for proteolytic degradation of mitotic substrates. Although the in vivo checkpoint inhibitor has not been identified, the checkpoint protein Mad2 is a likely candidate. Several Mad2 mutants have been developed that are unable to undergo binding events and conformational changes that occur during Mad2 activation; however, their behavior in a cellular environment has not been extensively studied. In this study, a panel of Mad2 mutants was evaluated in Xenopus laevis extracts to determine which Mad2 properties are critical to the activation and maintenance of the mitotic checkpoint.;Studying the regulation of the mitotic checkpoint is clinically relevant, as compounds that disrupt the mitotic spindle and interfere with mitosis are amongst the most successful pharmacological agents being used in cancer treatment. One promising target for chemotherapeutic agents is Eg5, which is required for bipolar spindle assembly. Eg5 inhibition activates the mitotic checkpoint by inducing monopolar spindle formation, typically resulting in either prolonged mitotic arrest or apoptosis following mitotic slippage. In this study, the effects of the Eg5 inhibitor SB-743921 were evaluated in a series of diffuse large B-cell lymphoma (DLBCL) cell lines. DLBCL is comprised of two clinically distinct subtypes: germinal center B cell-like (GCB) DLBCL and activated B cell-like (ABC) DLBCL. Although GCB DLBCL cell lines generally displayed greater sensitivity to SB-743921 when analyzing cell proliferation, mitotic arrest, and the expression of mitotic markers, ABC DLBCL cell lines were more sensitive to SB-743921 induced apoptosis. The effectiveness of current chemotherapeutic compounds to treat DLBCL patients is limited, particularly for ABC DLBCL patients. Therefore, the use of a chemotherapeutic agent that can effectively target both DLBCL subtypes could benefit patients by drastically improving survival rates.