The role of serine/threonine protein phosphatases in mitosis.

摘要

Previous studies of two naturally-occurring compounds suggested a correlation between protein phosphatase inhibition and anti-cancer qualities; however, the mechanism was unclear. Strong evidence suggested the mechanism involved inhibition of PPP phosphatases based on a common catalytic region, "inhibitor-binding domain"; however, identification of specific phosphatase(s) and associated functions was unclear. Collectively, this dissertation identifies PP2Aalpha and PP4 as the target phosphatases with critical roles in mitotic progression, whose selective inhibition is sufficient to induce comparable anti-mitotic effects associated with these compounds.;Initial studies characterized the anti-proliferative and cytotoxic effects of cantharidin demonstrating a dose-dependent inhibition of PPP-phosphatases at concentrations consistent with the dose/time-dependent decrease in viability and proliferation and increase in G2/M cell cycle arrest. Microscopic examination revealed disruptions in mitotic spindle formation supported by time-lapse studies revealing disruptions in attaining and maintaining metaphase alignment. Comparative examination of fostriecin at concentrations capable of the complete inhibition of PP2A/PP4 and 50% of PP1/PP5 activity (in vitro) revealed disruptions in metaphase alignment, mitotic exit and cytokinesis.;Subsequent studies identified PP2Aa and PP4 as the PPases with novel and critical mitotic roles, further implicating PP2Aalpha and PP4 as the target PPases responsible for the anti-mitotic effects attributed to cantharidin and fostriecin. Selective PPase inhibition using antisense oligonucleotide-mediated suppression was confirmed by Northern and Western analysis as an effective technique to suppress individual isoforms without altering other related PPases. Time-lapse microscopy revealed selective suppression of PP2Aalpha or PP4 resulted in prominent disruptions during mitosis consistent with those exhibited by cantharidin and fostriecin; comparatively, suppression of PP1gamma, PP2Abeta and PP5 exhibited typical mitotic progression. These results demonstrate novel roles for PP2Aalpha and PP4 as essential regulators in mitotic progression: PP2A via chromosome alignment and segregation, and PP4 via chromosome alignment/segregation, mitotic checkpoint fidelity and cytokinesis completion. Collectively, this dissertation identifies PP2Aalpha and PP4 as the target PPP phosphatases suppressed by cantharidin and fostriecin predominantly responsible for the cytotoxic and anti-proliferative effects. Furthermore, suppression of either PP2Aalpha or PP4 is sufficient to alter mitotic progression in a manner that emulates the anti-proliferative effects of cantharidin and fostriecin and thereby warrant further evaluation.