| Abstract |
CHK1 and WEE1 play pivotal roles in G2/M checkpoint following exogenous DNA damage and regulation of DNA replication under normal cellular conditions. Here, we monitored and compared the cell cycle kinetics of mitosis-associated events after CHK1 and WEE1 inhibitor treatments in a human tongue cancer cell line (SAS). A fluorescent ubiquitination-based cell cycle indicator (Fucci) that reflects SCFSKP2 and APCCDH1 E3 ligase activities was used to monitor cell cycle progression. Numerous γH2AX-positive cells were observed within the S phase population of cells following CHK1 inhibitor treatment, and polyploid cells exhibiting DNA damage emerged via abortive mitosis (endomitosis) at 24 h post treatment. While WEE1 inhibitor-treated cells exhibited similar polyploidy via endomitosis at later time points, they possessed fewer γH2AX foci during S phase, and polyploid cells exhibiting DNA damage were scarce. Instead, mitosis duration greatly extended and was accompanied by an abnormal emission of Fucci red fluorescence. Kinetic analysis of Fucci fluorescence revealed that abnormal emission occurred at early M phase in a manner independent of green fluorescence degradation as a marker of APCCDH1 activation. When an inhibitor of the essential spindle checkpoint factor MPS1 was co-treated with a WEE1 inhibitor, the elongated mitosis duration and abnormal red fluorescence were abrogated, and WEE1-induced reduction of clonogenic survival was offset. We demonstrate novel differential effects on mitosis-associated events following CHK1 and WEE1 inhibitor treatments.
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