01 and confirmed the inability of BxPC3 and CFPAC cells to override an S phase checkpoint arrest (Fig. S4B and data not shown). Thus, the failure to override the S phase arrest in certain cells is not idiosyncratic of gemcitabine but more likely a cellular defect. We next tested whether the limiting factors that prevented the BxPC3 and CFPAC cell lines to override the S phase checkpoint might also prevent them from overriding a G2 checkpoint arrest. We used the alkylating agent methyl methanesulfonate (MMS) as well as the topo II inhibitors etoposide and doxorubicin to induce a G2 arrest (Fig. S5A and data not shown). Interestingly, we found that BxPC3 cells arrested in G2 by MMS were able to enter mitosis upon addition of UCN-01. The same response was observed for PANC1 cells treated with MMS and UCN-01. Examination of the mitotic figures at the LM and EM levels in PANC1 and BxPC3 cells showed that they appeared to contain properly condensed chromosomes (Fig. 4A and B, top panel). We next treated PANC1 and BxPC3 with either etoposide or doxorubicin followed by UCN-01, and found that the G2-arrested cells were also forced into premature mitosis (Fig. 4B Figure 3. Biochemical purification of MUGs. (A) Lysates from MUGs were separated by and data not shown). Examination of the mitotic sucrose gradient fractionation and fractions were subjected to western blot analysis with figures showed extensive fragmentation and, the indicated antibodies. protein in each fraction was first concentrated by tCA and resuspended in the same volume, so that the fractions near the top of the gradient contained unexpectedly, evidence of centromere fragmenmore total protein than the bottom fractions. Fraction #1 represents the bottom of the tation as seen in cells that underwent MUGs gradient, while fraction #23 is the top. (B) Fraction 4 was fixed, pelleted onto coverslips (Fig. 4A and B). The presence of dissociated cenand used for the detection of Aurora B, Mis12 and DNA. Scale bar is 2 m. tromere/kinetochore complexes were confirmed by EM, which showed C-shaped kinetochore yielded kinetochore protein subcomplexes that migrated near the fragments that were indistinguishable from those generated from top of the gradient. The lack of detectable signal at the bottom of an S phase checkpoint override. the gradient suggested there were no MUGs (Fig. S3). Fractions Inhibitors of replication or topoisomerase II impair cenfrom the MUGs were also fixed and pelleted onto coverslips tromere replication.Peresolimab MUGs, according to the original obserand stained for kinetochore proteins Aurora B and Mis12.Mycophenolate Mofetil Only vations,15 were a consequence of forcing cells with unreplicated the bottom fractions contained material that produced discrete centromeres to enter mitosis.PMID:23546012 Thus, we were surprised as to how foci of staining (Fig. 3B). These complexes are likely MUGs, as topoII inhibitors, which do not appear to block DNA replicathey also contain DNA that was also detectable in the MUGs tion (Fig. S4), would generate MUGs when forced into mitoobserved in cells. These findings provide further evidence that sis. The EM images clearly show single C-shaped kinetochores, the centromere/kinetochore complex can be separated from bulk rather than a pair of kinetochores that would be expected to chromatin. assemble onto replicated centromeres. The discrepancy maybe Variable response of cell lines to S phase checkpoint over- resolved if one posits that centromere replication is uniquely ride. We n.
