RESUMO
When hydroxyurea and caffeine are added to Chinese hamster ovary cells, the cells bypass the S-phase checkpoint, and enter unscheduled mitosis. These cells build a morphologically normal spindle, and distribute unreplicated kinetochore fragments to daughters. We examined these cells and found that they undergo a full repertoire of mitotic stages, with the exception of anaphase B. Spindle elongation did not occur in these cells. When taxol was added, treated cells arrested indicating that microtubule turnover was necessary for kinetochore fragment separation. When released from taxol arrest, these cells divided. Finally, we determined that mitosis with unreplicated genome cells separated kinetochore fragments relatively equally. This mitosis is minimal, but still successful in kinetochore separation, which provides insight into the mechanism of anaphase movement.
Assuntos
Cinetocoros/metabolismo , Mitose/fisiologia , Anáfase/efeitos dos fármacos , Animais , Células CHO , Centrômero/efeitos dos fármacos , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cricetinae , Cricetulus , Genoma/genética , Hidroxiureia/farmacologia , Cinetocoros/efeitos dos fármacos , Cinetocoros/ultraestrutura , Microscopia Confocal , Microtúbulos/metabolismo , Mitose/efeitos dos fármacos , Paclitaxel/farmacologia , Fuso Acromático/ultraestruturaRESUMO
CHO cells can be arrested with hydoxyurea at the beginning of the DNA synthesis phase of the cell cycle. Subsequent treatment with the xanthine, caffeine, induces cells to bypass the S-phase checkpoint and enter unscheduled mitosis [Schlegel and Pardee,1986, Science 232:1264-1266]. These treated cells build a normal spindle and distribute kinetochores, unattached to chromosomes, to their daughter cells [Brinkley et al.,1988, Nature 336:251-254; Zinkowski et al.,1991, J Cell Biol 113:1091-1110; Wise and Brinkley,1997, Cell Motil Cytoskeleton 36:291-302; Balczon et al.,2003, Chromosoma 112:96-102]. To investigate how these cells distribute kinetochores to daughter cells, we analyzed the spindle checkpoint components, Mad2, CENP-E, and the 3F3 phosphoepitope, using immunofluorescence and digital microscopy. Even though the kinetochores were unpaired and DNA was fragmented, the tension, alignment, and motor components of the checkpoint were found to be present and localized as predicted in prometaphase and metaphase. This unusual mitosis proves that a cell can successfully localize checkpoint proteins and divide even when kinetochores are unpaired and fragmented.
