title: Centromere fragmentation is a common mitotic defect of S and G2 checkpoint override.
abstract: DNA damaging agents, including those used in the clinic, activate cell cycle checkpoints, which blocks entry into mitosis. Given that checkpoint override results in cell death via mitotic catastrophe, inhibitors of the DNA damage checkpoint are actively being pursued as chemosensitization agents. Here we explored the effects of gemcitabine in combination with Chk1 inhibitors in a panel of pancreatic cancer cell lines and found variable abilities to override the S phase checkpoint. In cells that were able to enter mitosis, the chromatin was extensively fragmented, as assessed by metaphase spreads and Comet assay. Notably, electron microscopy and high-resolution light microscopy showed that the kinetochores and centromeres appeared to be detached from the chromatin mass, in a manner reminiscent of mitosis with unreplicated genomes (MUGs). Cell lines that were unable to override the S phase checkpoint were able to override a G2 arrest induced by the alkylator MMS or the topoisomerase II inhibitors doxorubicin or etoposide. Interestingly, checkpoint override from the topoisomerase II inhibitors generated fragmented kinetochores (MUGs) due to unreplicated centromeres. Our studies show that kinetochore and centromere fragmentation is a defining feature of checkpoint override and suggests that loss of cell viability is due in part to acentric genomes. Furthermore, given the greater efficacy of forcing cells into premature mitosis from topoisomerase II-mediated arrest as compared with gemcitabine-mediated arrest, topoisomerase II inhibitors maybe more suitable when used in combination with checkpoint inhibitors.
authors.affiliation: Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, PA, USA. Neil.Beeharry@fccc.edu
authors.forename: Neil
authors.initials: N
authors.lastname: Beeharry
authors.forename: Jerome B
authors.initials: JB
authors.lastname: Rattner
authors.forename: Juliane P
authors.initials: JP
authors.lastname: Caviston
authors.forename: Tim
authors.initials: T
authors.lastname: Yen
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000633:toxicity; D017209:Apoptosis / Q000187:drug effects; D016466:CHO Cells; D045744:Cell Line, Tumor; D002503:Centromere / Q000378:metabolism*; D000071877:Checkpoint Kinase 1; D020552:Comet Assay; D006224:Cricetinae; D003412:Cricetulus; D004249:DNA Damage / Q000187:drug effects; D004250:DNA Topoisomerases, Type II / Q000737:chemistry / Q000378:metabolism; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000633:toxicity; D004317:Doxorubicin / Q000633:toxicity; D005047:Etoposide / Q000633:toxicity; D059565:G2 Phase Cell Cycle Checkpoints / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D018386:Kinetochores / Q000378:metabolism; D008938:Mitosis; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism; D059807:S Phase Cell Cycle Checkpoints / Q000187:drug effects; D019311:Staurosporine / Q000031:analogs & derivatives / Q000494:pharmacology; D059005:Topoisomerase II Inhibitors / Q000494:pharmacology; D000093542:Gemcitabine
chemical_list: D000970:Antineoplastic Agents; D059005:Topoisomerase II Inhibitors; D003841:Deoxycytidine; D005047:Etoposide; C054852:7-hydroxystaurosporine; D004317:Doxorubicin; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D004250:DNA Topoisomerases, Type II; D019311:Staurosporine; D000093542:Gemcitabine
keywords: DNA damage; MUGs; cell cycle checkpoints; centromere; mitotic catastrophe