U2OS cells lacking Chk1 undergo aberrant mitosis and fail to activate the spindle checkpoint.
Chk1 is a conserved protein kinase originally identified in fission yeast, required to delay entry of cells with damaged or unreplicated DNA into mitosis. The requirement of Chk1 for both S and G2/M checkpoints has been elucidated while only few studies have connected Chk1 to the mitotic spindle checkpoint. We used a small interference RNA strategy to investigate the role of Chk1 in unstressed conditions. Chk1 depletion in U2OS human osteosarcoma cells inhibited cell proliferation and raised the percentage of cells with a 4N DNA content, which correlated with accumulation of giant polynucleated cells morphologically distinct from apoptotic cells, while no increased number of cells in G2 or mitosis could be detected. Down-regulation of Chk1 also caused accumulation of cells in the last step of cytokinesis, and of tetraploid cells in G1 phase, which coincided with activation of p53 and increased levels of p21. In addition, Chk1-depleted U2OS cells failed to arrest in mitosis after spindle disruption by nocodazole and showed decreased protein levels of Mad2 and BubR1. These studies show that U2OS cells lacking Chk1 undergo abnormal mitosis and fail to activate the spindle checkpoint, suggesting a role of Chk1 in this checkpoint.
Carrassa
Laura
L
Department of Oncology, Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy. carrassa@marionegri.it
Sanchez
Yolanda
Y
Erba
Eugenio
E
Damia
Giovanna
G
D017209:Apoptosis / Q000187:drug effects; D002135:Calcium-Binding Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000187:drug effects / Q000201:enzymology; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D000071877:Checkpoint Kinase 1; D004273:DNA, Neoplasm / Q000378:metabolism; D006801:Humans; D064246:Mad2 Proteins; D008938:Mitosis / Q000187:drug effects; D015739:Nocodazole / Q000494:pharmacology; D010641:Phenotype; D011494:Protein Kinases / Q000172:deficiency* / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D008941:Spindle Apparatus / Q000187:drug effects / Q000201:enzymology*
D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D004273:DNA, Neoplasm; C577697:MAD2L1 protein, human; D064246:Mad2 Proteins; D012097:Repressor Proteins; D011494:Protein Kinases; C584277:BUB1 protein, human; C090464:Bub1 spindle checkpoint protein; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases; D015739:Nocodazole