Forced activation of Cdk1 via wee1 inhibition impairs homologous recombination.
In response to DNA breaks, the 'DNA damage response' provokes a cell cycle arrest to facilitate DNA repair. Recent findings have indicated that cells can respond to DNA damage throughout the cell cycle, except during mitosis. Specifically, various mitotic kinases, including Cdk1, Aurora A and Plk1, were shown to inactivate key DNA damage checkpoint proteins when cells enter mitosis. Aberrant activation of mitotic kinases during interphase could therefore modulate cellular responses to DNA damage. In this study, our aim was to determine how aberrant activation of Cdk1 affects the cellular responses to DNA damage. We used Wee1 inhibition, using MK-1775, to force Cdk1 activation, which did not cause cytotoxicity in non-transformed cells. Instead, it accelerated mitotic entry and caused radio sensitization in p53-defective cancer cells, but not in p53-proficient cancer cells. Interestingly, we showed that Wee1 inhibition leads to elevation of Cdk1 activity in interphase cells. When we subsequently analyzed DNA damage responses in cells with forced Cdk1 activation, we observed a marked reduction of 53BP1 at sites of DNA damage along with an increase in γ-H2AX staining after irradiation, indicative of defective DNA repair. Indeed, when DNA repair was analyzed using in vivo endonuclease-induced homologous recombination (HR) assays, compromised DNA repair after Wee1 inhibition was confirmed. This defect in HR was accompanied by increased phosphorylation of BRCA2 at the Cdk1 phosphorylation site S3291. Taken together, our results indicate that Wee1 inhibition leads to forced Cdk1 activation in interphase cells, which interferes with normal DNA damage responses.
Department of Medical Oncology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
M
M
Krajewska
A M
AM
Heijink
Y J W M
YJ
Bisselink
R I
RI
Seinstra
H H W
HH
Silljé
E G E
EG
de Vries
M A T M
MA
van Vugt
D016203:CDC2 Protein Kinase / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors*; D004249:DNA Damage; D004789:Enzyme Activation / Q000187:drug effects; D059765:Homologous Recombination / Q000187:drug effects*; D006801:Humans; D061986:MCF-7 Cells; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D011744:Pyrimidinones; D011804:Quinolines / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology
D018797:Cell Cycle Proteins; D009687:Nuclear Proteins; D047428:Protein Kinase Inhibitors; D011720:Pyrazoles; D011743:Pyrimidines; D011744:Pyrimidinones; D011804:Quinolines; C512984:RO 3306; D013844:Thiazoles; D011505:Protein-Tyrosine Kinases; C499349:WEE1 protein, human; D016203:CDC2 Protein Kinase; C549567:adavosertib