title: BMI1 attenuates etoposide-induced G2/M checkpoints via reducing ATM activation.
abstract: The BMI1 protein contributes to stem cell pluripotency and oncogenesis via multiple functions, including its newly identified role in DNA damage response (DDR). Although evidence clearly demonstrates that BMI1 facilitates the repair of double-stranded breaks via homologous recombination (HR), it remains unclear how BMI1 regulates checkpoint activation during DDR. We report here that BMI1 has a role in G2/M checkpoint activation in response to etoposide (ETOP) treatment. Ectopic expression of BMI1 in MCF7 breast cancer and DU145 prostate cancer cells significantly reduced ETOP-induced G2/M arrest. Conversely, knockdown of BMI1 in both lines enhanced the arrest. Consistent with ETOP-induced activation of the G2/M checkpoints via the ATM pathway, overexpression and knockdown of BMI1, respectively, reduced and enhanced ETOP-induced phosphorylation of ATM at serine 1981 (ATM pS1981). Furthermore, the phosphorylation of ATM targets, including γH2AX, threonine 68 (T68) on CHK2 (CHK2 pT68) and serine 15 (S15) on p53 were decreased in overexpression and increased in knockdown BMI1 cells in response to ETOP. In line with the requirement of NBS1 in ATM activation, we were able to show that BMI1 associates with NBS1 and that this interaction altered the binding of NBS1 with ATM. BMI1 consists of a ring finger (RF), helix-turn-helix-turn-helix-turn (HT), proline/serine (PS) domain and two nuclear localization signals (NLS). Although deletion of either RF or HT did not affect the association of BMI1 with NBS1, the individual deletions of PS and one NLS (KRMK) robustly reduced the interaction. Stable expression of these BMI1 mutants decreased ETOP-induced ATM pS1981 and CHK2 pT68, but not ETOP-elicited γH2AX in MCF7 cells. Furthermore, ectopic expression of BMI1 in non-transformed breast epithelial MCF10A cells also compromised ETOP-initiated ATM pS1981 and γH2AX. Taken together, we provide compelling evidence that BMI1 decreases ETOP-induced G2/M checkpoint activation via reducing NBS1-mediated ATM activation.
authors.affiliation: 1] Division of Nephrology, Department of Medicine, McMaster University, Hamilton, Ontario, Canada [2] Father Sean O'Sullivan Research Institute, Hamilton, Ontario, Canada [3] The Hamilton Center for Kidney Research, St. Joseph's Hospital, Hamilton, Ontario, Canada [4] The Genetics Laboratory, Institute of Women and Children's Health, Longgang District, Shenzhen, Guangdong, P.R. China.
authors.forename: F
authors.initials: F
authors.lastname: Wei
authors.affiliation: 1] Division of Nephrology, Department of Medicine, McMaster University, Hamilton, Ontario, Canada [2] Father Sean O'Sullivan Research Institute, Hamilton, Ontario, Canada [3] The Hamilton Center for Kidney Research, St. Joseph's Hospital, Hamilton, Ontario, Canada.
authors.forename: D
authors.initials: D
authors.lastname: Ojo
authors.affiliation: 1] Division of Nephrology, Department of Medicine, McMaster University, Hamilton, Ontario, Canada [2] Father Sean O'Sullivan Research Institute, Hamilton, Ontario, Canada [3] The Hamilton Center for Kidney Research, St. Joseph's Hospital, Hamilton, Ontario, Canada.
authors.forename: X
authors.initials: X
authors.lastname: Lin
authors.affiliation: 1] Division of Nephrology, Department of Medicine, McMaster University, Hamilton, Ontario, Canada [2] Father Sean O'Sullivan Research Institute, Hamilton, Ontario, Canada [3] The Hamilton Center for Kidney Research, St. Joseph's Hospital, Hamilton, Ontario, Canada.
authors.forename: N
authors.initials: N
authors.lastname: Wong
authors.affiliation: 1] Division of Nephrology, Department of Medicine, McMaster University, Hamilton, Ontario, Canada [2] Father Sean O'Sullivan Research Institute, Hamilton, Ontario, Canada [3] The Hamilton Center for Kidney Research, St. Joseph's Hospital, Hamilton, Ontario, Canada [4] Massachusetts General Hospital (MGH), Harvard Medical School, Boston, MA, USA.
authors.forename: L
authors.initials: L
authors.lastname: He
authors.affiliation: 1] Division of Nephrology, Department of Medicine, McMaster University, Hamilton, Ontario, Canada [2] Father Sean O'Sullivan Research Institute, Hamilton, Ontario, Canada [3] The Hamilton Center for Kidney Research, St. Joseph's Hospital, Hamilton, Ontario, Canada.
authors.forename: J
authors.initials: J
authors.lastname: Yan
authors.affiliation: 1] Division of Nephrology, Department of Medicine, McMaster University, Hamilton, Ontario, Canada [2] Father Sean O'Sullivan Research Institute, Hamilton, Ontario, Canada [3] The Hamilton Center for Kidney Research, St. Joseph's Hospital, Hamilton, Ontario, Canada.
authors.forename: S
authors.initials: S
authors.lastname: Xu
authors.affiliation: Department of Oncology, McMaster University, Hamilton, Ontario, Canada.
authors.forename: P
authors.initials: P
authors.lastname: Major
authors.affiliation: 1] Division of Nephrology, Department of Medicine, McMaster University, Hamilton, Ontario, Canada [2] Father Sean O'Sullivan Research Institute, Hamilton, Ontario, Canada [3] The Hamilton Center for Kidney Research, St. Joseph's Hospital, Hamilton, Ontario, Canada.
authors.forename: D
authors.initials: D
authors.lastname: Tang
mesh_terms: D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D064007:Ataxia Telangiectasia Mutated Proteins / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D045744:Cell Line, Tumor; D004249:DNA Damage; D005047:Etoposide / Q000494:pharmacology*; D059565:G2 Phase Cell Cycle Checkpoints / Q000187:drug effects; D055785:Gene Knockdown Techniques; D006801:Humans; D061986:MCF-7 Cells; D048054:Mitogen-Activated Protein Kinase 7 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D018797:Cell Cycle Proteins; C112156:NBN protein, human; D009687:Nuclear Proteins; D005047:Etoposide; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C481581:MAPK7 protein, human; D048054:Mitogen-Activated Protein Kinase 7
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