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Spy1 Expression Prevents Normal Cellular Responses to DNA Damage and Enhances Cell Survival in Response to UV Irradiation in a U2OS Osteosarcoma Cell Line

Randy Gastwirt

Appointment Period: 2003-2007, Grant Years: [19,20,21,22]

Randy GastwirtVarious checkpoints exist to ensure that cells replicate without genetic errors and repair damaged DNA, to avoid both the uncoupling of replication from cell cycle control as well as to avoid the transmission of genetic mutations. I investigated the role of Spy1 expression in apoptosis and checkpoint activation to begin to understand the molecular mechanisms by which Spy1 may contribute to oncogenesis.

Spy1 is the originally identified member of the Speedy/Ringo family of cyclin-like cell cycle regulators, which can control cell proliferation and survival through the atypical activation of cyclin dependent kinases. We showed that Spy1 expression enhances cell survival in response to UV irradiation by preventing the activation of caspases and apoptosis in a U2OS osteosarcoma cell line. Using an inducible system allowing for regulated expression of Spy1, we show that Spy1 expression prevents activation of caspase-3 and suppresses apoptosis in response to UV irradiation. Spy1 expression also allows for UV irradiation-resistant DNA synthesis and permits cells to progress into mitosis, as demonstrated by phosphorylation on histone H3, indicating that Spy1 expression can inhibit the S-phase/replication and G2/M checkpoints. We demonstrate that Spy1 expression inhibits phosphorylation of Chk1, RPA, and histone H2A.X, which may directly contribute to the decrease in apoptosis and checkpoint bypass. The data presented indicate that Spy1 expression allows cells to evade checkpoints and apoptosis and suggest that Spy1 regulation of CDK2 is important for the response to DNA damage.

Meyer AN, Gastwirt RF, Schlaepfer DD, Donoghue DJ. The cytoplasmic tyrosine kinase Pyk2 as a novel effector of fibroblast growth factor receptor 3 activation. J Biol Chem. (2004) 279:28450-7. PMID: 15105428.

Gastwirt RF, Slavin DA, McAndrew CW, Donoghue DJ. Spy1 expression prevents normal cellular responses to DNA damage: inhibition of apoptosis and checkpoint activation. J Biol Chem. (2006) 281:35425-35. PMID: 16951407; PMC2782695.

Gastwirt RF, McAndrew CW, Donoghue DJ. Speedy/RINGO regulation of CDKs in cell cycle, checkpoint activation and apoptosis. Cell Cycle. (2007) 6:1188-93. Review. PMID: 17507798.

McAndrew CW, Gastwirt RF, Meyer AN, Porter LA, Donoghue DJ. Spy1 enhances phosphorylation and degradation of the cell cycle inhibitor p27. Cell Cycle. (2007) 6:1937-45. PMID: 17671428.

McAndrew CW, Gastwirt RF, Donoghue DJ. The atypical CDK activator Spy1 regulates the intrinsic DNA damage response and is dependent upon p53 to inhibit apoptosis. Cell Cycle. (2009) 8:66-75. PMID: 19106603; PMCID: PMC2782695