Synthetic lethality by inhibition of PARP in ATM-deficient lymphoid cells : reduced proliferation, replication induced DNA damage, G2 delay and cell death
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Cells that are deficient in proteins involved in homologous recombination repair (HRR) have been shown to be hypersensitive to PARP inhibitors. While a cell may tolerate PARP inhibition or HRR defects alone, the combination is lethal. This phenomenon is termed synthetic lethality. The function of HRR signaling protein ATM is lost in 50% of mantle cell lymphomas (MCL). MCL is an aggressive and currently incurable subtype of Non-Hodgkins lymphoma. PARP inhibition might prove to be a valuable treatment option for ATM deficient MCL cases. In this master thesis, we have investigated the alterations in cell growth, cell cycle distribution and DNA damage levels, as well as mode of cell death caused by PARP inhibitor treatment of ATM deficient lymphoid cells. Four lymphoid cancer cell lines (Reh, U698, JVM-2 and Granta-519) were continuously exposed to the clinically relevant PARP inhibitor (olaparib/AZD-2281), and/or ATM inhibitor (KU-55933). Cell growth was reduced or inhibited in all cell lines exposed to both ATM inhibitor and PARP inhibitor. The ATM inhibitor alone had little effect on the measured parameters in general, but increased the doubling time for all cell lines, and extended mitosis of U698 and Granta-519 cells. PARP inhibition caused a dose dependent-increase of DNA double strand breaks (DSB) during S phase. A G2 phase delay was induced by combined PARP and ATM inhibition. The cells repaired the DSBs associated with γH2AX foci during the prolonged G2 phase and entered mitosis without foci. Granta-519 and Reh cells became apoptotic from G2 or M in response to PARP and ATM inhibition, possibly because of high levels of DSBs. PARP and ATM inhibited U698 and JVM-2 cells suffered from mitotic catastrophe before necrosis. TP53 deficient U698 cells endoreduplicated extensively, while JVM-2 (wildtype TP53) cells arrested after failed cytokinesis. ATM deficient/inhibited lymphoid cells are sensitized to PARP inhibitors in a cell line specific manner, possibly because of other underlying genetic aberrations. We propose that the synthetic lethality of PARP and ATM inhibition was caused by repeated cycles of incorrect or failed repair of DNA DSBs that occurred during replication. Even though the HRR deficient cells have repaired the DSBs (possibly by error-prone non-homologous end joining), they may still accumulate translocations and/or other structural chromosome-defects that lead to apoptosis or mitotic catastrophe.
Master i biomedisin
PublisherHøgskolen i Oslo og Akershus. Fakultet for helsefag
Oslo universitetssykehus HF. Institutt for kreftforskning