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Brittany Townley, Merideth Kamradt Krevosky, Jeffery Bowen; A PAR-ting Gift: 3-Aminobenzamide attenuates PARP-1-dependent cell death induced by Melphalan in a Retinoblastoma cell line. Invest. Ophthalmol. Vis. Sci. 2016;57(12):3196.
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© ARVO (1962-2015); The Authors (2016-present)
Clinically, the alkylating agent, melphalan, is used to treat retinoblastoma, yet few studies have investigated the mechanism of cell death. Alkylating agents induce a poly(ADP-ribose) polymerase-1 (PARP-1)-dependent form of cell death characterized by PARP-1 hyperactivity, accumulation of poly(ADP-ribose) (PAR) polymers, phosphatidylserine (PS) externalization, and nuclear translocation of apoptosis-inducing factor (AIF). This study tested the hypothesis that PARP-1 mediates the cell death response to treatment with melphalan in the Rb143 retinoblastoma cell line.
Cells were pre-incubated for 1 hour with 5mM of the PARP-1 inhibitor, 3-Aminobenzamide (3-ABA) (n=4), or 10μM of the Caspase-3 inhibitor, Z-VAD-(OMe)-FMK (Z-VAD) (n=3). Melphalan was administered at 5, 10, 25, 50, or 100μg/mL over 4 hours and samples were collected after 24 hours (n=7). Cell viability was first analyzed morphologically, and then via flow cytometry following Annexin and PI staining. Western blot analysis was used to identify PARP-1, PAR, and AIF expression as evidence for PARP-1-dependent signaling, and Caspase-3 for apoptotic signaling.
3-ABA exhibited marked attenuation of cell death as compared to Z-VAD in cells treated with 50 and 100μg/mL melphalan. Nuclear condensation was observed after treatment with melphalan and with Z-VAD, but not with 3-ABA. Melphalan-treated samples demonstrated phosphatidylserine-positivity that was reversed by treatment with 3-ABA but not with Z-VAD. PAR accumulation was greatest in samples treated with 50 and 100μg/mL melphalan, coincident with increased PARP-1 activity, decreased full-length AIF, and decreased Caspase-3 activation.
These results support that the cellular response to melphalan is PARP-1-dependent. In low-dose administration of melphalan, cell death occurs via PARP-1 cleavage in a Caspase-3-dependent manner. However, in high-dose administration, PARP-1 activity increases, PAR polymer accumulates, and nuclear translocation of AIF occurs. Hydrolysis of PAR by poly(ADP-ribose) glycohydrolase (PARG) may abrogate PAR accumulation in low doses of melphalan. As PARP-1 and PARG mediate a metabolic cycle that centers on PAR synthesis and degradation, inhibition of PARG presents an attractive avenue for increasing the efficacy of melphalan at lower doses.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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