Effects of 3-aminobenzamide on cell cycle arrest after gamma-irradiation

Poly(ADP-ribose) polymerase (Parp) inhibitor 3-aminobenzamide (3-AB) pretreatment suppresses G1 arrest and enhances G2 arrest after gamma-irradiation in mouse embryonic fibroblast C3D2F1 3T3-a cells. 3-AB partially inhibits Waf1/Cip1/p21 and Mdm2 mRNA inductions, which are transcriptionally activated by p53, suggesting that poly(ADP-ribosylation) is involved in the downstream of p53 dependent signal transduction after gamma-irradiation in C3D2F1 3T3-a cells. In this study we further examined the involvement of poly(ADP-ribosylation) in cell cycle arrest. Effect on G1 arrest suppression was lost when 3-AB was added 6 hrs after irradiation. When C3D2F1 3T3-a cells were synchronized by serum starvation, and gamma-irradiated, the peak time of DNA synthesis was not changed but the ratio of DNA synthesis was decreased in gamma-irradiated cells, where 3-AB pretreatment slightly enhanced the decrease of this ratio. 3-AB decreased G1 arrest in mouse embryonic fibroblast Swiss3T3 cells dose-independent manner whereas G1 arrest was not affected at low doses in FM3A and NRF49F cells. To confirm the inhibitory effect of 3-AB on Parp activity, NAD level change was measured after gamma-irradiation. We observed NAD decrease induced by gamma-irradiation was prevented by the 4 mM 3-AB, suggesting sufficient inhibition of cellular Parp activity at 4 mM concentration. These results suggested that the effect of Parp inhibitor on G1 arrest after gamma-irradiation depends on cell phenotypes. out by incubation of the cells with medium containing 0.25% FBS for 44 hrs and released cells by changing the medium containing 10% FBS and 4 hrs later, cells were treated with chemicals. DNA synthesis was measured by pulse-labeling with [ 3 H]-thymidine (New England Nuclear). and trichloroacetic acid precipitation method. Cells were irradiated with a 60 Co gamma-irradiator at 1 Gy/min. Cell cycle states were analyzed by pulsing cells with 10 µM bromodeoxyuridine (BrdU, Sigma) for 30 min at a selected period after gamma-irradiation. After irradiation, cells were harvested, fixed with 70% ethanol, denatured with 4 N hydrochloric acid, treated with RNase A, and stained with FITC-conjugated anti-BrdU antibody and with propidium iodide (Sigma), by two-dimensional flow cytometry using FACScan (Beckton Dickinson). pattern of the intracellular nucleotide pool in C3D2F1 3T3-a cells, as determined by HPLC.