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The radiosensitization of tumor cells is one of the promising approaches for enhancing radiation damage to cancer cells and limiting radiation effects on normal tissue. In this study, we performed a comprehensive screening of radiosensitization targets in human lung cancer cell line A549 using an shRNA library and identified apolipoprotein B mRNA editing enzyme catalytic subunit 3G (APOBEC3G: A3G) as a candidate target. APOBEC3G is an innate restriction factor that inhibits HIV-1 infection as a cytidine deaminase. APOBEC3G knockdown with siRNA showed an increased radiosensitivity in several cancer cell lines, including pancreatic cancer MIAPaCa2 cells and lung cancer A549 cells. Cell cycle analysis revealed that APOBEC3G knockdown increased S-phase arrest in MIAPaCa2 and G2/M arrest in A549 cells after γ-irradiation. DNA double-strand break marker γH2AX level was increased in APOBEC3G-knocked-down MIAPaCa2 cells after γ-irradiation. Using a xenograft model of A549 in mice, enhanced radiosensitivity by a combination of X-ray irradiation and APOBEC3G knockdown was observed. These results suggest that the functional inhibition of APOBEC3G sensitizes cancer cells to radiation by attenuating the activation of the DNA repair pathway, suggesting that APOBEC3G could be useful as a target for the radiosensitization of cancer therapy.

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.

Poly(ADP-ribose) polymerase (PARP) is involved in DNA repair and chromatin regulation. 5-Aza-2′-deoxycytidine (5-aza-dC) inhibits DNA methyltransferases, induces hypomethylation, blocks DNA replication, and causes DNA single strand breaks (SSBs). As the PARP inhibitor is expected to affect both DNA repair and transcriptional regulations, we investigated the effect of combinational use of PARP inhibitors on cytotoxicity of 5-aza-dC in human cancer cell lines. The combinational treatment of 5-aza-dC and PARP inhibitor PJ-34 exhibited a stronger cytotoxicity compared with their treatment alone in blood cancer HL-60, U937, and colon cancer HCT116 and RKO cells. Treatment with 5-aza-dC but not PJ-34 caused SSBs in HCT116 cell lines. Global genome DNA demethylation was observed after treatment with 5-aza-dC but not with PJ-34. Notably, in microarray analysis, combinational treatment with PJ-34 and 5-aza-dC caused dissimilar broad changes in gene expression profiles compared with their single treatments in both HCT116 and RKO cells. The profiles of reactivation of silenced genes were also different in combination of PJ-34 and 5-aza-dC and their single treatments. The results suggest that the combinational use of 5-aza-dC and PARP inhibitor may be useful by causing distinct transcriptional profile changes.

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