Naoyuki Shigematsu

Inhibition of topoisomerase IIα activity in CHO K1 cells by 2-[(aminopropyl)amino]ethanethiol (WR-1065)

The aminothiol 2-[(aminopropyl)amino]ethanethiol (WR-1065) is the active thiol of the clinically studied radioprotective agent S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721). WR-1065 is an effective radiation protector when it is administered 30 min prior to exposure of Chinese hamster ovary K1 cells to radiation (i.e., a dose modification factor of 1.4) at a concentration of 4 mM. Under these exposure conditions, topoisomerase (Topo) I and II alpha activities and associated protein contents were measured in cells of the K1 cell line using the DNA relaxation assay, the P4 unknotting assay and immunoblotting, respectively. WR-1065 was ineffective in modifying Topo I activity, but it did reduce Topo II alpha activity by an average of 50%. The magnitude of Topo II alpha protein content, however, was not affected by these exposure conditions. The effects on the cell cycle were monitored by the method of flow cytometry. Exposure of cells to 4 mM WR-1065 for up to 6 h resulted in a build-up of cells in the G2/M-phase compartment. However, under these conditions and in contrast to Topo II inhibitors used in chemotherapy, WR-1065 is an effective radioprotective agent capable of protecting against both radiation-induced cell lethality and mutagenesis. One of several mechanisms of action attributed to aminothiol compounds such as WR-1065 has been their ability to affect endogenous enzymatic reactions involved in DNA synthesis and repair and progression of cells through the phases of the cell cycle. These results are consistent with such a proposed mechanism and demonstrate in particular a modifying effect by WR-1065 on Topo II, which is involved in DNA synthesis.

A Comparison of Chromosome Repair Kinetics in G0 and G1 Reveals that Enhanced Repair Fidelity under Noncycling Conditions Accounts for Increased Potentially Lethal Damage Repair

Abstract Potentially lethal damage (PLD) and its repair were studied in confluent human fibroblasts by analyzing the kinetics of chromosome break rejoining and misrejoining in irradiated cells that were either held in noncycling G0 phase or allowed to enter G1 phase of the cell cycle immediately after 6 Gy irradiation. Virally mediated premature chromosome condensation (PCC) methods were combined with fluorescence in situ hybridization (FISH) to study chromosomal aberrations in interphase. Flow cytometry revealed that the vast majority of cells had not yet entered S phase 15 h after release from G0. By this time some 95% of initially produced prematurely condensed chromosome breaks had rejoined, indicating that most repair processes occurred during G1. The rejoining kinetics of prematurely condensed chromosome breaks was similar for each culture condition. However, under noncycling conditions misrepair peaked at 0.55 exchanges per cell, while under cycling conditions (G1) it peaked at 1.1 exchanges per cell. At 12 h postirradiation, complex-type exchanges were sevenfold more abundant for cycling cells (G1) than for noncycling cells (G0). Since most repair in G0/G1 occurs via the non-homologous end-joining (NHEJ) process, increased PLD repair may result from improved cell cycle-specific rejoining fidelity of the NHEJ pathway.