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Genomic Maintenance & Repair

(PP313) Aminothiols can reduce -H2AX formation against X-irradiation in human microvascular endothelial cells.

Kataoka, Yasushi^*,1, Murley, Jeffrey¹, Grdina, David¹, ¹ Radiation and Cellular Oncology, Chicago, IL, USA

ABSTRACT- -H2AX foci formation is a sensitive and specific marker for the detection of DNA DSBs. Thiol-containing radioprotective drugs such as WR-1065 (active form of amifostine, WR-2721) and WR-255591 (active form of phosphonol, WR-3689) are well known to reduce radiation-induced cell killing. We applied flow cytometric analysis to assess the effect of thiol-containing drugs on radiation-induced -H2AX formation. Human microvascular endothelial cells (HMEC) were irradiated with X-rays (RT250, Phillips) ranging from 2 Gy to 16 Gy at 1.65 Gy/min. -H2AX was assessed 1 h after X-irradiation unless otherwise stated. Thiol-containing drugs included WR-1065, WR-255591, N-acetyl-L-cysteine (NAC), captopril and mesna at 4 mM concentration 30 min before irradiation. WR-1065 was further studied either 30 min before (B), during (D) and/or after (A) irradiation. After radiation- and/or drug-treatment, cells were trypsinized, fixed, permeabilized, and stained with FITC-conjugated anti--H2AX antibody (Upstate) combined with DAPI staining. Flow cytometric analysis was carried out by LSR II System (BD Bioscience). -H2AX positive cells increased as a function of radiation dose (2-16 Gy) at 1 h and decreased in a radiation dose dependent manner at 4 h, followed by a further decrease to almost the control level at 24 h. Although all the thiol-containing drugs were effective in protecting against -H2AX formation, the relative effectiveness was WR-255591≥WR-1065>NAC, captopril and mesna. The effect of WR-1065 exposure was BDA≥BDBA>DA≥A≥D≥B. The anti-mutagenic dose of WR-1065 (40 micro M) was not effective in reducing -H2AX. Thiols effectiveness was observed in all phases of the cell cycle. Effectiveness of drug treatment suggests that more than just a radical scavenging mechanism by these agents accounts for this effect. This work was supported by NIH/NCI grants R01-CA37435 and CA99005.

Key words: -H2AX, aminothiols, radioprotector, flow cytometry