DNA damage response in resting and proliferating peripheral blood lymphocytes treated by camptothecin or X-ray

Abstract

DNA damage response (DDR) in different cell cycle status of human peripheral blood lymphocytes (PBLs) and the role of H2AX in DDR were investigated. The PBLs were stimulated into cell cycle with phytohemagglutinin (PHA). The apoptotic ratio and the phosphorylation H2AX (S139) were flow cytometrically measured in resting and proliferating PBLs after treatment with camptothecin (CPT) or X-ray. The expressions of γH2AX, Bcl-2, caspase-3 and caspase-9 were detected by Western blotting. DDR in 293T cells was detected after H2AX was silenced by RNAi method. Our results showed that DNA double strand breaks (DSBs) were both induced in quiescent and proliferating PBLs after CPT or X-ray treatment. The phosphorylation of H2AX and apoptosis were more sensitive in proliferating PBLs compared with quiescent lymphocytes (P<0.05). The expression levels of anti-apoptotic proteins Bcl-2 were reduced and cleaved caspase-3 and caspase-9 were increased. No significant changes were observed in CPT-induced apoptosis in 293T cells between H2AX knocking down group and controls. It was concluded that proliferating PBLs were more vulnerable to DNA damage compared to non-stimulated lymphocytes and had higher apoptosis rates. γH2AX may only serve as a marker of DNA damage but exert no effect on apoptosis regulation.

Applicability of flow cytometry γH2AX assay in population studies: suitability of fresh and frozen whole blood samples

“…As a whole, fresh whole blood samples exhibited a more intense response to BLM and Act-D treatments when cells had been stimulated with PHA. Although both quiescent and cycling cells (in all cell cycle phases, including mitosis) have shown generation of γH2AX in response to DNA damage (Giunta and Jackson 2011), the levels of phosphorylation have been reported to be higher in PHA stimulated lymphocytes than in unstimulated cells when exposed, for instance, to camptothecin (Tian et al 2011) or to X-rays (Hamasaki et al 2007;Tian et al 2011). This is probably related to the fact that in cycling cells DSB are also indirectly produced from other less relevant types of DNA damage.…”