Induction and inhibition of the pan-nuclear gamma-H2AX response in resting human peripheral blood lymphocytes after X-ray irradiation

Abstract

Human peripheral blood lymphocytes (HPBLs) are one of the most sensitive cells to ionizing radiation (IR) in the human body, and IR-induced DNA damage and functional impairment of HPBLs are the adverse consequences of IR accidents and major side effects of radiotherapy. Phosphorylated H2AX (γH2AX) is a sensitive marker for DNA double-strand breaks, but the role and regulation of the pan-nuclear γH2AX response in HPBLs after IR remain unclear. We herein demonstrated that the pan-nuclear γH2AX signals were increased in a time- and dose-dependent manner, colocalized with >94% of TUNEL apoptotic staining, and displayed a typical apoptotic pattern in resting HPBLs after low LET X-ray IR. In addition, the X-irradiation-induced pan-nuclear p-ATM and p-DNA-PKcs responses also occurred in resting HPBLs, and were colocalized with 92–95% of TUNEL staining and 97–98% of the pan-nuclear γH2AX signals, respectively, with a maximum at 6 h post irradiation, but disappeared at 24 h post irradiation. Moreover, ATM/DNA-PKcs inhibitor KU55933, p53 inhibitor PFT-μ and pan-caspase inhibitor ZVAD-fmk significantly decreased X-irradiation-induced pan-nuclear γH2AX signals and TUNEL staining, protected HPBLs from apoptosis, but decreased the proliferative response to mitogen in X-irradiated HPBLs. Notably, whereas both KU55933 and PFT-μ increased the IR-induced chromosome breaks and mis-repair events through inhibiting the formation of p-ATM, p-DNA-PKcs and γH2AX foci in X-irradiated HPBLs, the ZVAD-fmk did not increase the IR-induced chromosomal instability. Taken together, our data indicate that pan-nuclear γH2AX response represents an apoptotic signal that is triggered by the transient pan-nuclear ATM and DNA-PKcs activation, and mediated by p53 and pan-caspases in X-irradiated HPBLs, and that caspase inhibitors are better than ATM/DNA-PKcs inhibitors and p53 inhibitors to block pan-nuclear γH2AX response/apoptosis and protect HPBLs from IR.

The NEDD8-activating enzyme inhibitor MLN4924 induces DNA damage in Ph+ leukemia and sensitizes for ABL kinase inhibitors

“…In agreement with these data, MLN4924-treatment of BV173 and SUPB15 cells resulted in increased nuclear γH2AX foci formation, which is an established marker of DNA damage (Figure 4(b)). In addition, a pan-nuclear γH2AX staining pattern was frequently observed in MLN4924-treated cells, which most likely represents apoptotic cells [51]. Consistently, a greater fraction of cells with >10 γH2AX foci in the nucleus were found in the presence of the pan-caspase inhibitor QVD-OPh (Figure 4(c)).…”

γ-H2AX/53BP1/pKAP-1 foci and their linear tracks induced by in vitro exposure to radon and its progeny in human peripheral blood lymphocytes

“…It has been reported that IR-induced pan-nuclear γ-H2AX response represents an apoptotic signal33343536. In the present study, we further observed that the pan-nuclear γ-H2AX response in HPBLs induced by α-particle irradiation from 241 Am source was co-localized with apoptotic TUNEL staining accompanying with γ-H2AX foci in neighboring cells; exposure to radon also induced the apoptotic pan-nuclear γ-H2AX response accompanying with intermittent linear γ-H2AX foci track and individual foci in neighboring cells (Fig.…”

Impact of ATM and DNA-PK Inhibition on Gene Expression and Individual Response of Human Lymphocytes to Mixed Beams of Alpha Particles and X-Rays

“…This is supported by results from lymphocytes where numbers of γH2AX, phospho-ATM and phospho-DNA-PK foci were reduced by ca. 50-70% upon ATM inhibition [39].…”