Spontaneous and Bleomycin-Induced <i>&gamma;</i>H2AX Signals in CHO9 Metaphase Chromosomes

Abstract

In eukaryotes, a cascade of events named DNA damage response (DDR) has evolved to handle DNA lesions. DDR engages the recruitment of signaling, checkpoint control, repair and chromatin remodeling protein complexes, allowing cell cycle delay, DNA repair or induction of apoptosis. An early DDR event involves the phosphorylation of the histone variant γ H2AX on serine 139 (H2AX 139 phosphorylation) originating the so-called γ H2AX. DDR-related H2AX 139 phosphorylation have been extensively studied in interphase nuclei. More recently, γ H2AX signals on mitotic chromosomes of asynchronously growing cell cultures were observed. We performed a quantitative analysis of γ H2AX signals on γ H2AX immunolabeled cytocentrifuged metaphase spreads, analyzing the γ H2AX signal distributions of CHO9 chromosomes harboring homologous regions both in control and bleomycin (BLM)-treated cultures. We detected γ H2AX signals in CHO9 chromosomes of controls which significantly increase after BLM-exposure. γ H2AX signals were uniformly distributed in chromosomes of controls. However, the γ H2AX signal distribution in BLM exposed cells was significantly different between chromosomes and among chromosome regions, with few signals near the centromeres and a tendency to increase towards the telomeres. Interestingly, both basal and BLM-induced γ H2AX signal distribution were statistically equal between CHO9 homologous chromosome regions. Our results suggest that BLM exerts an effect on H2AX 139 phosphorylation, prevailing towards acetylated and gene-rich distal chromosome segments. The comparable H2AX 139 phosphorylation of homologous regions puts forward its dependence on chromatin structure or function and its independence of the position in the karyotype.