Cytometric assessment of DNA damage in relation to cell cycle phase and apoptosis

Abstract

Reviewed are the methods aimed to detect DNA damage in individual cells, estimate its extent and relate it to cell cycle phase and induction of apoptosis. They include the assays that reveal DNA fragmentation during apoptosis, as well as DNA damage induced by genotoxic agents. DNA fragmentation that occurs in the course of apoptosis is detected by selective extraction of degraded DNA. DNA in chromatin of apoptotic cells shows also increased propensity to undergo denaturation. The most common assay of DNA fragmentation relies on labelling DNA strand breaks with fluorochrome-tagged deoxy-nucleotides. The induction of double-strand DNA breaks (DSBs) by genotoxic agents provides a signal for histone H2AX phosphorylation on Ser139; the phosphorylated H2AX is named γH2AX. Also, ATM-kinase is activated through its autophosphorylation on Ser1981. Immunocytochemical detection of γH2AX and/or ATM-Ser1981(P) are sensitive probes to reveal induction of DSBs. When used concurrently with analysis of cellular DNA content and caspase-3 activation, they allow one to correlate the extent of DNA damage with the cell cycle phase and with activation of the apoptotic pathway. The presented data reveal cell cycle phase-specific patterns of H2AX phosphorylation and ATM autophosphorylation in response to induction of DSBs by ionizing radiation, topoisomerase I and II inhibitors and carcinogens. Detection of DNA damage in tumour cells during radio-or chemotherapy may provide an early marker predictive of response to treatment. DNA FRAGMENTATION DURING APOPTOSIS Involvement of different nucleases in DNA fragmentationCondensation of chromatin and internucleosomal DNA fragmentation, together with cell shrinkage and shedding of apoptotic bodies ('blebbing'), are widely recognized hallmarks of apoptosis (Kerr et al. 1972;Arends et al. 1990;Nagata 2000;Nagata et al. 2003). Several nucleases have been identified as contributing towards DNA degradation; their activity is modulated by divalent cations. Depending on cation concentration, three distinct steps of DNA fragmentation, likely mediated by different enzymes, can be identified: (i) in the presence of Mg 2+ (2 mM, DNA is fragmented to about 0.05-1 megabase (Mb)-size sections (type-I, high molecular weight DNA fragmentation)); (ii) at low (nanomolar) Ca 2+ concentration, nuclear DNA is cleaved into intermediate (∼300 kb) fragments (type-II, intermediate DNA fragmentation); (iii) at micromolar levels of Ca 2+ , internucleosomal (type-III) DNA fragmentation takes place leading to formation of DNA sections of the size of mono and oligonucleosomes, which form a characteristic 'DNA-ladder' pattern during electrophoresis (Arends et al. 1990 NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptAmong the nucleases associated with DNA fragmentation during apoptosis, the best characterized is CAD (caspase-activated DNase) with its inhibitor ICAD (inhibitor of CAD) in mice, and its human homologue DFF40/DFF45 (DNA fragmentation factor) (Enari et al. 1998). CAD and ICAD (or ...

H2AX phosphorylation marks gemcitabine-induced stalled replication forks and their collapse upon S-phase checkpoint abrogation

“…This links elevated H2AX phosphorylation with a decreased capacity for cellular reproduction after checkpoint abrogation. Other reports have made similar correlations with g-H2AX and apoptosis (27,49). Therefore, intense pan-nuclear staining of g-H2AX may signal that either DNA breaks are prevalent throughout the nucleus or DNA structure has been significantly compromised.…”

Ubiquitous marine bacterium inhibits diatom cell division

“…A significant proportion of cells without a nucleus were also observed on day 6 using microscopy ( Figure 3d). This is suggestive of nuclear disintegration and DNA fragmentation, which accompanies apoptosis (Huang et al, 2005). C. atlanticus thus appears to manipulate the cell cycle of the diatom, stimulating both polyploidy and nuclear disintegration.…”

Constitutive Histone H2AX Phosphorylation and ATM Activation, the Reporters of DNA Damage by Endogenous Oxidants

“…[28][29][30][31] Likewise, DSBs generated during DNA fragmentation in apoptotic cells induce phosphorylation of H2AX, the extent of which, however, is much greater compared to its phosphorylation induced by exogenous genotoxic agents. [32][33][34] In fact, the large difference in the degree of H2AX phosphorylation makes it possible, based on the intensity of γH2AX IF, to discriminate between the apoptotic cells and the cells with primary DSBs generated by DNA damaging agents such as ionizing radiation or by DNA topoisomerase inhibitors. [33][34][35] DNA damage triggers activation of Ataxia telangiectasia mutated (ATM) through its phosphorylation on Ser1981, and, as noted, H2AX is a substrate of this kinase.…”

“…[32][33][34] In fact, the large difference in the degree of H2AX phosphorylation makes it possible, based on the intensity of γH2AX IF, to discriminate between the apoptotic cells and the cells with primary DSBs generated by DNA damaging agents such as ionizing radiation or by DNA topoisomerase inhibitors. [33][34][35] DNA damage triggers activation of Ataxia telangiectasia mutated (ATM) through its phosphorylation on Ser1981, and, as noted, H2AX is a substrate of this kinase. 18,[36][37][38][39][40] Whereas H2AX can be phosphorylated by ATR in response to other types of DNA damage, its phosphorylation when mediated by ATM appears to be strongly linked to the induction of DSBs.…”