Liudmila Solovjeva

H2AX phosphorylation at the sites of DNA double-strand breaks in cultivated mammalian cells and tissues

A sequence variant of histone H2A called H2AX is one of the key components of chromatin involved in DNA damage response induced by different genotoxic stresses. Phosphorylated H2AX (γH2AX) is rapidly concentrated in chromatin domains around DNA double-strand breaks (DSBs) after the action of ionizing radiation or chemical agents and at stalled replication forks during replication stress. γH2AX foci could be easily detected in cell nuclei using immunofluorescence microscopy that allows to use γH2AX as a quantitative marker of DSBs in various applications. H2AX is phosphorylated in situ by ATM, ATR, and DNA-PK kinases that have distinct roles in different pathways of DSB repair. The γH2AX serves as a docking site for the accumulation of DNA repair proteins, and after rejoining of DSBs, it is released from chromatin. The molecular mechanism of γH2AX dephosphorylation is not clear. It is complicated and requires the activity of different proteins including phosphatases and chromatin-remodeling complexes. In this review, we summarize recently published data concerning the mechanisms and kinetics of γH2AX loss in normal cells and tissues as well as in those deficient in ATM, DNA-PK, and DSB repair proteins activity. The results of the latest scientific research of the low-dose irradiation phenomenon are presented including the bystander effect and the adaptive response estimated by γH2AX detection in cells and tissues.

Inhibition of transcription at radiation-induced nuclear foci of phosphorylated histone H2AX in mammalian cells

Double-strand DNA breaks (DSBs) induced by ionizing radiation can be visualized in human cells using antibodies against Ser-139 phosphorylated histone H2AX (γ-H2AX). Large γ-H2AX foci are seen in the nucleus fixed 1 hour after irradiation and their number corresponds to the number of DSBs, allowing analysis of these genome lesions after low doses. We estimated whether transcription is affected in chromatin domains containing γ-H2AX by following in vivo incorporation of 5-bromouridine triphosphate (BrUTP) loaded by cell scratching (run-on assay). We found that BrUTP incorporation is strongly suppressed at γ-H2AX foci, suggesting that H2AX phosphorylation inhibits transcription. This is not caused by preferential association of γ-H2AX foci with constitutive or facultative heterochromatin, which was visualized in irradiated cells using antibodies against histone H3 trimethylated at lysine-9 (H3-K9m3) or histone H3 trimethylated at lysine-27 (H3-K27m3). Apparently, formation of γ-H2AX induces changes of chromatin that inhibit assembly of transcription complexes without heterochromatin formation. Inhibition of transcription by phosphorylation of histone H2AX can decrease chromatin movement at DSBs and frequency of misjoining of DNA ends.

Reduced extractability of the XPA DNA repair protein in ultraviolet light-irradiated mammalian cells

The XPA protein is essential for both of the known modes of nucleotide excision repair (NER) in human cells: transcription‐coupled repair (TCR) and global genome repair (GGR). In TCR, this protein is thought to be recruited to lesion sites in DNA at which RNA polymerase II is blocked and in GGR, by direct recognition of damages by repair protein complex containing XPC/HR23B or DNA damage‐binding protein. However, details of the recruitment of the XPA protein in vivo are unknown. It was shown earlier that a portion of another NER protein, PCNA, which is completely extractable from non‐S‐phase mammalian nuclei, becomes insoluble after ultraviolet (UV) light irradiation and cannot be extracted by methanol or buffer containing Triton X‐100. In the present study, we have found that UV light irradiation of human or Chinese hamster cells leads to decrease of extractability of the XPA protein by Triton X‐100. Maximal insolubilization of the XPA protein is observed 1–4 h after irradiation but it is not detectable by 22 h. This effect is dose‐dependent for UV light from 2.5 to 15 J/m2 and is unaffected by the pre‐treatment of cells with sodium butyrate, an inhibitor of histone deacetylation. The UV light‐induced insolubilization of the XPA protein was also observed in two lines of Cockayne syndrome complementation group A cells, indicating that the effect is not dependent upon TCR. The results are discussed in relation to possible mechanisms of NER.

Characterization of telomeric repeats in metaphase chromosomes and interphase nuclei of Syrian Hamster Fibroblasts

BackgroundRodents have been reported to contain large arrays of interstitial telomeric sequences (TTAGGG)n (ITS) located in pericentromeric heterochromatin. The relative sizes of telomeric sequences at the ends of chromosomes (TS) and ITS in Syrian hamster (Mesocricetus auratus) cells have not been evaluated yet, as well as their structural organization in interphase nuclei.ResultsFISH signal distribution analysis was performed on DAPI-banded metaphase chromosomes of Syrian hamster fibroblasts, and relative lengths of telomere signals were estimated. Besides well-distinguished FISH signals from ITS located on chromosomes ##2, 4, 14, 20 and X that we reported earlier, low-intensity FISH signals were visualized with different frequency of detection on all other metacentric chromosomes excluding chromosome #21. The analysis of 3D-distribution of TS in interphase nuclei demonstrated that some TS foci formed clearly distinguished associations (2–3 foci in a cluster) in the nuclei of cells subjected to FISH or transfected with the plasmid expressing telomeric protein TRF1 fused with GFP. In G0 and G1/early S-phase, the average total number of GFP-TRF1 foci per nucleus was less than that of PNA FISH foci in the corresponding cell cycle phases suggesting that TRF1 overexpression might contribute to the fusion of neighboring telomeres. The mean total number of GFP-TRF1 and FISH foci per nucleus was increased during the transition from G0 to G1/early S-phase that might be the consequence of duplication of some TS.ConclusionsThe relative lengths of TS in Syrian hamster cells were found to be moderately variable. All but one metacentric chromosomes contain ITS in pericentromeric heterochromatin indicating that significant rearrangements of ancestral genome occurred in evolution. Visualization of GFP-TRF1 fibrils that formed bridges between distinct telomeric foci allowed suggesting that telomere associations observed in interphase cells are reversible. The data obtained in the study provide the further insight in the structure and dynamics of telomeric sequences in somatic mammalian cells.

Chapter 6. Application of new methods for detection of DNA damage and repair.

New methods for detecting DNA damage and repair are reviewed and their potential significance is discussed. These include methods based on analysis of DNA damage-induced chromatin modifications, cytological detection of DNA repair synthesis, damage-induced immobilization of repair proteins and living cell imaging. Special attention is paid to current methods of detection of modifications of histones and other proteins associated with DNA double-strand breaks which represent most dangerous genome damage. New methods of analysis of DNA damage and repair may be useful in biodosimetry, early cancer diagnostics and in the analysis of efficiency of cancer radiation therapy and chemotherapy.

Immunofluorescence Analysis of γ-H2AX Foci in Mammalian Fibroblasts at Different Phases of the Cell Cycle

H2AX phosphorylation at Ser139 (formation of γ-H2AX) is an indicator of double-strand breaks in DNA (DSBs) after the action of different genotoxic stresses, including ionizing radiation, environmental agents, and chemotherapy drugs. The sites of DSBs can be visualized as focal sites of γ-H2AX using antibodies and immunofluorescence microscopy. The microscopy technique is the most sensitive method of DSB detection in individual cells. It is useful for experimental research, radiation biodosimetry, and clinical practice. In this chapter, we provide an immunochemical protocol for γ-H2AX labeling and analysis by confocal microscopy. The advantage of the assay is that it enables the quantitation of γ-H2AX foci in individual cells in different phases of the cell cycle.

Rapid Detection of Apoptosis in Cultured Mammalian Cells

Flow cytometry is a powerful tool for the analysis of apoptosis, the process that directly determines cell fate after the action of different stresses. Here, we describe a flow cytometry method for the assessment of early and late stages of apoptosis in non-fixed cultured cells using SYTO16, DRAQ7, and PO-PRO1 dyes simultaneously. This multicolor flow cytometry procedure requires 45 min for completion and provides a quantitative assessment of cell viability. It can be useful in evaluating the cytotoxic properties of new drugs, and antitumor interventions.

Rapid Detection of γ-H2AX by Flow Cytometry in Cultured Mammalian Cells

Methods commonly used for detection of DNA double-strand breaks (DSBs) and analysis of cell death are generally time-consuming, and, therefore, any improvements in these techniques are important for researchers and clinicians. At present, flow cytometry is the most rapid method for detection of DSBs and cell viability. In this chapter, we provide our experience and methodological modification of flow cytometry protocol for the detection of γ-H2AX, a well-known marker of DSBs, in fixed mammalian fibroblasts. The modifications permit a reduction in the time required for DSB detection by flow cytometry.

Organization of Chromosomes in Human Sperm Nucleus

Chromosomes in the human sperm nucleus adopt a hierarchy of structures starting from protamine toroids, the elementary units of DNA packaging, and up to the higher-order organization of chromosome territories. Nonrandom intranuclear positioning of individual chromosomes, with centromeres aggregated in the internally located chromocenter and preferential location of telomeres at the nuclear periphery, suggests a highly defined architecture of the sperm nucleus at the level of genome. Such an ordered chromatin organization in the sperm nucleus may have a functional significance and determine the onset of paternal gene activity at early stages of embryonic development. This chapter describes relevant experimental data with primary attention to studies of human spermatozoa and discusses the implications of sperm chromosome organization for male reproductive health.