Alena Bačíková

HMGB1 interacts with human topoisomerase IIα and stimulates its catalytic activity

DNA topoisomerase IIα (topo IIα) is an essential nuclear enzyme and its unique decatenation activity has been implicated in many aspects of chromosome dynamics such as chromosome replication and segregation during mitosis. Here we show that chromatin-associated protein HMGB1 (a member of the large family of HMG-box proteins with possible functions in DNA replication, transcription, recombination and DNA repair) promotes topo IIα-mediated catenation of circular DNA, relaxation of negatively supercoiled DNA and decatenation of kinetoplast DNA. HMGB1 interacts with topo IIα and this interaction, like the stimulation of the catalytic activity of the enzyme, requires both HMG-box domains of HMGB1. A mutant of HMGB1, which cannot change DNA topology stimulates DNA decatenation by topo IIα indistinguishably from the wild-type protein. Although HMGB1 stimulates ATP hydrolysis by topo IIα, the DNA cleavage is much more enhanced. The observed abilities of HMGB1 to interact with topo IIα and promote topo IIα binding to DNA suggest a mechanism by which HMGB1 stimulates the catalytic activity of the enzyme via enhancement of DNA cleavage.

Two New Faces of Amifostine: Protector from DNA Damage in Normal Cells and Inhibitor of DNA Repair in Cancer Cells

Amifostine protects normal cells from DNA damage induction by ionizing radiation or chemotherapeutics, whereas cancer cells typically remain uninfluenced. While confirming this phenomenon, we have revealed by comet assay and currently the most sensitive method of DNA double strand break (DSB) quantification (based on γH2AX/53BP1 high-resolution immunofluorescence microscopy) that amifostine treatment supports DSB repair in γ-irradiated normal NHDF fibroblasts but alters it in MCF7 carcinoma cells. These effects follow from the significantly lower activity of alkaline phosphatase measured in MCF7 cells and their supernatants as compared with NHDF fibroblasts. Liquid chromatography-mass spectrometry confirmed that the amifostine conversion to WR-1065 was significantly more intensive in normal NHDF cells than in tumor MCF cells. In conclusion, due to common differences between normal and cancer cells in their abilities to convert amifostine to its active metabolite WR-1065, amifostine may not only protect in multiple ways normal cells from radiation-induced DNA damage but also make cancer cells suffer from DSB repair alteration.

Heterochromatinization associated with cell differentiation as a model to study DNA double strand break induction and repair in the context of higher-order chromatin structure

Cell differentiation is associated with extensive gene silencing, heterochromatinization and potentially decreasing need for repairing DNA double-strand breaks (DSBs). Differentiation stages of blood cells thus represent an excellent model to study DSB induction, repair and misrepair in the context of changing higher-order chromatin structure. We show that immature granulocytes form γH2AX and 53BP1 foci, contrary to the mature cells; however, these foci colocalize only rarely and DSB repair is inefficient. Moreover, specific chromatin structure of granulocytes probably influences DSB induction.

Function of chromatin structure and dynamics in DNA damage, repair and misrepair: γ-rays and protons in action.

According to their physical characteristics, protons and ion beams promise a revolution in cancer radiotherapy. Curing protocols however reflect rather the empirical knowledge than experimental data on DNA repair. This especially holds for the spatio-temporal organization of repair processes in the context of higher-order chromatin structure-the problematics addressed in this work. The consequences for the mechanism of chromosomal translocations are compared for gamma rays and proton beams.

Granulocyte maturation determines ability to release chromatin NETs and loss of DNA damage response; these properties are absent in immature AML granulocytes.

Terminally-differentiated cells cease to proliferate and acquire specific sets of expressed genes and functions distinguishing them from less differentiated and cancer cells. Mature granulocytes show lobular structure of cell nuclei with highly condensed chromatin in which HP1 proteins are replaced by MNEI. These structural features of chromatin correspond to low level of gene expression and the loss of some important functions as DNA damage repair, shown in this work and, on the other hand, acquisition of a new specific function consisting in the release of chromatin extracellular traps in response to infection by pathogenic microbes. Granulocytic differentiation is incomplete in myeloid leukemia and is manifested by persistence of lower levels of HP1γ and HP1β isoforms. This immaturity is accompanied by acquisition of DDR capacity allowing to these incompletely differentiated multi-lobed neutrophils of AML patients to respond to induction of DSB by γ-irradiation. Immature granulocytes persist frequently in blood of treated AML patients in remission. These granulocytes contrary to mature ones do not release chromatin for NETs after activation with phorbol myristate-12 acetate-13 and do not exert the neutrophil function in immune defence. We suggest therefore the detection of HP1 expression in granulocytes of AML patients as a very sensitive indicator of their maturation and functionality after the treatment. Our results show that the changes in chromatin structure underlie a major transition in functioning of the genome in immature granulocytes. They show further that leukemia stem cells can differentiate ex vivo to mature granulocytes despite carrying the translocation BCR/ABL.

Accumulation of DNA damage and cell death after fractionated irradiation.

Abstract The purpose of this work was to determine how fractionated radiation used in the treatment of tumors affects the ability of cancer as well as normal cells to repair induced DNA double-strand breaks (DSBs) and how cells that have lost this ability die. Lymphocytic leukemia cells (MOLT4) were used as an experimental model, and the results were compared to those for normal cell types. The results show that cancer and normal cells were mostly unable to repair all DSBs before the next radiation dose induced new DNA damage. Accumulation of DSBs was observed in normal human fibroblasts and healthy lymphocytes irradiated in vitro after the second radiation dose. The lymphocytic leukemia cells irradiated with 4 × 1 Gy and a single dose of 4 Gy had very similar survival; however, there was a big difference between human fibroblasts irradiated with 4 × 1.5 Gy and a single dose of 6 Gy. These results suggest that exponentially growing lymphocytic leukemia cells, similar to rapidly proliferating tumors, are not very sensitive to fraction size, in contrast to the more slowly growing fibroblasts and most late-responding (radiation therapy dose-limiting) normal tissues, which have a low proliferation index.

Chromatin differentiation of white blood cells decreases DSB damage induction, prevents functional assembly of repair foci, but has no influence on protrusion of heterochromatic DSBs into the low-dense chromatin

Purpose: Higher order chromatin structure progressively changes with cell differentiation and seems to play an important role in DNA double-strand break (DSB) induction and repair (reviewed in [1]). We compared DNA damage in heterochromatin (Hc) upon the action of qualitatively different radiations. We also studied, how is the sensitivity to DSB induction, assembly of repair foci and processing of DSBs influenced by the differentiation-induced changes in chromatin structure and composition. Materials and methods: Formation, localization (relative to higher-order chromatin domains) and mutual colocalization of γH2AX and p53BP1 repair foci have been studied together with DSB repair kinetics in spatially fixed human skin fibroblast and differently differentiated white blood cells (WBC) irradiated with gamma rays, protons of different energies [2, 3], and 20Ne ions (submitted). Immunostaining and ImmunoFISH were used in combination with high-resolution confocal microscopy [2, 3] and living cell imaging [4]. Results: We found that less DSBs appear in Hc after irradiating cells with gamma rays and protons but not 20Ne ions (preliminary results). In addition, contrary to γ-irradiated human skin fibroblasts and lymphocytes, mature granulocytes neither express DSB repair proteins nor form functional repair foci [5]. At least some DSB repair proteins (e.g. 53BP1) are expressed and γH2AX foci still occur in immature granulocytes and monocytes [2, 5]; however, the colocalization of γH2AX with 53BP1 is low and the majority of DSBs are not repaired. Despite this fact, γH2AX foci protrude from Hc into nuclear subcompartments with low chromatin density. Our living cell observations suggest that 53BP1 can penetrate into the interior of dense Hc domains only after their decondensation [2]. Conclusions: We show that Hc is less sensitive to DSB induction by gamma rays but not heavy ions; lower Hc hydratation and higher protein density (when compared with euchromatin) probably reduce formation of free radicals and increase their sequestration, respectively. This mechanism can protect cells against the indirect effect of ionizing radiation (marked for gamma rays and protons but not heavy ions). Hc features, however, preclude DSB repair, which is best illustrated by its absence in differentiated WBC but not their immature precursors. The protrusion of Hc-DSBs into low-density chromatin nuclear subdomains, however, appears also in differentiated WBC, so the process might simply follow physical forces (e.g. as suggested by M Durantes group). There is no Clinical Trial Registration number.

Chromatin architecture changes and DNA replication fork collapse are critical features in cryopreserved cells that are differentially controlled by cryoprotectants

In this work, we shed new light on the highly debated issue of chromatin fragmentation in cryopreserved cells. Moreover, for the first time, we describe replicating cell-specific DNA damage and higher-order chromatin alterations after freezing and thawing. We identified DNA structural changes associated with the freeze-thaw process and correlated them with the viability of frozen and thawed cells. We simultaneously evaluated DNA defects and the higher-order chromatin structure of frozen and thawed cells with and without cryoprotectant treatment. We found that in replicating (S phase) cells, DNA was preferentially damaged by replication fork collapse, potentially leading to DNA double strand breaks (DSBs), which represent an important source of both genome instability and defects in epigenome maintenance. This induction of DNA defects by the freeze-thaw process was not prevented by any cryoprotectant studied. Both in replicating and non-replicating cells, freezing and thawing altered the chromatin structure in a cryoprotectant-dependent manner. Interestingly, cells with condensed chromatin, which was strongly stimulated by dimethyl sulfoxide (DMSO) prior to freezing had the highest rate of survival after thawing. Our results will facilitate the design of compounds and procedures to decrease injury to cryopreserved cells.

Changes in cryopreserved cell nuclei serve as indicators of processes during freezing and thawing

The mechanisms underlying cell protection from cryoinjury are not yet fully understood. Recent biological studies have addressed cryopreserved cell survival but have not correlated the cryoprotection effectiveness with the impact of cryoprotectants on the most important cell structure, the nucleus, and the freeze/thaw process. We identified changes of cell nuclei states caused by different types of cryoprotectants and associate them with alterations of the freeze/thaw process in cells. Namely, we investigated both higher-order chromatin structure and nuclear envelope integrity as possible markers of freezing and thawing processes. Moreover, we analyzed in detail the relationship between nuclear envelope integrity, chromatin condensation, freeze/thaw processes in cells, and cryopreservation efficiency for dimethyl sulfoxide, glycerol, trehalose, and antifreeze protein. Our interdisciplinary study reveals how changes in cell nuclei induced by cryoprotectants affect the ability of cells to withstand freezing and thawing and how nuclei changes correlate with processes during freezing and thawing. Our results contribute to the deeper fundamental understanding of the freezing processes, notably in the cell nucleus, which will expand the applications and lead to the rational design of cryoprotective materials and protocols.

Primary and secondary clustering of DSB repair foci and repair kinetics compared for γ-rays, protons of different energies and high-LET 20Ne ions

Purpose: Ionizing radiations of different qualities (e.g. high-LET and low-LET) might differently interact with structurally and functionally distinct higher order chromatin domains (discussed in [ 1] and citations therein); this might be reflected by DNA double strand break (DSB) repair efficiency and the mechanism of how cancerogenous chromosomal translocations (CHT) form. Therefore, we compared the DSB repair kinetics and formation of γH2AX/p53BP1 repair clusters upon the action of γ-rays [ 2, 3], protons (15 and 30 MeV) [ 4], and 20Ne ions (preliminary data). Consequently, we discuss biological impacts of these clusters. Material and methods: Immunostaining methods in combination with high-resolution confocal microscopy, performed on 3D-fixed normal human skin fibroblasts [ 2– 4], were used to study initial distributions of γH2AX and p53BP1 repair foci and their changes during the post-irradiation (PI) time, with a special concern on foci clustering. Irradiations with γ-rays, protons of different energies (15 and 30 MeV), and high-LET 20Ne ions was performed in IBP ASCR Brno (CR), NPI AVCR Řež (CR) and JINR Dubna (Russia), respectively. Results: Upon irradiating cells with 20Ne ions, tracks of multiple clustered γH2AX and p53BP1 repair foci appeared immediately after the irradiation; these clusters, called here as the ‘primary clusters’, were rare in cells irradiated with γ-rays or protons (submitted). Though γH2AX/p53BP1 foci were positionally quite stable [ 2], ‘secondary clusters’ occasionally appeared after all kinds of irradiation during about 30 min PI. The formation of secondary clusters usually appeared due to the heterochromatin decondensation at the sites of heterochromatic DNA double-strand breaks (hcDSBs), followed by their protrusion into a limited space of nuclear subdomains of low density-chromatin (discussed in [ 1, 2, 5]). Conclusions: Primary clusters appear in cell nuclei immediately PI as the consequence of highly localized energy deposition, while secondary clusters develop during (and because of) DSB repair. Primary DSB clusters probably represent the main cause of chromosomal translocations induced with high-LET radiations while secondary clusters seem to be more important for low-LET γ-rays and protons. Secondary clusters of primary clusters (higher-order clusters) observed for 20Ne ions might explain frequent formation of complex translocations upon the action of high-LET radiations. Finally, we suggest [ 1, 2, 4] a model that describes the relationship between the higher order chromatin structure, DSB formation, repair and misrepair.