Eva Pagáčová

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.

Post-Translational Modifications of Histones in Human Sperm

We examined the levels and distribution of post‐translationally modified histones and protamines in human sperm. Using western blot immunoassay, immunofluorescence, mass spectrometry (MS), and FLIM‐FRET approaches, we analyzed the status of histone modifications and the protamine P2. Among individual samples, we observed variability in the levels of H3K9me1, H3K9me2, H3K27me3, H3K36me3, and H3K79me1, but the level of acetylated (ac) histones H4 was relatively stable in the sperm head fractions, as demonstrated by western blot analysis. Sperm heads with lower levels of P2 exhibited lower levels of H3K9ac, H3K9me1, H3K27me3, H3K36me3, and H3K79me1. A very strong correlation was observed between the levels of P2 and H3K9me2. FLIM‐FRET analysis additionally revealed that acetylated histones H4 are not only parts of sperm chromatin but also appear in a non‐integrated form. Intriguingly, H4ac and H3K27me3 were detected in sperm tail fractions via western blot analysis. An appearance of specific histone H3 and H4 acetylation and H3 methylation in sperm tail fractions was also confirmed by both LC‐MS/MS and MALDI‐TOF MS analysis. Taken together, these data indicate that particular post‐translational modifications of histones are uniquely distributed in human sperm, and this distribution varies among individuals and among the sperm of a single individual. J. Cell. Biochem. 116: 2195–2209, 2015.

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.