PubMed · 8856992
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R I Morimoto. 1996. Outlook.. https://doi.org/10.1007/978-3-0348-9088-5_33
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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.
Certain nickel compounds including crystalline nickel sulfide (NiS) and subsulfide (Ni3S2) are potent human and animal carcinogens. In Chinese hamster embryo cells, an X-linked senescence gene was inactivated following nickel-induced DNA methylation. Nickel also induced the inactivation of the gpt reporter gene by chromatin condensation and a DNA methylation process in a transgenic gpt+ Chinese hamster cell line (G12), which is located near a heterochromatic region. To determine if nickel can cause gene silencing independently of DNA methylation, based only on the induction of changes in chromatin structure, we measured its effect on gene silencing in Saccharomyces cerevisiae. Growth of yeast in the presence of nickel chloride repressed a telomeric marker gene (URA3) and resulted in a stable epigenetic switch. This phenomenon was dependent on the number of cell doubling prior to selection and also on the distance of the marker gene from the end of the chromosome. The level of TPE (telomeric position effect) increased linearly with elevations of nickel concentration. Addition of magnesium inhibited this effect, but magnesium did not silence the reporter gene by itself. The level of silencing was also assessed following treatment with other transition metals: cobalt, copper and cadmium. In the sublethal range, cobalt induced similar effects as nickel, while copper and cadmium did not change the basal level of gene expression. Silencing by copper and cadmium were evident only at concentrations of those metals where the viability was very low.
Xeroderma pigmentosum (XP) and trichothiodystrophy (TTD) are rare genodermatoses transmitted as recessive and autosomal traits that result in reduced capacity to repair UV-induced DNA lesions. Although XP, but not TTD, patients are prone to basal and squamous cell carcinomas, to date no comparative studies of the XP and TTD phenotypes have included epidermal keratinocytes. We compared the DNA repair capacity (by unscheduled DNA synthesis) and cell survival (by clonal analysis) of epidermal keratinocytes and dermal fibroblasts grown from normal individuals and patients with xeroderma pigmentosum and trichothiodystrophy following UVA and UVB irradiation. The same dose of UVB (1000 J/m2) induced twice as many DNA lesions in normal fibroblasts as in normal keratinocytes. UV survival rates were always higher in keratinocytes than in fibroblasts. Normal and TTD keratinocytes survived better following UVA and UVB irradiation than XP-C and XP-D keratinocytes. XP-C keratinocytes exhibited exacerbated sensitivity toward UVA radiation. Unscheduled DNA synthesis at UV doses leading to 50% cell survival indicated that the ratio of DNA repair capacity to cell survival is higher in keratinocytes than in fibroblasts. In addition, UVA and UVB irradiation induced a transition from proliferative to abortive keratinocyte colonies. This transition varied between donors and was in part correlated with their cancer susceptibility. Altogether these data provide the first evidence of the differential behaviors of normal, XP, and TTD keratinocytes toward UV radiation.