PubMed · 3539461
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J Conway, A P Anderson. 1986. Electromagnetic techniques in hyperthermia.. https://doi.org/10.1088/0143-0815%2F7%2F4%2F001
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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.
To study the pathways associated with genomic instability in cancer, we examined UV-induced and spontaneous mutagenesis in clonal cell lines expressing human papillomavirus (HPV) proteins, either high-risk (HPV16) E6 or E7 or low-risk (HPV11) E6, in comparison to the parental RKO cells, a colon carcinoma cell line expressing only normal p53. High-risk E6 and E7 bind and functionally inactivate tumor suppressor proteins p53 and Rb, respectively, and both disrupt the G1 arrest in response to DNA damage. Low-risk HPV E6 proteins bind p53 with much lower affinity than high-risk E6 and fail to mediate p53 degradation or to disrupt the G1 checkpoint. We found that cells expressing HPV16 E6 had reduced survival and increased mutagenesis at the hprt locus when treated with low doses of UV. However, this analysis was complicated by the unexpected observation of a very high background of spontaneous mutagenesis in the unirradiated cells expressing the HPV16 E6 gene. Fluctuation analysis revealed a 5-fold elevated mutation rate in the cells expressing HPV16 E6. HPV11 E6 conferred a 2-fold elevation in the mutation rate, but HPV16 E7 had no effect. The increased spontaneous mutagenesis, therefore, appeared to be mediated by p53 inactivation and to be independent of Rb (which acts downstream of p53 in the G1 arrest pathway following DNA damage). Taken together, these findings suggest that the effect of p53 inactivation on spontaneous mutagenesis is manifested at the level of DNA repair, recombination, or coupling of transcription with one of these processes instead of by an alteration in G1 arrest.
BACKGROUND: The nitroxide Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) is a stable, free radical that exhibits protection from ionizing radiation damage and from oxidative stress mediated through exposure of cells to superoxide or hydrogen peroxide. Radiation protection has been observed in both in vivo and in vitro models. To understand the mechanism of Tempol-mediated radioprotection better, the production of radiation-induced chromosome aberrations was evaluated. This study analyzed Tempol-mediated radioprotection of human peripheral blood lymphocytes (PBLs). METHODS: Peripheral blood lymphocytes were exposed to control (0mM), 10 mM (Tp10), and 50 mM (Tp50) concentrations of Tempol for 20 minutes before irradiation with 0, 150, 300, and 450 cGy. One quarter ml whole blood was cultured in F12 medium and phytohemagglutinin at 37 degrees C for 49, 54, 59, and 64 hours. Colcemide was added to each sample for the last 5 hours before harvest. Cells were harvested, treated with hypotonic solution, and fixed before dropping on cold clean slides. Mitotic indices and frequency of dicentric, ring, and triradial chromosomal aberrations were determined at 1000x magnification for each treatment group at each collection point. RESULTS: Treatment of cells with Tempol alone did not induce the chromosomal aberration frequency above that for unirradiated controls. Radiation dose response curves for total chromosome aberration production revealed radioprotection for Tempol treatment for both 10 and 50 mM exposures. Tempol protection factors (assessed at 0.2 aberrations/cell level) for Tp 10 and Tp 50 were 2.2 and 2.8, respectively. CONCLUSIONS: Tempol protects against radiation-induced chromosome aberrations in human PBLs. This finding is consistent with and lends support to previous studies in which Tempol was reported to enhance cell survival and reduce radiation-induced DNA double strand breaks.