PubMed · 7938479
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H H Rossi. 1994. Interactive DNA damage.. https://pubmed.ncbi.nlm.nih.gov/7938479/
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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.
Sixty % of clones isolated from HeLa cells treated with toxic concentrations of a methylating carcinogen showed increased resistance to the cytotoxicity of N-methyl-N-nitrosourea. D37 values were 6- to 100-fold higher than in the parental cell population. The absence of detectable levels of the repair enzyme O6-methylguanine-DNA methyltransferase indicated that the resistant clones were able to tolerate the presence of O6-methylguanine in their DNA. Analysis of N-methyl-N-nitrosourea survival in the hybrids between tolerant clones and HeLa cells showed that tolerance can be either recessive or codominant. Fusion between tolerant clones indicated two complementation groups. We measured spontaneous mutation rates at microsatellites and at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in several tolerant clones. All the clones of Complementation Group I showed unstable microsatellites and 4-8-fold increases in mutation rates at hprt. No significant alterations in spontaneous mutation rates were found in clones of Complementation Group II. The data indicate that tolerance to methylation damage can be conferred by alterations in at least two different gene products and that one of the two groups has the mutator phenotype typical of mismatch correction defective cells.