In situ DNAse I sensitivity assay indicates DNA conformation differences between CHO cells and the radiation-sensitive CHO mutant IRS-20.
The radiosensitive mutant cell line IRS-20, its wild type counterpart CHO and a derivative of IRS-20 with a transfected YAC clone (YAC-IRS) that restores radioresistance were tested for DNAse I sensitivity. The three cell lines were cultured under the same conditions and had a mitotic index of 2-5%. One drop of fixed cells from the three lines was always spread on the same microscopic slide. After one day of ageing, slides were exposed to DNAse I and stained with DAPI. Images from every field were captured and the intensity of blue fluorescence was measured with appropriate software. For untreated cells, the fluorescence intensity was similar for all of the cell lines. After DNAse I treatment, CHO and YAC-IRS had an intensity of 85% but IRS-20 had an intensity of 60%, when compared with the controls. DNAse I sensitivity differences between the cell lines indicate that overall conformation of chromatin might contribute to radiation sensitivity of the IRS-20 cells.
Marañon
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National Commission of Atomic Energy (CNEA), Centro Atómico Constituyentes, Buenos Aires, Argentina.
Laudicina
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Muhlmann
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D000818:Animals; D016466:CHO Cells / Q000378:metabolism / Q000528:radiation effects*; D018244:Chromosomes, Artificial, Yeast / Q000235:genetics; D002898:Chromosomes, Human, Pair 8 / Q000235:genetics; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000187:drug effects / Q000528:radiation effects*; D004249:DNA Damage; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D003850:Deoxyribonuclease I / Q000494:pharmacology*; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D005816:Genetic Complementation Test; D006801:Humans; D008940:Mitotic Index; D009687:Nuclear Proteins; D009690:Nucleic Acid Conformation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000502:physiology; D011836:Radiation Tolerance / Q000235:genetics*; D014162:Transfection
D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D009687:Nuclear Proteins; D004247:DNA; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D003850:Deoxyribonuclease I; D016650:Fluorescein-5-isothiocyanate