title: Increased sensitivity of damaged DNA to digestion with nuclease S1 as assessed in single cells by flow cytometry.
abstract: DNA sensitivity to digestion with nuclease S1 was investigated in cells irradiated with gamma rays, or treated with the antitumor drug adriamycin (Adr). The nuclease-resistant DNA fraction was determined by propidium iodide staining. Treated cells were found to be more sensitive to nuclease digestion than the undamaged controls. Gamma ray-induced strand breaks were detectable at doses up to 10 Gy; an increase in the reaction temperature, from 37 degrees to 63 degrees C, was necessary in order to detect higher levels of damage. Nuclease S1 sensitivity in Adr-treated cells showed a single-peak, concentration-dependent relationship, in agreement with the known self-inhibitory effect exerted by high drug doses. Determination of DNA digestion could be performed in combination with other cellular parameters (e.g., protein content). Detection of drug-resistant cells in a heterogeneous population of small-cell lung carcinoma was achieved on the basis of the different sensitivity of the cells to enzymatic digestion. These results indicate that nuclease S1 may be a useful probe for studying in single cells DNA alterations induced by drugs or radiation.
authors.affiliation: Centro di Studio per l'Istochimica del C.N.R., Dipartimento di Biologia Animale, Università di Pavia, Italy.
authors.forename: E
authors.initials: E
authors.lastname: Prosperi
authors.forename: R
authors.initials: R
authors.lastname: Supino
authors.forename: G
authors.initials: G
authors.lastname: Bottiroli
mesh_terms: D002469:Cell Separation / Q000379:methods; D004247:DNA / Q000032:analysis* / Q000187:drug effects / Q000528:radiation effects; D004249:DNA Damage; D004317:Doxorubicin / Q000494:pharmacology; D005434:Flow Cytometry; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D011419:Propidium; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism
chemical_list: D011419:Propidium; D004317:Doxorubicin; D004247:DNA; D015719:Single-Strand Specific DNA and RNA Endonucleases