Increased sensitivity of damaged DNA to digestion with nuclease S1 as assessed in single cells by flow cytometry.
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.
Prosperi
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E
Centro di Studio per l'Istochimica del C.N.R., Dipartimento di Biologia Animale, Università di Pavia, Italy.
Supino
R
R
Bottiroli
G
G
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
D011419:Propidium; D004317:Doxorubicin; D004247:DNA; D015719:Single-Strand Specific DNA and RNA Endonucleases