Induction of chromosomal damage by restriction endonuclease in CHO cells porated with streptolysin O.
A procedure is described for the poration of living CHO cells with the bacterial cytotoxin streptolysin O (SLO) which allows the introduction into cells of the restriction endonuclease Pvu II to mimic and model the effects of ionising radiation in causing chromosomal damage. The dependence of this clastogenic effect of Pvu II on SLO concentration was measured by assaying the formation of micronuclei in cytokinesis-blocked binucleate cells. The optimum concentration was found to be 0.045 U/ml. Using the micronucleus assay, the time-course of expression of chromosome damage was investigated and found to show a biphasic kinetic with time. Using a sampling time of 30 h, a dose-effect curve for micronucleus induction by Pvu II was generated. Using this optimized SLO treatment protocol, the frequency of metaphase chromosome damage was subsequently investigated and found to be also linearly related to Pvu II concentration and total aberrations were approximately double the frequency of micronuclei. The induction and repair kinetics of DNA double-strand breaks were investigated in CHO cells treated with SLO and Pvu II using the neutral filter elution technique at pH 9.6. The data presented show that SLO can be used as an alternative method for porating cells to allow the introduction of restriction endonucleases into cells.
Bryant
P E
PE
Department of Biology and Preclinical Medicine, University of St. Andrews, Fife, Great Britain.
D000818:Animals; D001426:Bacterial Proteins; D016466:CHO Cells; D002463:Cell Membrane Permeability / Q000187:drug effects*; D002869:Chromosome Aberrations; D006224:Cricetinae; D004249:DNA Damage / Q000187:drug effects*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000494:pharmacology*; D048629:Micronuclei, Chromosome-Defective / Q000187:drug effects; D013301:Streptolysins / Q000494:pharmacology*
D001426:Bacterial Proteins; D013301:Streptolysins; C025964:streptolysin O; C025630:CAGCTG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific