title: Transfection of the Escherichia coli nth gene into radiosensitive Chinese hamster cells: effects on sensitivity to radiation, hydrogen peroxide, and bleomycin sulfate.
abstract: The Escherichia coli nth gene encodes endonuclease III, which catalyses the glycolytic removal of various oxidized thymine residues from DNA. A truncated version of nth, with the prokaryotic regulatory sequences removed, was ligated into the retrovirus-based vector pZipneoSV(X)1 and transfected into the radiosensitive Chinese hamster ovary cell line, xrs7. Following selection with G418, two clones (x7nth1 and x7nth6) were shown by Southern analysis to contain the nth gene. No substantial difference in gamma-ray sensitivity was detected between xrs7, clones x7nth1 and x7nth6, and the parent vector transfected clone (x7neo1). However, clones containing the nth gene were more resistant to hydrogen peroxide cytotoxicity [D0's for x7nth1 and x7nth6 were 0.072 microgram/ml (4 microM) and 0.046 microgram/ml, respectively, compared with D0's of 0.034 and 0.027 microgram/ml for xrs7 and x7neo1, respectively] but markedly more sensitive to bleomycin sulfate cytotoxicity than xrs7 and x7neo1 (e.g., 1D0's for x7nth6 and xrs7 were 0.05 and 0.12 microgram/ml, while 2D0's for x7nth1 and xrs7 were 0.35 and 0.48 microgram/ml, respectively). Alterations in sensitivity to hydrogen peroxide and bleomycin sulfate could not be explained by differences in the distribution of the cell-cycle phases and growth rate of nth-containing clones and control cell lines. These results are consistent with the hypothesis that modified thymine lesions are potentially cytotoxic. Hence, when cells incur a high level of endonuclease III-repairable damage relative to strand breakage, such as after treatment with hydrogen peroxide, increased repair capacity increases survival. Gamma radiation produces a lower level of endonuclease III-repairable damage relative to all the other types of lesions produced; hence increased repair capacity has no measurable effect on cell survival. The increased sensitivity of x7nth1 and x7nth6 to bleomycin sulfate toxicity may indicate that, when thymine damage and single-strand breaks are in close proximity on opposite strands of the DNA, endonuclease III, which incises DNA at the site of damaged residues, can increase the number of double-strand breaks and hence decrease the level of cell survival.
authors.affiliation: Cancer Research Campaign Department of Experimental Radiation Oncology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, United Kingdom.
authors.forename: L
authors.initials: L
authors.lastname: Harrison
authors.forename: M
authors.initials: M
authors.lastname: Skorvaga
authors.forename: R P
authors.initials: RP
authors.lastname: Cunningham
authors.forename: J H
authors.initials: JH
authors.lastname: Hendry
authors.forename: G P
authors.initials: GP
authors.lastname: Margison
mesh_terms: D000818:Animals; D001761:Bleomycin / Q000494:pharmacology*; D016466:CHO Cells / Q000187:drug effects / Q000528:radiation effects; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004307:Dose-Response Relationship, Radiation; D004926:Escherichia coli / Q000235:genetics*; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D011836:Radiation Tolerance / Q000235:genetics*; D014162:Transfection
chemical_list: D001761:Bleomycin; D006861:Hydrogen Peroxide