Rejoining of DNA double-strand breaks after the introduction of chromosome 11 into a radiosensitive bladder carcinoma cell line.
Insertion of a normal chromosome 11 into tumour cell lines can protect against a sensitivity to irradiation and oxidative stress. A possible mechanism underlying this effect is that there is a correction of a defect in the rejoining of double-strand breaks (dsb) by the chromosome insertion. In order to explore this hypothesis, three cell lines were evaluated for their ability to rejoin dsb: (1) a bladder carcinoma cell line ('parent') previously shown to be sensitive to irradiation and radical generating species; (2) a derivative of this cell line into which a normal chromosome 11 had been inserted by microcell fusion ('hybrid') showing corrected radiosensitivity; and (3) a 'revertant' cell line that had spontaneously lost the insert and reverted to the radiosensitive phenotype. Nuclear extracts from the 3 lines were isolated and evaluated for their capacity to rejoin plasmid (pUC18) DNA broken at defined restriction sites (SalI, EcoRI, KpnI, SmaI) in the lacZ gene. The extent of rejoining was determined by gel electrophoresis and the fidelity of rejoining determined by expression of the lacZ gene in E. coli DH5 alpha bacteria. Results suggest there is no difference between the 'parent', 'hybrid' and 'revertant' nuclear extracts in the fidelity and the total extent of rejoining, regardless of the type of break. However, there is an alteration in the distribution of rejoined products. Nuclear extracts from 'hybrid' cells tend to rejoin linear DNA into circular monomers with a greater efficiency than extracts from both 'parent' and 'revertant' cells. This alteration in distribution is observed when 3'- or 5'-protruding ends are rejoined but not in the rejoining of blunt ends. The results suggest that loci on chromosome 11 are involved in the rejoining of dsb, affecting the relative amount of the different rejoined products. Whether this alteration plays a role in the 'parent' cell's radiosensitivity is yet to be determined.
Hofseth
L J
LJ
Division of Epidemiology and Cancer Prevention, British Columbia Cancer Agency, Vancouver, Canada.
Tsang
S S
SS
Rosin
M P
MP
D002459:Cell Fusion; D002880:Chromosomes, Human, Pair 11; D004249:DNA Damage; D004260:DNA Repair; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007763:Lac Operon; D011836:Radiation Tolerance / Q000235:genetics; D014170:Transformation, Genetic; D014407:Tumor Cells, Cultured; D001749:Urinary Bladder Neoplasms / Q000235:genetics*
D004262:DNA Restriction Enzymes