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 d efect 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 ce ll line ('parent') previously shown to be sensitive to irradiation and radical generating species; (2) a derivative of this cell line into w hich a normal chromosome Il had been inserted by microcell fusion ('hy brid') showing corrected radiosensitivity; and (3) a 'revertant' cell line that had spontaneously lost the insert and reverted to the radios ensitive phenotype. Nuclear extracts from the 3 lines were isolated an d 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 betwe en the 'parent', 'hybrid' and 'revertant' nuclear extracts in the fide lity and the total extent of rejoining, regardless of the type of brea k. However, there is an alteration in the distribution of rejoined pro ducts. Nuclear extracts from 'hybrid' cells tend to rejoin linear DNA into circular monomers with a greater efficiency than extracts from bo th 'parent' and 'revertant' cells. This alteration in distribution is observed when 3'- or 5'-protruding ends are rejoined but not in the re joining 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.