Differential level of DSB repair fidelity effected by nuclear protein extracts derived from radiosensitive and radioresistant human tumour cells

Abstract

Summary A cell-free plasmid reactivation assay was used to determine the fidelity of DNA double-strand break (DSB) repair in a panel of eight DSB repair-proficient human tumour cell lines. Nuclear protein extracts derived from radiosensitive tumour cells were less capable of correctly rejoining EcoRI-induced DSBs than were similar extracts from radioresistant tumour cells. Linear regression analysis suggests that there was a significant (r2 = 0.84, P = 0.001, d.f. = 6) correlation between the fidelity of DSB rejoining and the SF2 values of the cell lines studied. This cell-free assay is clearly sensitive to differences in the nuclear protein composition that reflect the clinically relevant radiosensitivity of these cell lines. The fact that our cell-free assay yielded similar results to previous studies that used intracellular plasmid reactivation assays suggests that those differences in DSB mis-rejoining frequencies in radiosensitive and radioresistant cell lines may be due to inherent differences in nuclear protein composition and are not directly attributable to differences in proliferation rates between cell lines. The underlying cause for this association between DSB mis-rejoining frequencies and radiosensitivity is presently unknown, however restriction endonuclease mapping and polymerase chain reaction (PCR) amplification analysis revealed that approximately 40% of the misrejoined DSBs arose as a result of the deletion of between 40 and 440 base pairs. These data raise the possibility that the radiosensitivity of DSB repair-proficient human tumour cell lines may be partly determined by the predisposition of these cell lines to activate non-conservative DSB rejoining pathways.

Modification of non-conservative double-strand break (DSB) rejoining activity after the induction of cisplatin resistance in human tumour cells

“…It has been suggested that restriction endonucleasegenerated DSBs act as substrates for proteins that are involved in the (abnormal) conversion of DSBs into chromosomal aberrations (Bryant and Liu, 1994). This conclusion is supported by the fact that ataxia telangiectasia (AT) and irs cells exhibit high DSB misrejoining activity (Cox et al, 1984;Thacker and Ganesh, 1990;Ganesh et al, 1993;Powell et al, 1993;Lou et al, 1996), and generate high levels of chromatid-type aberrations in response to restriction endonuclease-induced DSBs (Liu and Bryant, 1993;Bryant, and Liu, 1994).The biochemical basis for the differential level of DSB misrejoining activity in nuclear protein extracts from radiosensitive and radioresistant tumour cells is presently unknown, although we have shown that the high DSB misrejoining activity in radiosensitive tumour cells was not a consequence of non-specific endonuclease activity but rather due to an increase in non-conservative DSB rejoining activity that results in losses of between 40 and 440 bp of DNA (Britten et al, 1997). Furthermore, previous studies have Modification of non-conservative double-strand break (DSB) rejoining activity after the induction of cisplatin resistance in human tumour cells…”

“…The biochemical basis for the differential level of DSB misrejoining activity in nuclear protein extracts from radiosensitive and radioresistant tumour cells is presently unknown, although we have shown that the high DSB misrejoining activity in radiosensitive tumour cells was not a consequence of non-specific endonuclease activity but rather due to an increase in non-conservative DSB rejoining activity that results in losses of between 40 and 440 bp of DNA (Britten et al, 1997). Furthermore, previous studies have Modification of non-conservative double-strand break (DSB) rejoining activity after the induction of cisplatin resistance in human tumour cells…”

“…The level of DSB misrejoining activity in CHO (strain AA8) cells has been previously characterized (Britten et al, 1997), and we have also established their susceptibility to DSB induction after exposure to 60 Co γ-rays . The CHO-AA8 cells were maintained in monolayer culture in McCoy's 5A medium (Hsu's modification; Life Technologies) supplemented with 10% FCS and antibiotics and passaged every 2-3 days to ensure exponential growth.…”

“…The fidelity of DSB repair effected by nuclear protein extracts from the cell lines was assessed using a slightly modified version of our cell-free plasmid reactivation assay (Britten et al, 1997). Briefly, this plasmid reactivation assay determines the ability of nuclear protein extracts to correctly rejoin a simple staggered (EcoRI-induced) DSB within the lacZα complementation gene of the pUC18 plasmid.…”

“…SF 2 , of eight human tumour cell lines (Britten et al, 1997). Nuclear protein extracts from radiosensitive, yet overtly DSB rejoining proficient, tumour cells were found to be less capable of correctly rejoining EcoRI-induced DSBs than were similar extracts from radioresistant tumour cells.…”