A role for topoisomerase II alpha in the formation of radiation-induced chromatid breaks

Abstract

Chromatid breaks in cells exposed to low dose irradiation are thought to be initiated by DNA double-strand breaks (DSB), and the frequency of chromatid breaks has been shown to increase in DSB rejoining deficient cells. However, the underlying causes of the wide variation in frequencies of G2 chromatid breaks (or chromatid 'radiosensitivity') in irradiated T-lymphocytes from different normal individuals and cancer cases are as yet unclear. Here we report evidence that topoisomerase II alpha expression level is a factor determining chromatid radiosensitivity. We have exposed the promyelocytic leukaemic cell line (HL60) and two derived variant cell lines (MX1 and MX2) that have acquired resistance to mitoxantrone and low expression of topoisomerase II alpha, to low doses of gamma-radiation and scored the induced chromatid breaks. Chromatid break frequencies were found to be significantly lower in the variant cell lines, compared with their parental HL60 cell line. Rejoining of DSB in the variant cell lines was similar to that in the parental HL60 strain. Our results indicate the indirect involvement of topoisomerase II alpha in the formation of radiation-induced chromatid breaks from DSB, and suggest topoisomerase II alpha as a possible factor in the inter-individual variation in chromatid radiosensitivity.

Figure 1

Frequency of chromatid breaks in control and irradiated HL60, MX1, and MX2 cells using the G2 assay.

Figure 2

Western blots showing expression of topoisomerase IIα in HL60, MX1 and MX2 cells.

Figure 3

Relative topoisomerase IIα expression level in HL60, MX1 and MX2 as estimated from western blots. Bars represent s.e.m. values from several experiments.

Figure 4

Chromatid break frequency as a function of relative topoisomerase IIα level in HL60, MX1 and MX2 cells. Error bars represent standard errors of mean values.

Figure 5

Mitotic index, measured by FACS in cells labelled with phospho-histone H3 labelled HL60 (squares), MX1 (circles) and MX2 (triangles) following treatment with mAMSA.

Figure 6

Relationship between relative topoisomerase IIα and relative mitotic index (measured by FACS in cells labelled with phospho-histone H3) derived from data in Figure 5, at the highest mAMSA concentration used. Error bars represent s.e.m. values.

Figure 7

Rejoining of DSB following irradiation of HL60, MX1, and MX2 cell strains.