A role for topoisomerase IIα 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 IIa 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 a, to low doses of g-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 IIa in the formation of radiation-induced chromatid breaks from DSB, and suggest topoisomerase IIa as a possible factor in the inter-individual variation in chromatid radiosensitivity. British Journal of Cancer (2008) Human response to low doses of ionising radiation shows a wide variation as exemplified by the different frequencies of radiationinduced chromatid breaks observed in metaphase chromosomes in phytohaemagglutinin-stimulated peripheral blood T-lymphocytes (PBL) from different normal individuals and sporadic cancer cases, and elevated frequencies of such chromatid breaks have been linked to cancer susceptibility (Scott et al, 1994(Scott et al, , 1996Roberts et al, 1999;Baria et al, 2001Baria et al, , 2002Buchholz and Wu, 2001;Riches et al, 2001;Papworth et al, 2001;Smart et al, 2003;Baeyens et al, 2002Baeyens et al, , 2005. Using a short time interval (1-2 h) between radiation exposure and sampling, the metaphase cells (blocked with colcemid) collected are those that were in the G2-phase of the cell cycle at the time of exposure and show frequent chromatid breaks (discontinuities or terminal deletions) that have been shown to be induced as a linear function of radiation dose (Bryant, 1998).Cell-cycle arrest is a factor that could possibly influence observed frequencies of chromatid breaks, and at least one study of human tumour cell lines in vitro (Schwartz et al, 1996) reports an inverse relationship between mitotic inhibition and chromatid break frequency. Also, using premature chromosome condensation (PCC) in G2-phase cells with phosphatase inhibitor calyculin, G2 cells were distinguished from normal mitotic metaphases by their split centromeres and by the loss of centromeric constriction (Terzoudi et al, 2005). Using this feature of calyculin-induced PCC to distinguish G2 cells from mitotic metaphase cells, these authors reported that cells from individuals with ataxia telangiectasia (A -T), a homozy...

Radioresistance in a human laryngeal squamous cell carcinoma cell line is associated with DNA methylation changes and topoisomerase II α

“…[32][33][34][35] A previous study showed that TOPO2A contributes to forming chromatid breaks in radiation-exposed cells. 27 Here, we report that TOPO2A protein levels were increased by 5-Aza treatment of RR-Hep-2 cells (Fig. 6C and E), and that the promoter demethylation of TOPO2A was closely mirrored by an increase of its decatenation activity in Hep-2 and RR-Hep-2 cells (Fig.…”

“…27 To examine the cellular effects of 5-Aza (5 mM)-induced TOPO2A expression in Hep-2 and RR-Hep-2 cells, we conducted clonogenic survival assays. Our results revealed that 5-Aza treatment significantly reduced the survival of Hep-2 and RRHep2 cells exposed to various doses of radiation (Fig.…”

Lymphovascular invasion in breast cancer: improved methods of detection and clinical significance

“…The chromatid break frequency and topoisomerase IIa expression (ELISA assay) are being compared in 3-day-stimulated peripheral blood T lymphocytes from a group of breast cancer patients and control individuals exposed to a low dose of γ-radiation. Results We show that chromatid radiosensitivity (based on the frequency of metaphase chromatid breaks in irradiated G 2 cells) is significantly lower in a topoisomerase IIa underexpressing variant cell lines [5], and preliminary results show that reducing expression with SiRNA also reduces chromatid radiosensitivity. In a pilot study we are currently comparing the chromatid radiosensitivity and topoisomerase IIa expression in stimulated peripheral blood lymphocytes of a group of Tayside breast cancer patients and a similar number of normal noncancer control individuals.…”

Regulation of DNA damage response and cell cycle in radiation-resistant HL60 myeloid leukemia cells

“…There are very few studies investigating DNA repair and DSBs in HL60 cells (14). Res-HL60 cells established in our study were different from the cells from which they originated in terms of size, and DNA repair and proliferation activities.…”