Alpha Radiation as a Way to Target Heterochromatic and Gamma Radiation-Exposed Breast Cancer Cells

Abstract

Compact chromatin is linked to a poor tumour prognosis and resistance to radiotherapy from photons. We investigated DNA damage induction and repair in the context of chromatin structure for densely ionising alpha radiation as well as its therapeutic potential. Chromatin opening by histone deacetylase inhibitor trichostatin A (TSA) pretreatment reduced clonogenic survival and increased γH2AX foci in MDA-MB-231 cells, indicative of increased damage induction by free radicals using gamma radiation. In contrast, TSA pretreatment tended to improve survival after alpha radiation while γH2AX foci were similar or lower; therefore, an increased DNA repair is suggested due to increased access of repair proteins. MDA-MB-231 cells exposed to fractionated gamma radiation (2 Gy × 6) expressed high levels of stem cell markers, elevated heterochromatin H3K9me3 marker, and a trend towards reduced clonogenic survival in response to alpha radiation. There was a higher level of H3K9me3 at baseline, and the ratio of DNA damage induced by alpha vs. gamma radiation was higher in the aggressive MDA-MB-231 cells compared to hormone receptor-positive MCF7 cells. We demonstrate that heterochromatin structure and stemness properties are induced by fractionated radiation exposure. Gamma radiation-exposed cells may be targeted using alpha radiation, and we provide a mechanistic basis for the involvement of chromatin in these effects.

Keywords: DNA damage; alpha radiation; breast cancer; chromatin; gamma radiation.

Figure 1

(A) Levels of acetylated lysine 8 of histone H4 (H4K8ac) were analysed in MDA-MB-231 cells using Western blot after treatment with 0.25–1 µM trichostatin A (TSA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. (B) Effects of TSA alone on clonogenic survival using 0.5 and 1 µM TSA relative to untreated control cells. * p < 0.05 and ** p < 0.01, for 0.5 or 1 versus 0 µM TSA, respectively. (C) Clonogenic survival analysis of MDA-MB-231 cells pretreated with 0.5 or 1 µM TSA for 18 h before exposure to 1–3 Gy of gamma or 0.125–0.5 Gy of alpha radiation: All TSA groups were set to 1 for 0 Gy. ** p < 0.01 for 1 versus 0.5 µM TSA and *** p < 0.001 versus 0 µM TSA. *.

Figure 2

(A) The dose response of gamma and alpha radiation was evaluated at 30 min post-irradiation by analysis of the γH2AX foci number in MDA-MB-231 cells. (B) The repair kinetics of γH2AX foci are presented from 15 min up to 24 h postexposure to 2 Gy of gamma or 0.75 Gy of alpha radiation in MDA-MB-231 cells pretreated with 1 µM TSA for 18 h. (C) Focus areas per cell in pixels were plotted as a histogram, using the relative frequencies (where the sum is 1) on the Y-axis, to show the discrimination between small and large foci. Data was pooled from the 30 min time point of all experiments (0 and 1 µM TSA). The numbers of small (D) and large (E) foci are displayed, using the data from Figure 2C. The foci numbers for controls (0 Gy) were subtracted from sample foci numbers in all graphs to allow for comparisons between 0 and 1 µM TSA; therefore, negative values are also seen. * p < 0.05 versus 0 µM TSA.

Figure 3

(A) Two parallel cell replicates of MDA-MB-231 (denoted A and B) were exposed to 6 × 0.5, 1.0, or 1.5 Gy fractions of gamma radiation, 3 days per week. (B) Cell growth was assayed and presented as number of doublings for 14 days, with the ionising radiation (IR) fractions indicated by arrows. (C) The frequency of micronuclei (MN) per 1000 binucleated cells (BNC) were scored at day 15 in replicate A and B. (D) The mRNA levels of cancer/normal stem cell markers CD44, CD133, Sox2, Oct4, and Nanog were analysed by real-time PCR at days 2, 4, 7, 9, 11, and 14 in replicate A.

Figure 4

(A) Two parallel cell replicates (denoted 1 and 2) were exposed to 6 × 2 Gy fractions of gamma radiation, 2 days per week, aiming to establish gamma radiation-exposed (RE) MDA-MB-231 cells. Analysis was performed at early (2 replicates) and late time points (from replicate 1, three independent experiments). (B) Cell growth was assayed and presented as number of doublings during the first 9 days, with the 2 Gy fractions indicated as arrows. The mRNA levels of cancer/normal stem cell markers CD44, CD133, Sox2, Oct4, and Nanog were analysed by real-time PCR at 24 h after 2 Gy or in total 6 Gy, i.e., in samples collected on day 2 and 9 (C) or in samples 4–6 weeks after 12 Gy (D). Trimethylated lysine 9 of histone H3 (H3K9me3) levels, normalised to GAPDH, were assayed using Western blot (E), and clonogenic survival was analysed in RE cells versus control cells in response to gamma and alpha radiation in samples 4–6 weeks after 12 Gy (F). * p < 0.05, ** p < 0.01, and *** p < 0.001 for RE versus control cells.

Figure 5

Sphere-forming MCF7 tumour-initiating cells (TICs) versus MCF7 were analysed at the level of CD44, CD133, Sox2, Oct4, and Nanog mRNA by (A) real-time PCR; (B) H3K9me3, normalised to GAPDH, was assayed using Western blot; and (C) clonogenic survival was analysed in MCF7 TICs versus MCF7 cells in response to gamma and alpha radiation. *** p < 0.001 for MCF7 TICs versus MCF7 cells.

Figure 6

(A) Basal levels of H3K9me3 and total protein levels of histone H3 were analysed in MDA-MB-231 and MCF7 cells using Western blot. GAPDH was used as a loading control. (B) γH2AX foci numbers are presented at 30 min and 24 h postexposure to 6 Gy of gamma or 2 Gy of alpha radiation in MDA-MB-231 and MCF7 cells. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus MDA-MB-231.