Thiopurine-induced mitotic catastrophe in Rad51d-deficient mammalian cells

Abstract

Thiopurines are part of a clinical regimen used for the treatment of autoimmune disorders and childhood acute lymphoblastic leukemia. However, despite these successes, there are also unintended consequences such as therapy-induced cancer in long-term survivors. Therefore, a better understanding of cellular responses to thiopurines will lead to improved and personalized treatment strategies. RAD51D is an important component of homologous recombination (HR), and our previous work established that mammalian cells defective for RAD51D are more sensitive to the thiopurine 6-thioguanine (6TG) and have dramatically increased numbers of multinucleated cells and chromosome instability. 6TG is capable of being incorporated into telomeres, and interestingly, RAD51D contributes to telomere maintenance, although the precise function of RAD51D at the telomeres remains unclear. We sought here to investigate: (1) the activity of RAD51D at telomeres, (2) the contribution of RAD51D to protect against 6TG-induced telomere damage, and (3) the fates of Rad51d-deficient cells following 6TG treatment. These results demonstrate that RAD51D is required for maintaining the telomeric 3' overhangs. As measured by γ-H2AX induction and foci formation, 6TG induced DNA damage in Rad51d-proficient and Rad51d-deficient cells. However, the extent of γ-H2AX telomere localization following 6TG treatment was higher in Rad51d-deficient cells than in Rad51d-proficient cells. Using live-cell imaging of 6TG-treated Rad51d-deficient cells, two predominant forms of mitotic catastrophe were found to contribute to the formation of multinucleated cells, failed division and restitution. Collectively, these findings provide a unique window into the role of the RAD51D HR protein during thiopurine induction of mitotic catastrophe. Environ. Mol. Mutagen. 59:38-48, 2018. © 2017 Wiley Periodicals, Inc.

Keywords: 6-thioguanine; H2AX; homologous recombination; mitotic catastrophe; telomeres.

Figure 1

Comparison of relative telomeric 3′ overhang lengths in primary MEFs. (A) Radioactively labeled oligonucleotide in-gel hybridizations to MboI digested genomic DNA isolated from primary MEFs. A native gel was first hybridized with the (CCCTAA)₄ probe, and (B) the same gel was alkali denatured and hybridized with the (TTAGGG)₄ probe. (C) Relative G-strand overhang lengths were determined using the following equation: RS_N/[(TL_C/TL_E) * RS_D], where RS_N is the radioactive signal from native gels hybridized with the (CCCTAA)₄ oligonucleotide, TL_C is the estimated telomere lengths of the control (homozygous wild-type) cells, TL_E is the estimated telomere lengths of the primary wild-type, Rad51d^+/− Trp53^+/−, Trp53^−/−, or Rad51d^−/− Trp53^−/− cells, and RS_D is the total radioactive signal from the denatured gels hybridized with the (TTAGGG)₄ oligonucleotide. Telomere lengths were estimated as described [Harley et al 1990]. Error bars are the SEM from at least three independent experiments. (D) Localization of γ-H2AX foci at telomeres in primary MEFs. Telomeric DNA was identified by the peptide nucleic acid (PNA) probe (red), γ-H2AX was immunolabeled by indirect immunofluorescence (green), and DNA stained with DAPI (blue). White arrowheads demonstrate the localization of γ-H2AX foci at telomeres. (E) Percentage of γ-H2AX foci at telomeres in primary wild-type MEFs (n = 58 nuclei scored) and primary Rad51d ^−/− MEFs (n = 59 nuclei scored). Statistical significance was calculated by ANOVA; *indicated p=0.011.

Figure 2

Induction of γ-H2AX following treatment with 6TG. (A) The γ-H2AX signal (lower band) was determined by Western blot analysis after 6TG treatment for 48 and 72 h at the doses indicated in Rad51d^+/+ (lanes 1 – 3) and Rad51d^−/− (lanes 4 – 6) and Rad51d^−/−Mlh1^−/− MEFs (lanes 7 – 9). (B & C) Quantification of γ-H2AX band intensities from untreated cells (□), or cells treated for 48 h (■) or 72 h (■) that were normalized to GAPDH (*p<0.05) after 50 nM (B) or 100 nM (C) 6TG treatment. (D) Quantitation of cellular γ-H2AX foci from untreated cells (□), or cells treated with 50 (■) or 100 nM (■) 6TG. Nuclei with ten or more γ-H2AX foci were scored as positive, and at least 100 nuclei were counted for each sample.

Figure 3

(A). Localization of γ-H2AX foci co-localized with telomere signal at telomeres in immortalized MEFs after treatment with vehicle alone, 50 nM, or 100 nM 6TG. The three categories are 0 to 2 co-localized foci (□), 3 to 7 co-localized foci (■), or ≥8 co-localized foci (■) per nuclei. (B). Representative images of Rad51d^−/− cells mock treated or treated with 50 or 100 nM 6TG. Blue panels are DAPI stained nuclei. Green panels are stained with anti- γ-H2AX antibody. Red panels are stained with the telomere probe. Merge is the overlay of each panel.

Figure 4

6TG induced chromosome fusions in Rad51d-deficient immortalized MEFs. After treatment for 72 hours, chromosomes were stained with DAPI. The number of chromosome fusions was scored as a percent of the total number of chromosomes in vehicle, 50 nM, or 100 nM 6TG-treated Rad51d^+/+ (□), and Rad51d^−/− (■) and Rad51d^−/−Mlh1^−/− MEFs (■) Statistical significance was determined by calculating a z-score; *indicates p<0.05.

Figure 5

Morphological analysis of Rad51d-deficient MEFs following 6TG treatment. The average time of immortalized Rad51d^−/− MEFs spent in (A) interphase and (B) mitosis is shown. Statistical significance was determined by one-tailed T test; error bars indicate the standard deviation; *** indicates p<0.001. * indicates p<0.05. (C) Cellular outcomes of nine grids from three independent experiments after no treatment, treatment with 50 nM or 100 nM 6TG scored as division, apoptosis, restitution, failed division, and arrest. The total number of cells scored were 112 for untreated (□), 68 for cells treated with 50 nM 6TG (■) and 123 for cells treated with 100 nM 6TG (■). (D & E) Representative still images show multi-nucleation events as a result of restitution (D) or failed division (E). The arrow in the second panel of (D) indicates the beginning of cytokinesis, and the arrows in the fourth panel indicate each nucleus within a single cell. The arrows in the fourth panel of (E) indicate each nucleus within a single cell.