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 therapyinduced 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 0 overhangs. As measured by g-H2AX induction and foci formation, 6TG induced DNA damage in Rad51d-proficient and Rad51d-deficient cells. However, the extent of g-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.

Collateral damage of NUDT15 deficiency in cancer provides a cancer pharmacogenetic therapeutic window with thiopurines

“…The induction of DNA damage is consistent with the known mechanism of action for thiopurines, further suggesting that the loss of NUDT15 exacerbates the DNA-directed effects of thiopurines. Of note, there was a striking difference in the level of 6-TG induced chromosomal aberrations, including dramatic increases in complex exchange events that were previously observed in HR-defective cells 23 . The work with cancer cells demonstrates that NUDT15-deficient cancer cells are sensitized to thiopurines and that the DNA-directed effects of thiopurines are exacerbated by NUDT15 deficiency.…”

“…This supports previous findings that cells progress though mitosis with doses of 6-TG, while higher doses of 6-TG cause a G 2 arrest. 23…”

“…While NUDT15 deficiency is known from clinical experience to cause hematopoietic toxicity 25 , and NUDT15-defective cancer cells were shown to be sensitized to 6-TG treatments 5 , the cytotoxic mechanism of action for 6-TG treatments in NUDT15-deficient cells remained unexplored. Because the established cytotoxic mechanism of action for thiopurines occurs through the induction of DNA damage 21, 23, 24, 26 , markers of DNA damage were measured and compared in the NUDT15-proficient and deficient cells. The induction of DNA damage is consistent with the known mechanism of action for thiopurines, further suggesting that the loss of NUDT15 exacerbates the DNA-directed effects of thiopurines.…”

“…This supports previous findings that cells progress though mitosis with doses of 6-TG, while higher doses of 6-TG cause a G2 arrest. 23 Induction of the DNA damage marker γ-H2AX was assessed by western blotting after 6-TG treatments in parental and NUDT15-deficient OVCAR-8 cells. Following treatments of 0.3 µM and 1 µM of 6-TG, increases in γ-H2AX are induced in the NUDT15 KO cells after 48 and 72 h exposure to 6-TG (Figure 6A, lanes 7 and 8).…”

Effect of Macleaya cordata extract on the growth performance and growth-related expression gene pathways in rats was investigated by liver transcriptome technology

“…This protein-coding gene translates into a protein complex that promotes homologous pairing between single-and double-stranded DNA, homologous recombinant repair (HRR) pathways that can participate in the DNA replication process, and double-stranded DNA breaks occur during the overrun or are induced by DNA damage agents (Baldock et al 2019). Because RAD51D is an important part of homologous recombination, if this gene is defective, it will have a huge negative impact on cell mitosis; therefore, this gene plays an indispensable role in various biological processes of DNA (Wyatt et al 2018). Through transcriptome high-throughput sequencing and quantitative uorescence PCR experiments, we found that the expression level of RAD51D in the low-dose and high-dose groups was signi cantly higher than that in the blank group.…”