Repopulation Advantage of Blm−/− Cells in the Primary Recipients Can Be Reversed by Cisplatin Treatment.

Abstract

Deficiencies in DNA repair-related genes such RecQ DNA helicases lead to human genetic disorders such as Bloom's syndrome, Werner's syndrome, and Rothmund-Thomson syndrome characterized by premature aging and cancer predisposition. We hypothesized that deficiency in RecQ DNA helicases may lead to drug hypersensitivity and/or stem cell failure after serial transplantation. Several genotoxic agents were tested on the bone marrow cells isolated from Bloom (Blm)−/− and RecQ4−/− knockout mice and compared to wildtype bone marrow cells. RecQ4−/− bone marrow showed mild sensitivity to γ-ray irradiation and very mild sensitivity to cisplatin. RecQ4−/− bone marrow did not show sensitivity to etoposide. However, Blm−/− bone marrow cells did not show hypersensitivity to either γ-ray irradiation or etoposide treatment, implying each RecQ DNA helicase may have different roles for DNA repair and/or proliferation in bone marrow cells.To assess how deficiency in RecQ DNA helicase affects hematopoietic stem cell function, serial transplantation capacity into lethally irradiated recipients was compared between bone marrow cells isolated from Blm−/− (Ly5.2) and wildtype (Ly5.1) mice. Competitive repopulating capacity was monitored by Ly5.1 / Ly5.2 marker analysis of peripheral blood every three weeks. There was no difference in early repopulating capacity between Blm−/− and wildtype in primary transplants at 3, 6 and 9 weeks post-transplantation (wt 46.6% ± 11.9% vs. Blm−/− 53.5% ± 11.9%, n=6, p=0.33 at 9 weeks post-transplantation). However, at 15 weeks post-transplantation, Blm−/− cells showed higher repopulation than wildtype bone marrow cells (wt 30.7 % ± 7.1 % vs. Blm−/− 69.3% ± 7.1%, n=3, p=0.003), implying Blm−/− cells might gradually accumulate a proliferative advantage over wildtype cells. To assess whether drug treatment may cause sensitivity in Blm−/− cells after transplantation, primary recipients of an equal mixture of Blm−/− and wildtype bone marrow were treated with 1 mg/kg cisplatin biweekly i.p. between 9 to 15 weeks post-transplantation and repopulation capacity was monitored. The repopulation advantage of Blm−/− cells in primary transplants was abolished by cisplatin treatment (wt 61.3 % ± 17.3 % vs. Blm−/− 38.7% ± 17.3%, n=3, p=0.19, in the cisplatin-treated cohort compared to wt 30.7 % ± 7.1 % vs. Blm−/− 69.3% ± 7.1%, n=3, p=0.003, in the control cohort). Thus, a proliferative advantage of progenitors is apparent in Blm−/− bone marrow, but lost after cisplatin-mediated DNA damage. This suggests that defective DNA repair of Blm−/− cell may promote deregulated proliferation of hematopoietic progenitors.