DNA Repair and Longevity: A Possible Link in the Mouse Hematopoietic System.

Abstract DNA repair efficiency has been postulated to play a role in aging-associated phenotypes as well as in the generation of a variety of cancers. This is especially pertinent in highly proliferative tissues such as the lymphohematopoietic system, since the stem and progenitor compartments are responsible for maintaining proliferative demands within a restricted range for the lifetime of an individual. Hydroxyurea (HU) is a chemotherapeutic drug that targets DNA synthesis by inhibiting the synthesis of the nucleotide substrate resulting in stalled replication forks and single- and double-stranded breaks (DSBs) in the DNA. Recently, our lab has mapped a locus on mouse chromosome 7 that is involved in both organismal lifespan determination and HU sensitivity of bone marrow stem and progenitor cells in the HU-sensitive (25.9% killing), short-lived (540 days) DBA/2J (D2) and HU-insensitive (11.8% killing), long-lived (816 days) C57Bl/6J (B6) strains of mouse. To confirm that this locus is responsible for hydroxyurea sensitivity we generated congenic mice where the locus-containing interval was moved from B6 to D2 (D2. B6 chr. 7) and vice versa (B6. D2 chr. 7). When these animals were treated with HU it was found that the D2 locus imparts a high killing phenotype (38.0%) and the B6 locus confers a low killing phenotype (−4.2%). Using a flow cytometry-based in vivo Bromodeoxyuridine (BrdU) incorporation assay, we measured the recovery of DNA synthesis in the bone marrow in D2 and B6 mice after IP injection of HU (2mg/g). We first determined that DNA synthesis was completely inhibited within 15 minutes of injection and persisted for at least 3 hours in both mouse strains. At 4 hours, bone marrow cells of both strains began to incorporate BrdU, with B6 recovery more rapid than D2, 2.9+/−.5 vs. 7.9+/−3.9 percent BrdU positive cells (p=.01), respectively. Because HU has been used in the past to synchronize cells in G0/G1 and to measure cells in S phase, it was expected that BrdU incorporation would re-initiate within the G0/G1 compartment of cells. Indeed, bone marrow cells from D2 mice incorporated BrdU exclusively within the G0/G1 population. Surprisingly it was found that cells from B6 mice that had an S phase content of DNA prior to HU survived the insult and began to synthesize DNA. It was concluded that B6 bone marrow might have a more robust DNA damage response than that of D2. To study the DNA damage response in the bone marrow we treated mice with HU followed by BrdU and stained the bone marrow cells with an anti-BrdU antibody and an antibody to gamma-H2AX (gH2AX), a histone variant that becomes phosphorylated in the vicinity of DNA DSBs. In both D2 and B6 bone marrow cells it was shown that maximal gH2AX phosphorylation occurred within 1 hour and only occurred in the BrdU+ fraction of the bone marrow cells. Thus it can be concluded that HU causes DNA damage and these two strains of mouse differ in their response due in part to a locus on chromosome 7. Current studies are aimed at identifying the gene(s) of interest in the congenic interval, which include Tfpt and Prkcc.