Abstract
Intact function of DNA repair gene is required for maintenance of genomic stability and long term survival of stem cells. We hypothesize that DNA-PKcs, a key factor for DNA double-strand break (DSB) repair, is critical for hematopoietic stem cell (HSC) function. Expression level of DNA-PKcs mRNA monitored by RT-PCR was high in kit+lin− and sca+lin− cells, low in sca+kit+lin− cells and not seen in lin+ cells, implying its role in highly proliferative progenitors. To assess the function of HSCs deficient in DSB repair, serial transplantation capacity of scid (DNA-PKcs−/−) BM cells into lethally irradiated recipients was compared to wildtype BM. Primary transplants of scid BM died after treatment with 2Gy irradiation 4 wks post-transplantation (n=3). In contrast, parental scid mice survived 3Gy irradiation, implying radiation hypersensitivity of scid BM cells after transplantation. No changes were found in the telomere length, cell cycle distribution and apoptosis between the wildtype and scid BM cells after primary transplantation. Scid BM cells failed to repopulate recipients after the third round of transplantation (n=8). To assess competitive repopulating capacity, mixtures of wildtype and scid cells were transplanted into lethally irradiated recipients. BM CFU of primary recipients were predominantly wildtype (8 mice for C3H background, total CFU=262; 5 mice for C56B/6 background, total CFU=336; n>15 per mouse). Scid cells with two independent genetic backgrounds caused consistent repopulation defects, confirming repopulation defect is caused by DNA-PKcs deficiency. All five primary recipients with C56B/6 background was repopulated predominantly by wildtype CFU (wt CFU 93±5% vs. wt CFU of input; 60±31%, p<10−4). Six of eight primary recipients with C3H background had BM cells repopulated by wildtype CFUs (wt CFU 93±9 % vs. wt CFU of input; 65+13 %, p<10−4), and two of eight primary recipients (wt CFU 67±10 %, p>0.05) had BM cells repopulated similar to donor mixture of wildtype and scid. BM cells of all eight primary recipient mice with C3H background were transplanted into secondary recipients. In all cases, including recipients of the primary cells with the mixed chimera, most BM CFU of secondary recipients originated from wildtype (wt CFU 96±7.8 %, total 16 mice, total CFU=511, and CFU=192 from the mixed chimera). Sca+kit+lin− cells were isolated from the secondary recipients, cultured for 2wks and genotyped. All sca+kit+lin− cells were originated from wildtype (total n=73, 6 mice), implying DNA-PKcs function for HSC proliferation. This confirmed that primary recipients had reconstituted with 100% wildtype HSCs and that the mixed chimera reverted to 100% wildtype. Frequency of sca+kit+lin− cells in scid BM was significantly higher than wildtype (scid 1.94±0.5x10−4, n=4 vs. wt 0.92±0.4x10−4, n=4; p=0.017). Frequency of sca+kit+lin− cells in scid secondary recipients became similar to wildtype secondary recipients (scid 0.61±0.2x10−4, n=4 vs. wt 0.48±0.02x10−4, n=3; p=0.25), implying decreased self-renewal of scid HSCs during repetitive transplantation. This indicates that deficiency in DNA double-strand break repair (scid) leads to HSC failure during repetitive transplantation. Thus, intact DNA repair is essential for maintenance and genomic stability of HSCs.
MCL-1 Depletion Impairs DNA Double-Strand Break Repair and Reinitiation of Stalled DNA Replication Forks
