Wortmannin, a specific inhibitor of phosphatidylinositol-3-kinase, induces accumulation of DNA double-strand breaks

Wortmannin, a fungal metabolite, is a specific inhibitor of the phosphatidylinositol 3-kinase (PI3K) family, which includes double-stranded DNA dependent protein kinase (DNA-PK) and ataxia telangiectasia mutated kinase (ATM). We investigated the effects of wortmannin on DNA damage in DNA-PK-deficient cells obtained from severe combined immunodeficient mice (SCID cells). Survival of wortmannin-treated cells decreased in a concentration-dependent manner. After treatment with 50 μM wortmannin, survival decreased to 60% of that of untreated cells. We observed that treatment with 20 and 50 μM wortmannin induced DNA damage equivalent to that by 0.37 and 0.69 Gy, respectively, of γ-ray radiation. The accumulation of DNA double-strand breaks (DSBs) in wortmannin-treated SCID cells was assessed using pulsed-field gel electrophoresis. The maximal accumulation was observed 4 h after treatment. Moreover, the presence of DSBs was confirmed by the ability of nuclear extracts from γ-ray-irradiated SCID cells to produce in vitro phosphorylation of histone H2AX. These results suggest that wortmannin induces cellular toxicity by accumulation of spontaneous DSBs through inhibition of ATM.

Lack of Functional DNA-PKcs and DNA Double-Strand Break Repair Leads to a Repopulating Stem Cell Defect in Scid Mice.

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.

Irradiation Promotes V(D)J Joining and RAG-Dependent Neoplastic Transformation in SCID T-Cell Precursors

ABSTRACT Defects in the nonhomologous end-joining (NHEJ) pathway of double-stranded DNA break repair severely impair V(D)J joining and selectively predispose mice to the development of lymphoid neoplasia. This connection was first noted in mice with the severe combined immune deficient (SCID) mutation in the DNA-dependent protein kinase (DNA-PK). SCID mice spontaneously develop thymic lymphoma with low incidence and long latency. However, we and others showed that low-dose irradiation of SCID mice dramatically increases the frequency and decreases the latency of thymic lymphomagenesis, but irradiation does not promote the development of other tumors. We have used this model to explore the mechanistic basis by which defects in NHEJ confer selective and profound susceptibility to lymphoid oncogenesis. Here, we show that radiation quantitatively and qualitatively improves V(D)J joining in SCID cells, in the absence of T-cell receptor-mediated cellular selection. Furthermore, we show that the lymphocyte-specific endonuclease encoded by the recombinase-activating genes (RAG-1 and RAG-2) is required for radiation-induced thymic lymphomagenesis in SCID mice. Collectively, these data suggest that irradiation induces a DNA-PK-independent NHEJ pathway that facilitates V(D)J joining, but also promotes oncogenic misjoining of RAG-1/2-induced breaks in SCID T-cell precursors.

Activation of V(D)J Recombination Induces the Formation of Interlocus Joints and Hybrid Joints in scid Pre-B-Cell Lines

ABSTRACT V(D)J recombination is the mechanism by which antigen receptor genes are assembled. The site-specific cleavage mediated by RAG1 and RAG2 proteins generates two types of double-strand DNA breaks: blunt signal ends and covalently sealed hairpin coding ends. Although these DNA breaks are mainly resolved into coding joints and signal joints, they can participate in a nonstandard joining process, forming hybrid and open/shut joints that link coding ends to signal ends. In addition, the broken DNA molecules excised from different receptor gene loci could potentially be joined to generate interlocus joints. The interlocus recombination process may contribute to the translocation between antigen receptor genes and oncogenes, leading to malignant transformation of lymphocytes. To investigate the underlying mechanisms of these nonstandard recombination events, we took advantage of recombination-inducible cell lines derived from scid homozygous (s/s) and scid heterozygous (s/+) mice by transforming B-cell precursors with a temperature-sensitive Abelson murine leukemia virus mutant (ts-Ab-MLV). We can manipulate the level of recombination cleavage and end resolution by altering the cell culture temperature. By analyzing various recombination products in scid and s/+ts-Ab-MLV transformants, we report in this study that scid cells make higher levels of interlocus and hybrid joints than their normal counterparts. These joints arise concurrently with the formation of intralocus joints, as well as with the appearance of opened coding ends. The junctions of these joining products exhibit excessive nucleotide deletions, a characteristic of scid coding joints. These data suggest that an inability of scid cells to promptly resolve their recombination ends exposes the ends to a random joining process, which can conceivably lead to chromosomal translocations.