Impaired elimination of DNA double-strand break-containing lymphocytes in ataxia telangiectasia and Nijmegen breakage syndrome

Abstract

The repair of DNA double-strand breaks is critical for genome integrity and tumor suppression. Here we show that following treatment with the DNA-intercalating agent actinomycin D (ActD), normal quiescent T cells accumulate double-strand breaks and die, whereas T cells from ataxia telangiectasia (AT) and Nijmegen breakage syndrome (NBS) patients are resistant to this death pathway despite a comparable amount of DNA damage. We demonstrate that the ActD-induced death pathway in quiescent T lymphocytes follows DNA damage and H2AX phosphorylation, is ATM- and NBS1-dependent and due to p53-mediated cellular apoptosis. In response to genotoxic 2-Gy γ-irradiation, on the other hand, quiescent T cells from normal donors survive following complete resolution of the damage thus induced. T cells from AT and NBS patients also survive, but retain foci of phosphorylated H2AX due to a subtle double-strand break (DSB) repair defect. A common consequence of these two genetic defects in the DSB response is the apparent tolerance of cells containing DNA breaks. We suggest that this tolerance makes a major contribution to the oncogenic risk of patients with chromosome instability syndromes.

Introduction

DSB repair is critical for genome integrity and tumor suppression. DSBs are produced either by exogenous agents or by endogenous events, such as collapsed replication forks and gene rearrangements during lymphocyte development. Antigen receptor gene recombination is initiated by the recombinase-activating gene 1/2 (RAG1/2) endonuclease during the G₀/G₁ phase of the cell cycle [1]. This generates DSBs adjacent to the V, D and J segments [2] that are subsequently compiled as complete V(D)J coding exons by nonhomologous end joining [3].

V(D)J recombination is now known to be active in precursor bone marrow and thymic cells, and in a fraction of mature B and T cells that undergo receptor revision in the periphery [4], [5]. We and others have observed functional RAG gene products in mature T cells featuring defective TCR expression [6], [7]. The frequency of this T cell subset is increased more than 10-fold in patients with AT and NBS [8], [9], two autosomal recessive genomic instability syndromes characterized by immunodeficiency, hypersensitivity to ionizing radiation (IR) and DSB-inducing agents, defective DNA repair, and an increased risk of lymphoid malignancies [10], [11], [12], [13]. The role of V(D)J recombination in the onset of tumorigenic translocations is well established [14], [15], [16], [17], and a similar role has been suggested for secondary V(D)J rearrangements [18]. Several mechanisms have evolved in normal cells to counter the dangers of V(D)J recombination, i.e. the specific targeting of DNA DSBs to the intended sites, their efficient rejoining and the activation by unresolved DSBs of a general DNA damage-response [19]. Alterations of any of these processes, together with increased RAG activity, in lymphocytes defective for the ATM or NBS1 proteins would provide an explanation of their increased risk of leukemia/lymphoma [20].

We here assessed the response of normal, AT and NBS T cells kept in the G0/G1 phase to two genotoxic agents: ActD and IR. ActD is an anti-tumor antibiotic used in many chemotherapeutical protocols that intercalates into DNA and thus inhibits transcription and induces damage [21], [22]. We show that ActD produces DSBs in normal quiescent T lymphocytes and targets them to apoptosis, whereas T cells from AT and NBS patients resist this apoptosis and survive. Following IR (2-Gy) quiescent T cells from normal donors resolve virtually all damage within 72 h and survive. T cells from AT and NBS patients also survive, despite the maintenance of γH2AX foci.

Inefficient elimination of DSB-containing cells may be supposed to make a major contribution to the oncogenic risk of patients with chromosome instability syndromes.