Rnf8 deficiency impairs class switch recombination, spermatogenesis, and genomic integrity and predisposes for cancer

Signaling and repair of DNA double-strand breaks (DSBs) are critical for preventing immunodeficiency and cancer. These DNA breaks result from exogenous and endogenous DNA insults but are also programmed to occur during physiological processes such as meiosis and immunoglobulin heavy chain (IgH) class switch recombination (CSR). Recent studies reported that the E3 ligase RNF8 plays important roles in propagating DNA DSB signals and thereby facilitating the recruitment of various DNA damage response proteins, such as 53BP1 and BRCA1, to sites of damage. Using mouse models for Rnf8 mutation, we report that Rnf8 deficiency leads to impaired spermatogenesis and increased sensitivity to ionizing radiation both in vitro and in vivo. We also demonstrate the existence of alternative Rnf8-independent mechanisms that respond to irradiation and accounts for the partial recruitment of 53bp1 to sites of DNA damage in activated Rnf8−/− B cells. Remarkably, IgH CSR is impaired in a gene dose-dependent manner in Rnf8 mutant mice, revealing that these mice are immunodeficient. In addition, Rnf8−/− mice exhibit increased genomic instability and elevated risks for tumorigenesis indicating that Rnf8 is a novel tumor suppressor. These data unravel the in vivo pleiotropic effects of Rnf8

Inactivation of Chk2 and Mus81 Leads to Impaired Lymphocytes Development, Reduced Genomic Instability, and Suppression of Cancer

Chk2 is an effector kinase important for the activation of cell cycle checkpoints, p53, and apoptosis in response to DNA damage. Mus81 is required for the restart of stalled replication forks and for genomic integrity. Mus81Δex3-4/Δex3-4 mice have increased cancer susceptibility that is exacerbated by p53 inactivation. In this study, we demonstrate that Chk2 inactivation impairs the development of Mus81Δex3-4/Δex3-4 lymphoid cells in a cell-autonomous manner. Importantly, in contrast to its predicted tumor suppressor function, loss of Chk2 promotes mitotic catastrophe and cell death, and it results in suppressed oncogenic transformation and tumor development in Mus81Δex3-4/Δex3-4 background. Thus, our data indicate that an important role for Chk2 is maintaining lymphocyte development and that dual inactivation of Chk2 and Mus81 remarkably inhibits cancer

Cytoplasmic ATM protein kinase: An emerging therapeutic target for diabetes, cancer and neuronal degeneration

Ataxia–telangiectasia (A–T) is an autosomal recessive disorder characterized by cerebellar ataxia and oculocutaneous telangiectasias. The gene mutated in this disease, Atm (A–T mutated), encodes a serine/threonine protein kinase that has been traditionally considered to be a nuclear protein controlling cell cycle progression. However, many of the growth abnormalities observed in patients with A–T, including neuronal degeneration and insulin resistance, remain difficult to explain with nuclear localization of ATM. Here, recent advances in elucidating the cytoplasmic localization and function of ATM are reviewed. Particular attention is given to the role of ATM in insulin signaling and Akt activation. The potential for cytoplasmic ATM protein kinase to be an emerging therapeutic target for treating diabetes, cancer and neuronal degeneration is discussed

Translational Control Protein 80 Stimulates IRES-Mediated Translation of p53 mRNA in Response to DNA Damage

Synthesis of the p53 tumor suppressor increases following DNA damage. This increase and subsequent activation of p53 are essential for the protection of normal cells against tumorigenesis. We previously discovered an internal ribosome entry site (IRES) that is located at the 5′-untranslated region (UTR) of p53 mRNA and found that the IRES activity increases following DNA damage. However, the mechanism underlying IRES-mediated p53 translation in response to DNA damage is still poorly understood. In this study, we discovered that translational control protein 80 (TCP80) has increased binding to the p53 mRNA in vivo following DNA damage. Overexpression of TCP80 also leads to increased p53 IRES activity in response to DNA damage. TCP80 has increased association with RNA helicase A (RHA) following DNA damage and overexpression of TCP80, along with RHA, leads to enhanced expression of p53. Moreover, we found that MCF-7 breast cancer cells with decreased expression of TCP80 and RHA exhibit defective p53 induction following DNA damage and diminished expression of its downstream target PUMA, a proapoptotic protein. Taken together, our discovery of the function of TCP80 and RHA in regulating p53 IRES and p53 induction following DNA damage provides a better understanding of the mechanisms that regulate IRES-mediated p53 translation in response to genotoxic stress

Synergistic Interaction of Rnf8 and p53 in the Protection against Genomic Instability and Tumorigenesis

<div><p>Rnf8 is an E3 ubiquitin ligase that plays a key role in the DNA damage response as well as in the maintenance of telomeres and chromatin remodeling. <em>Rnf8^−/−</em> mice exhibit developmental defects and increased susceptibility to tumorigenesis. We observed that levels of p53, a central regulator of the cellular response to DNA damage, increased in <em>Rnf8^−/−</em> mice in a tissue- and cell type–specific manner. To investigate the role of the p53-pathway inactivation on the phenotype observed in <em>Rnf8^−/−</em> mice, we have generated <em>Rnf8^−/−p53^−/−</em> mice. Double-knockout mice showed similar growth retardation defects and impaired class switch recombination compared to <em>Rnf8^−/−</em> mice. In contrast, loss of p53 fully rescued the increased apoptosis and reduced number of thymocytes and splenocytes in <em>Rnf8^−/−</em> mice. Similarly, the senescence phenotype of <em>Rnf8^−/−</em> mouse embryonic fibroblasts was rescued in p53 null background. <em>Rnf8^−/−p53^−/−</em> cells displayed defective cell cycle checkpoints and DNA double-strand break repair. In addition, <em>Rnf8^−/−p53^−/−</em> mice had increased levels of genomic instability and a remarkably elevated tumor incidence compared to either <em>Rnf8^−/−</em> or <em>p53^−/−</em> mice. Altogether, the data in this study highlight the importance of p53-pathway activation upon loss of Rnf8, suggesting that Rnf8 and p53 functionally interact to protect against genomic instability and tumorigenesis.</p> </div

Radioresistance and increased proliferation of <i>Rnf8^−/−</i> cells in the absence of p53.

<p>(A) Freshly isolated thymocytes from <i>Rnf8^−/−p53^−/−</i> mice and control littermates were irradiated with 2 Gy and cell death was determined 12 hours later. Data are presented as the means SD of at least 5 independent experiments. * represents significant difference (P<0.05; student t-test) compared to <i>WT</i> and <i>Rnf8^−/−</i> controls. No difference was observed between <i>p53^−/−</i> and <i>Rnf8^−/−p53^−/−</i> thymoctes. (B) Freshly isolated splenocytes were irradiated (2 Gy) and cell death was determined 24 hours later. Data are presented as the means SD of at least 5 independent experiments. * represents statistical significance (P<0.05; student t-test) compared to <i>WT</i> and <i>Rnf8^−/−</i> controls. No significant difference was observed between <i>p53^−/−</i> and <i>Rnf8^−/−p53^−/−</i> splenocytes. (C) Cumulative growth curve of <i>Rnf8^−/−p53^−/−</i> MEFs. Passage 2 <i>Rnf8^−/−p53^−/−</i> MEFs and controls were plated in 60 mm plates at a density of 3×10⁵ cells/plate. Cells were replated at the same density every 3 days and cumulative cell growth was calculated. Data are presented as the log10 of means ± SD of at least 5 independent experiments.</p

Increased p53 expression and cleaved caspase-3 levels in <i>Rnf8^−/</i>⁻ thymocytes.

<p>(A) p53 IHC staining of thymus sections of <i>Rnf8^−/−</i> mice and <i>WT</i> littermates. (B) Quantification of p53 positive cells in thymus. An average of 20 randomly chosen fields was counted at 63× magnifications. (C) Cleaved caspase-3 levels in the thymus of <i>Rnf8^−/−p53^−/−</i> mice and control littermates. Anti-cleaved caspase-3 IHC staining of thymus of <i>Rnf8^−/−p53^−/−</i>, <i>Rnf8^−/−</i>, <i>p53^−/−</i> and <i>WT</i> mice. (D) Quantification of cleaved caspase-3 positive cells in the thymus. An average of 20 fields counted at 63× magnification. Data is representative of 3 different experiments. * indicates statistical significance (P<0.05). Bar: 50 µm.</p

Increased basal and residual γ-H2ax foci in <i>Rnf8^−/−p53^−/−</i> MEFs.

<p>(A) γ-H2ax staining for <i>Rnf8^−/−p53^−/−</i> MEFs and controls. <i>Rnf8^−/−p53^−/−</i>, <i>Rnf8^−/−</i>, <i>p53^−/−</i> and <i>WT</i> early passage primary MEFs were either mock-treated (UT) or irradiated (8 Gy) and allowed to recover for 0.5, 4 and 24 hours. Cells were then fixed, stained using anti-γ-H2ax antibody and counterstained with DAPI. (B) Percentage of untreated cells that contained 10 or more γ-H2ax foci. Data are presented as the means SD of at least 3 independent experiments. At least 100 cells were quantified per experiment. * denotes statistical significance (P<0.05; student t-test). (C, D) Percentage of cells that showed more than 10 γ-H2ax foci at 0.5 and 4 hours post-irradiation respectively. At least 100 cells were quantified for each experiment. (E) Percentage of cells showing 10 or more γ-H2ax foci 24 hours after irradiation. Data are presented as the means SD of at least 3 independent experiments. At least 100 cells were quantified per experiment. * indicates statistical significance (P<0.05; student t-test).</p