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  • Synergistic cytotoxicity of the DNA alkylating agent busulfan, nucleoside analogs and suberoylanilide hydroxamic acid in lymphoma cell lines. 22023523

    Hematopoietic stem cell transplant (HSCT) is a promising treatment for lymphomas. Its success depends on effective pre-transplant conditioning regimens. We previously reported on the efficacy of DNA alkylating agent-nucleoside analog (NA) combinations for conditioning in acute myeloid leukemia (AML). We hypothesized that a similar combinatory approach can be used for lymphomas. A combination of busulfan (Bu) with two NAs - clofarabine (Clo), fludarabine (Flu) or gemcitabine (Gem) - resulted in synergistic cytotoxicity in lymphoma cell lines. We demonstrated that the [2 NAs + Bu] combination activates a DNA damage response through the ATM-CHK2 and ATM-CHK1 pathways, leading to cell cycle checkpoint activation and apoptosis. Histone modifications and KAP1 phosphorylation are indicative of chromatin relaxation mediated by the nucleoside analogs, which sequentially increase Bu alkylation. Addition of suberoylanilide hydroxamic acid (SAHA) enhanced chromatin relaxation through increased histone acetylation and further augmented the cytotoxicity of [2 NAs + Bu]. Our results provide a preclinical basis for a clinical trial on using [2 NAs + Bu ± SAHA] combinations as conditioning therapy for patients with chemotherapy-refractory lymphoma undergoing HSCT.
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  • Wip1 phosphatase is associated with chromatin and dephosphorylates gammaH2AX to promote checkpoint inhibition. 20101220

    DNA double-stranded breaks (DSBs) elicit a checkpoint response that causes a delay in cell cycle progression. Early in the checkpoint response, histone H2AX is phosphorylated in the chromatin region flanking the DSB by ATM/ATR and DNA-PK kinases. The resulting foci of phosphorylated H2AX (gamma-H2AX) serve as a platform for recruitment and retention of additional components of the checkpoint-signaling cascade that enhance checkpoint signaling and DSB repair. Upon repair, both the assembled protein complexes and the chromatin modifications are removed to quench the checkpoint signal. In this study, we show that the DNA damage-responsive Wip1 phosphatase is bound to chromatin. Moreover, Wip1 directly dephosphorylates gamma-H2AX and cells depleted of Wip1 fail to dephosphorylate gamma-H2AX during checkpoint recovery. Conversely, premature activation of Wip1 leads to displacement of MDC1 from damage foci and prevents activation of the checkpoint. Taken together, our data show that Wip1 has an essential role in dephosphorylation of gamma-H2AX to silence the checkpoint and restore chromatin structure once DNA damage is repaired.
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  • Human parvovirus B19 DNA replication induces a DNA damage response that is dispensable for cell cycle arrest at phase G2/M. 22837195

    Human parvovirus B19 (B19V) infection is highly restricted to human erythroid progenitor cells, in which it induces a DNA damage response (DDR). The DDR signaling is mainly mediated by the ATR (ataxia telangiectasia-mutated and Rad3-related) pathway, which promotes replication of the viral genome; however, the exact mechanisms employed by B19V to take advantage of the DDR for virus replication remain unclear. In this study, we focused on the initiators of the DDR and the role of the DDR in cell cycle arrest during B19V infection. We examined the role of individual viral proteins, which were delivered by lentiviruses, in triggering a DDR in ex vivo-expanded primary human erythroid progenitor cells and the role of DNA replication of the B19V double-stranded DNA (dsDNA) genome in a human megakaryoblastoid cell line, UT7/Epo-S1 (S1). All the cells were cultured under hypoxic conditions. The results showed that none of the viral proteins induced phosphorylation of H2AX or replication protein A32 (RPA32), both hallmarks of a DDR. However, replication of the B19V dsDNA genome was capable of inducing the DDR. Moreover, the DDR per se did not arrest the cell cycle at the G(2)/M phase in cells with replicating B19V dsDNA genomes. Instead, the B19V nonstructural 1 (NS1) protein was the key factor in disrupting the cell cycle via a putative transactivation domain operating through a p53-independent pathway. Taken together, the results suggest that the replication of the B19V genome is largely responsible for triggering a DDR, which does not perturb cell cycle progression at G(2)/M significantly, during B19V infection.
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  • Development of a high-content high-throughput screening assay for the discovery of ATM signaling inhibitors. 22653913

    The genome is constantly exposed to DNA damage agents, leading up to as many as 1 million individual lesions per cell per day. Cells have developed a variety of DNA damage repair (DDR) mechanisms to respond to harmful effects of DNA damage. Failure to repair the damaged DNA causes genomic instability and, as a result, leads to cellular transformation. Indeed, deficiencies of DDR frequently occur in human cancers, thus providing a great opportunity for cancer therapy by developing anticancer agents that work by synthetic lethality-based mechanisms or enhancing the clinical efficacy of radiotherapy and existing chemotherapies. Ataxia-telangiectasia mutated (ATM) plays a key role in regulating the cellular response to DNA double-strand breaks. Ionizing radiation causes double-strand breaks and induces rapid ATM autophosphorylation on serine 1981 that initiates ATM kinase activity. Activation of ATM results in phosphorylation of many downstream targets that modulate numerous damage-response pathways, most notably cell-cycle checkpoints. We describe here the development and validation of a high-throughput imaging assay measuring levels of phospho-ATM Ser1981 in HT29 cells after exposure to ionizing radiation. We also examined activation of downstream ATM effectors and checked specificity of the endpoint using known inhibitors of DNA repair pathways.
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  • ROS-generating NADPH oxidase NOX4 is a critical mediator in oncogenic H-Ras-induced DNA damage and subsequent senescence. 21841825

    Activated Ras oncogene induces DNA-damage response by triggering reactive oxygen species (ROS) production and this is critical for oncogene-induced senescence. Until now, little connections between oncogene expression, ROS-generating NADPH oxidases and DNA-damage response have emerged from different studies. Here we report that H-RasV12 positively regulates the NADPH oxidase system NOX4-p22(phox) that produces H(2)O(2). Knocking down the NADPH oxidase with small interference RNA decreases H-RasV12-induced DNA-damage response detected by γ-H2A.X foci analysis. Using HyPer, a specific probe for H(2)O(2), we detected an increase in H(2)O(2) in the nucleus correlated with NOX4-p22(phox) perinuclear localization. DNA damage response can be caused not only by H-RasV12-driven accumulation of ROS but also by a replicative stress due to a sustained oncogenic signal. Interestingly, NOX4 downregulation by siRNA abrogated H-RasV12 regulation of CDC6 expression, an essential regulator of DNA replication. Moreover, senescence markers, such as senescence-associated heterochromatin foci, PML bodies, HP1β foci and p21 expression, induced under H-RasV12 activation were decreased with NOX4 inactivation. Taken together, our data indicate that NADPH oxidase NOX4 is a critical mediator in oncogenic H-RasV12-induced DNA-damage response and subsequent senescence.
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  • DNA replication fading as proliferating cells advance in their commitment to terminal differentiation. 22359734

    Terminal differentiation is the process by which cycling cells stop proliferating to start new specific functions. It involves dramatic changes in chromatin organization as well as gene expression. In the present report we used cell flow cytometry and genome wide DNA combing to investigate DNA replication during murine erythroleukemia-induced terminal cell differentiation. The results obtained indicated that the rate of replication fork movement slows down and the inter-origin distance becomes shorter during the precommitment and commitment periods before cells stop proliferating and accumulate in G1. We propose this is a general feature caused by the progressive heterochromatinization that characterizes terminal cell differentiation.
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  • Histone lysine methyltransferase SETD8 promotes carcinogenesis by deregulating PCNA expression. 22556262

    Although the physiologic significance of lysine methylation of histones is well known, whether lysine methylation plays a role in the regulation of nonhistone proteins has not yet been examined. The histone lysine methyltransferase SETD8 is overexpressed in various types of cancer and seems to play a crucial role in S-phase progression. Here, we show that SETD8 regulates the function of proliferating cell nuclear antigen (PCNA) protein through lysine methylation. We found that SETD8 methylated PCNA on lysine 248, and either depletion of SETD8 or substitution of lysine 248 destabilized PCNA expression. Mechanistically, lysine methylation significantly enhanced the interaction between PCNA and the flap endonuclease FEN1. Loss of PCNA methylation retarded the maturation of Okazaki fragments, slowed DNA replication, and induced DNA damage, and cells expressing a methylation-inactive PCNA mutant were more susceptible to DNA damage. An increase of methylated PCNA was found in cancer cells, and the expression levels of SETD8 and PCNA were correlated in cancer tissue samples. Together, our findings reveal a function for lysine methylation on a nonhistone protein and suggest that aberrant lysine methylation of PCNA may play a role in human carcinogenesis.
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  • Ataxia telangiectasia mutated- and Rad3-related kinase drives both the early and the late DNA-damage response to the monofunctional antitumour alkylator S23906. 21470188

    Numerous anticancer agents and environmental mutagens target DNA. Although all such compounds interfere with the progression of the replication fork and inhibit DNA synthesis, there are marked differences in the DNA-damage response pathways they trigger, and the relative impact of the proximal or the distal signal transducers on cell survival is mainly lesion-specific. Accordingly, checkpoint kinase inhibitors in current clinical development show synergistic activity with some DNA-targeting agents, but not with others. In the present study, we characterize the DNA-damage response to the antitumour acronycine derivative S23906, which forms monofunctional adducts with guanine residues in the minor groove of DNA. S23906 exposure is accompanied by specific recruitment of RPA (replication protein A) at replication sites and rapid Chk1 activation. In contrast, neither MRN (Mre11-Rad50-Nbs1) nor ATM (ataxia-telangiectasia mutated), contributes to the initial response to S23906. Interestingly, genetic attenuation of ATR (ATM- and Ras3-related) activity inhibits not only the early phosphorylation of histone H2AX and Chk1, but also interferes with the late phosphorylation of Chk2. Moreover, loss of ATR function or pharmacological inhibition of the checkpoint kinases by AZD7762 is accompanied by abrogation of the S-phase arrest and increased sensitivity towards S23906. These findings identify ATR as a central co-ordinator of the DNA-damage response to S23906, and provide a mechanistic rationale for combinations of S23906 and similar agents with checkpoint abrogators.
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  • Role for hACF1 in the G2/M damage checkpoint. 21745822

    Active chromatin remodelling is integral to the DNA damage response in eukaryotes, as damage sensors, signalling molecules and repair enzymes gain access to lesions. A variety of nucleosome remodelling complexes is known to promote different stages of DNA repair. The nucleosome sliding factors CHRAC/ACF of Drosophila are involved in chromatin organization during development. Involvement of corresponding hACF1-containing mammalian nucleosome sliding factors in replication, transcription and very recently also non-homologous end-joining of DNA breaks have been suggested. We now found that hACF1-containing factors are more generally involved in the DNA damage response. hACF1 depletion increases apoptosis, sensitivity to radiation and compromises the G2/M arrest that is activated in response to UV- and X-rays. In the absence of hACF1, γH2AX and CHK2ph signals are diminished. hACF1 and its ATPase partner SNF2H rapidly accumulate at sites of laser-induced DNA damage. hACF1 is also required for a tight checkpoint that is induced upon replication fork collapse. ACF1-depleted cells that are challenged with aphidicolin enter mitosis despite persistence of lesions and accumulate breaks in metaphase chromosomes. hACF1-containing remodellers emerge as global facilitators of the cellular response to a variety of different types of DNA damage.
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