05-740 Sigma-AldrichAnti-phospho-ATM (Ser1981) Antibody, clone 10H11.E12

Therapeutic resistance in glioblastoma multiforme (GBM) has been linked to a subpopulation of cells with stem cell-like properties, the glioma stem cells (GSCs), responsible for cancer progression and recurrence. This study investigated the in vitro cytotoxicity of three chemotherapeutics, temozolomide (TMZ), doxorubicin (Dox) and paclitaxel (PTX) on glioma cell lines, by analyzing the molecular mechanisms leading to DNA repair and cell resistance, or to cell death. The drugs were tested on 16 GBM cell lines, grown as neurospheres (NS) or adherent cells (AC), by studying DNA damage occurrence by Comet assay, the expression by immunofluorescence and western blotting of checkpoint/repair molecules and apoptosis. The three drugs were able to provoke a genotoxic injury and to inhibit dose- and time-dependently cell proliferation, more evidently in AC than in NS. The first cell response to DNA damage was the activation of the damage sensors (p-ATM, p-53BP1, γ-H2AX), followed by repair effectors; the expression of checkpoint/repair molecules appeared higher in NS than in AC. The non-homologous repair pathway (NHEJ) seemed more involved than the homologous one (HR). Apoptosis occurred after long treatment times, but only a small percentage of cells in NS underwent death, even at high drug concentration, whereas most cells survived in a quiescent state and resumed proliferation after drug removal. In tumor specimens, checkpoint/repair proteins were constitutively expressed in GBMs, but not in low-grade gliomas.

Telomeric repeat binding factor 2 (TRF2) is essential for telomere maintenance and has been implicated in DNA damage response and aging. Telomere dysfunction induced by TRF2 inhibition can accelerate cellular senescence in human fibroblasts. While previous work has demonstrated that a variety of factors can regulate TRF2 expression transcriptionally and post-translationally, whether microRNAs (miRNAs) also participate in post-transcriptionally modulating TRF2 levels remains largely unknown. To better understand the regulatory pathways that control TRF2, we carried out a large-scale luciferase reporter screen using a miRNA expression library and identified four miRNAs that could target human TRF2 and significantly reduce the level of endogenous TRF2 proteins. In particular, our data revealed that miR-23a could directly target the 3' untranslated region (3'UTR) of TRF2. Overexpression of miR-23a not only reduced telomere-bound TRF2 and increased telomere dysfunction-induced foci (TIFs), but also accelerated senescence of human fibroblast cells, which could be rescued by ectopically expressed TRF2. Our findings demonstrate that TRF2 is a specific target of miR-23a, and uncover a previously unknown role for miR-23a in telomere regulation and cellular senescence.

DNA damage repair (DDR) is an orchestrated process encompassing the injury detection to its complete resolution. DNA double-strand break lesions are repaired mainly by two distinct mechanisms: the error-free homologous recombination (HR) and the error-prone non-homologous end-joining. Galectin-3 (GAL3) is the unique member of the chimeric galectins subfamily and is reported to be involved in several cancer development and progression related events. Recently our group described a putative protein interaction between GAL3 and BARD1, the main partner of breast and ovarian cancer susceptibility gene product BRCA1, both involved in HR pathway. In this report we characterized GAL3/BARD1 protein interaction and evaluated the role of GAL3 in DDR pathways using GAL3 silenced human cells exposed to different DNA damage agents. In the absence of GAL3 we observed a delayed DDR response activation, as well as a decrease in the G 2/M cell cycle checkpoint arrest associated with HR pathway. Moreover, using a TAP-MS approach we also determined the protein interaction network of GAL3.

Wip1 (protein phosphatase Mg(2+)/Mn(2+)-dependent 1D, Ppm1d) is a nuclear serine/threonine protein phosphatase that is induced by p53 following the activation of DNA damage response (DDR) signaling. Ppm1d(-/-) mouse embryonic fibroblasts (MEFs) exhibit premature senescence under conventional culture conditions; however, little is known regarding the role of Wip1 in regulating cellular senescence. In this study, we found that even at a representative physiological concentration of 3% O2, Ppm1d(-/-) MEFs underwent premature cellular senescence that depended on the functional activation of p53. Interestingly, Ppm1d(-/-) MEFs showed increased H2AX phosphorylation levels without increased levels of reactive oxygen species (ROS) or DNA base damage compared with wild-type (Wt) MEFs, suggesting a decreased threshold for DDR activation or sustained DDR activation during recovery. Notably, the increased H2AX phosphorylation levels observed in Ppm1d(-/-) MEFs were primarily associated with S-phase cells and predominantly dependent on the activation of ATM. Moreover, these same phenotypes were observed when Wt and Ppm1d(-/-) MEFs were either transiently or chronically exposed to low levels of agents that induce replication-mediated double-stranded breaks. These findings suggest that Wip1 prevents the induction of cellular senescence at physiological oxygen levels by attenuating DDR signaling in response to endogenous double-stranded breaks that form during DNA replication.

The prevention of persistent human immunodeficiency virus type 1 (HIV-1) infection requires the clarification of the mode of viral transduction into resting macrophages. Recently, DNA double-strand breaks (DSBs) were shown to enhance infection by D64A virus, which has a defective integrase catalytic activity (IN-CA). However, the mechanism by which DSBs upregulate viral transduction was unclear. Here we analyzed the roles of DSBs during IN-CA-independent viral transduction into macrophages.We used cellular systems with rare-cutting endonucleases and found that D64A virus integrated efficiently into the sites of artificially induced DSBs. This IN-CA-independent viral transduction was blocked by an inhibitor of ataxia telangiectasia mutated protein (ATM) but was resistant to raltegravir (RAL), an inhibitor of integrase activity during strand transfer. Moreover, Vpr, an accessory gene product of HIV-1, induced DSBs in resting macrophages and significantly enhanced the rate of IN-CA-independent viral transduction into macrophages with concomitant production of secondary viruses.DSBs contribute to the IN-CA-independent viral infection of macrophages, which is resistant to RAL. Thus, the ATM-dependent cellular pathway and Vpr-induced DNA damage are novel targets for preventing persistent HIV-1 infection.

Increasing evidence suggests that tumor-initiating cells (TICs), also called cancer stem cells, are partly responsible for resistance to DNA-damaging treatment. Here we addressed if such a phenotype may contribute to radio- and cisplatin resistance in non-small cell lung cancer (NSCLC). We showed that four out of eight NSCLC cell lines (H125, A549, H1299 and H23) possess sphere-forming capacity when cultured in stem cell media and three of these display elevated levels of CD133. Indeed, sphere-forming NSCLC cells, hereafter called TICs, showed a reduced apoptotic response and increased survival after irradiation (IR), as compared with the corresponding bulk cell population. Decreased cytotoxicity and apoptotic signaling manifested by diminished poly (ADP-ribose) polymerase (PARP) cleavage and caspase 3 activity was also evident in TICs after cisplatin treatment. Neither radiation nor cisplatin resistance was due to quiescence as H125 TICs proliferated at a rate comparable to bulk cells. However, TICs displayed less pronounced G2 cell cycle arrest and S/G2-phase block after IR and cisplatin, respectively. Additionally, we confirmed a cisplatin-refractory phenotype of H125 TICs in vivo in a mouse xenograft model. We further examined TICs for altered expression or activation of DNA damage repair proteins as a way to explain their increased radio- and/or chemotherapy resistance. Indeed, we found that TICs exhibited increased basal γH2AX (H2A histone family, member X) expression and diminished DNA damage-induced phosphorylation of DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia-mutated (ATM), Krüppel-associated protein 1 (KAP1) and monoubiquitination of Fanconi anemia, complementation group D2 (FANCD2). As a proof of principle, ATM inhibition in bulk cells increased their cisplatin resistance, as demonstrated by reduced PARP cleavage. In conclusion, we show that reduced apoptotic response, altered DNA repair signaling and cell cycle perturbations in NSCLC TICs are possible factors contributing to their therapy resistance, which may be exploited for DNA damage-sensitizing purposes.

When DNA replication is stalled at sites of DNA damage, a cascade of responses is activated in the cell to halt cell cycle progression and promote DNA repair. A pathway initiated by the kinase Ataxia teleangiectasia and Rad3 related (ATR) and its partner ATR interacting protein (ATRIP) plays an important role in this response. The Fanconi anemia (FA) pathway is also activated following genomic stress, and defects in this pathway cause a cancer-prone hematologic disorder in humans. Little is known about how these two pathways are coordinated. We report here that following cellular exposure to DNA cross-linking damage, the FA core complex enhances binding and localization of ATRIP within damaged chromatin. In cells lacking the core complex, ATR-mediated phosphorylation of two functional response targets, ATRIP and FANCI, is defective. We also provide evidence that the canonical ATR activation pathway involving RAD17 and TOPBP1 is largely dispensable for the FA pathway activation. Indeed DT40 mutant cells lacking both RAD17 and FANCD2 were synergistically more sensitive to cisplatin compared with either single mutant. Collectively, these data reveal new aspects of the interplay between regulation of ATR-ATRIP kinase and activation of the FA pathway.

Many tumor suppressors play an important role in the DNA damage pathway. Zinc finger protein 668 (ZNF668) has recently been identified as one of the potential tumor suppressors in breast cancer, but its function in DNA damage response is unknown. Herein, we report that ZNF668 is a regulator of DNA repair. ZNF668 knockdown impairs cell survival after DNA damage without affecting the ATM/ATR DNA-damage signaling cascade. However, recruitment of repair proteins to DNA lesions is decreased. In response to IR, ZNF668 knockdown reduces Tip60-H2AX interaction and impairs IR-induced histone H2AX hyperacetylation, thus impairing chromatin relaxation. Impaired chromatin relaxation causes decreased recruitment of repair proteins to DNA lesions, defective homologous recombination (HR) repair and impaired cell survival after IR. In addition, ZNF668 knockdown decreased RPA phosphorylation and its recruitment to DNA damage foci in response to UV. In both IR and UV damage responses, chromatin relaxation counteracted the impaired loading of repair proteins and DNA repair defects in ZNF668-deficient U2OS cells, indicating that impeded chromatin accessibility at sites of DNA breaks caused the DNA repair defects observed in the absence of ZNF668. Our findings suggest that ZNF668 is a key molecule that links chromatin relaxation with DNA damage response in DNA repair control.

The translationally controlled tumor protein (TCTP) is essential for survival by mechanisms that as yet are incompletely defined. Here we describe an important role of TCTP in response to DNA damage. Upon exposure of normal human cells to low-dose γ rays, the TCTP protein level was greatly increased, with a significant enrichment in nuclei. TCTP up-regulation occurred in a manner dependent on ataxia-telangiectasia mutated (ATM) kinase and the DNA-dependent protein kinase and was associated with protective effects against DNA damage. In chromatin of irradiated cells, coimmunoprecipitation experiments showed that TCTP forms a complex with ATM and γH2A.X, in agreement with its distinct localization with the foci of the DNA damage-marker proteins γH2A.X, 53BP1, and P-ATM. In cells lacking TCTP, repair of chromosomal damage induced by γ rays was compromised significantly. TCTP also was shown to interact with p53 and the DNA-binding subunits, Ku70 and Ku80, of DNA-dependent protein kinase. TCTP knockdown led to decreased levels of Ku70 and Ku80 in nuclei of irradiated cells and attenuated their DNA-binding activity. It also attenuated the radiation-induced G(1) delay but prolonged the G(2) delay. TCTP therefore may play a critical role in maintaining genomic integrity in response to DNA-damaging agents.

Activation-induced cytidine deaminase (AID) initiates DNA double-strand breaks (DSBs) in the IgH gene (Igh) to stimulate isotype class switch recombination (CSR), and widespread breaks in non-Igh (off-target) loci throughout the genome. Because the DSBs that initiate class switching occur during the G₁ phase of the cell cycle, and are repaired via end joining, CSR is considered a predominantly G₁ reaction. By contrast, AID-induced non-Igh DSBs are repaired by homologous recombination. Although little is known about the connection between the cell cycle and either induction or resolution of AID-mediated non-Igh DSBs, their repair by homologous recombination implicates post-G₁ phases. Coordination of DNA breakage and repair during the cell cycle is critical to promote normal class switching and prevent genomic instability. To understand how AID-mediated events are regulated through the cell cycle, we have investigated G₁-to-S control in AID-dependent genome-wide DSBs. We find that AID-mediated off-target DSBs, like those induced in the Igh locus, are generated during G₁. These data suggest that AID-mediated DSBs can evade G₁/S checkpoint activation and persist beyond G₁, becoming resolved during S phase. Interestingly, DSB resolution during S phase can promote not only non-Igh break repair, but also Ig CSR. Our results reveal novel cell cycle dynamics in response to AID-initiated DSBs, and suggest that the regulation of the repair of these DSBs through the cell cycle may ensure proper class switching while preventing AID-induced genomic instability.