DNA Damage Checkpoint Signaling Pathway Research Articles

Bismuth Sulfide Nanoflowers Facilitated miR339 Delivery to Overcome Stemness and Radioresistance through Ubiquitin-Specific Peptidase 8 in Esophageal Cancer.

Despite the superior efficacy of radiotherapy in esophageal squamous cell carcinoma (ESCC), radioresistance by cancer stem cells (CSCs) leads to recurrence, metastasis, and treatment failure. Therefore, it is necessary to develop CSC-based therapies to enhance radiotherapy. miR-339-5p (miR339) is involved in stem cell division and DNA damage checkpoint signaling pathways based on ESCC cohort. miR339 inhibited ESCC cell stemness and enhanced radiation-induced DNA damage by targeting USP8, suggesting that it acts as a potential CSC regulator and radiosensitizer. Considering the limited circulating periods and poor tumor-targeting ability of miRNA, a multifunctional nanoplatform based on bismuth sulfide nanoflower (Bi@PP) is developed to efficiently deliver miR339 and improve radioresistance. Intriguingly, Bi@PP encapsulates more miR339 owing to their flower-shaped structure, delivering more than 1000-fold miR339 into cells, superior to free miR339 alone. Besides being used as a carrier, Bi@PP is advantageous for dynamically monitoring the distribution of delivered miR339 in vivo while simultaneously inhibiting tumor growth. Additionally, Bi@PP/miR339 can significantly enhance radiotherapy efficacy in patient-derived xenograft models. This multifunctional platform, incorporating higher miRNA loading capacity, pH responsiveness, hypoxia relief, and CT imaging, provides another method to promote radiosensitivity and optimize ESCC treatment.

Functional interplay between the oxidative stress response and DNA damage checkpoint signaling for genome maintenance in aerobic organisms.

The DNA damage checkpoint signaling pathway is a highly conserved surveillance mechanism that ensures genome integrity by sequential activation of protein kinase cascades. In mammals, the main pathway is orchestrated by two central sensor kinases, ATM and ATR, that are activated in response to DNA damage and DNA replication stress. Patients lacking functional ATM or ATR suffer from ataxia-telangiectasia (A-T) or Seckel syndrome, respectively, with pleiotropic degenerative phenotypes. In addition to DNA strand breaks, ATM and ATR also respond to oxidative DNA damage and reactive oxygen species (ROS), suggesting an unconventional function as regulators of intracellular redox status. Here, we summarize the multiple roles of ATM and ATR, and of their orthologs in Saccharomyces cerevisiae, Tel1 and Mec1, in DNA damage checkpoint signaling and the oxidative stress response, and discuss emerging ideas regarding the possible mechanisms underlying the elaborate crosstalk between those pathways. This review may provide new insights into the integrated cellular strategies responsible for maintaining genome stability in eukaryotes with a focus on the yeast model organism.

The inhibition of checkpoint activation by telomeres does not involve exclusion of dimethylation of histone H4 lysine 20 (H4K20me2).

DNA double-strand (DSBs) breaks activate the DNA damage checkpoint machinery to pause or halt the cell cycle. Telomeres, the specific DNA-protein complexes at linear eukaryotic chromosome ends, are capped DSBs that do not activate DNA damage checkpoints. This "checkpoint privileged" status of telomeres was previously investigated in the yeast Schizosaccharomyces pombe lacking the major double-stranded telomere DNA binding protein Taz1. Telomeric DNA repeats in cells lacking Taz1 are 10 times longer than normal and contain single-stranded DNA regions. DNA damage checkpoint proteins associate with these damaged telomeres, but the DNA damage checkpoint is not activated. This severing of the DNA damage checkpoint signaling pathway was reported to stem from exclusion of histone H4 lysine 20 dimethylation (H4K20me2) from telomeric nucleosomes in both wild type cells and cells lacking Taz1. However, experiments to identify the mechanism of this exclusion failed, prompting our re-evaluation of H4K20me2 levels at telomeric chromatin. In this short report, we used an extensive series of controls to identify an antibody specific for the H4K20me2 modification and show that the level of this modification is the same at telomeres and internal loci in both wild type cells and those lacking Taz1. Consequently, telomeres must block activation of the DNA Damage Response by another mechanism that remains to be determined.

Mechanisms of a novel anticancer therapeutic strategy involving atmospheric pressure plasma-mediated apoptosis and DNA strand break formation.

Atmospheric pressure plasma has been developed for a variety of biomedical applications due to its chemically reactive components. Recently, the plasma has emerged as a promising novel cancer therapy based on its ability to selectively ablate cancer cells while leaving normal cells essentially unaffected. The therapeutic effect of plasma is attributed to intracellular generation of reactive oxygen/nitrogen species (ROS/RNS) leading to mitochondria-mediated apoptosis and to activation of the DNA damage checkpoint signaling pathway via severe DNA strand break formation. However, the biochemical mechanisms responsible for appropriate activation of these physiological events and which pathway is more crucial for plasma-mediated cytotoxicity have not been clarified. Understanding the molecular link between ROS/RNS-mediated apoptosis and DNA damage-involved chromosome instability is critical for the development of more efficacious therapeutic strategies for selective killing of diverse cancer cells.

The Expression of DNA Damage Checkpoint Proteins and Prognostic Implication in Metastatic Brain Tumors

The most important therapeutic tool in brain metastasis is radiation therapy. However, resistance to radiation is a possible cause of recurrence or treatment failure. Recently, DNA damage checkpoint signaling pathway activation after irradiation has received increasing attention. The association between the expression levels and survival outcome was evaluated to find possible therapeutic targets in brain metastasis. Radiosensitivity of human non-small cell lung cancer cell lines was determined by checking their viability after treatment with varying doses of ionizing radiation (IR). The expression of DNA checkpoint proteins was analyzed by Western blots and immunohistochemistry. On the basis of the clinical data for the patients, the association between the expression of the components and patients' survival was investigated. The expression levels of TopBP1 and phosphorylated Chk1 (P-Chk1) protein were higher in radioresistant lung cancer cell lines compared to radiosensitive cell lines. We previously assessed radiation survival of lung cancer cell lines after treating them with Chk1 inhibitor, AZD7762. AZD7762 significantly sensitized both radioresistant and radiosensitive cells to IR. We also observed a strong inverse relationship between progression-free survival (PFS) and expression level of P-Chk1 and TopBP1. This study, which is the first clinical report that connects DNA damage checkpoints and prognosis of brain metastasis, supports these two proteins to be promising targets for overcoming the radioresistance in brain metastasis.

The DNA Damage Response Kinases DNA-dependent Protein Kinase (DNA-PK) and Ataxia Telangiectasia Mutated (ATM) Are Stimulated by Bulky Adduct-containing DNA

A variety of environmental, carcinogenic, and chemotherapeutic agents form bulky lesions on DNA that activate DNA damage checkpoint signaling pathways in human cells. To identify the mechanisms by which bulky DNA adducts induce damage signaling, we developed an in vitro assay using mammalian cell nuclear extract and plasmid DNA containing bulky adducts formed by N-acetoxy-2-acetylaminofluorene or benzo(a)pyrene diol epoxide. Using this cell-free system together with a variety of pharmacological, genetic, and biochemical approaches, we identified the DNA damage response kinases DNA-dependent protein kinase (DNA-PK) and ataxia telangiectasia mutated (ATM) as bulky DNA damage-stimulated kinases that phosphorylate physiologically important residues on the checkpoint proteins p53, Chk1, and RPA. Consistent with these results, purified DNA-PK and ATM were directly stimulated by bulky adduct-containing DNA and preferentially associated with damaged DNA in vitro. Because the DNA damage response kinase ATM and Rad3-related (ATR) is also stimulated by bulky DNA adducts, we conclude that a common biochemical mechanism exists for activation of DNA-PK, ATM, and ATR by bulky adduct-containing DNA.

Ataxia telangiectasia-mutated-Rad3-related DNA damage checkpoint signaling pathway triggered by hepatitis B virus infection

To explore whether acute cellular DNA damage response is induced upon hepatitis B virus (HBV) infection and the effects of the HBV infection. We incubated HL7702 hepatocytes with HBV-positive serum, mimicking a natural HBV infection process. We used immunoblotting to evaluate protein expression levels in HBV-infected cells or in non-infected cells; immunofluorescence to show ATR foci ands Chk1 phosphorylation foci formation; flow cytometry to analyze the cell cycle and apoptosis; ultraviolet (UV) radiation and ionizing radiation (IR)-treated cells to mimic DNA damage; and Trypan blue staining to count the viable cells. We found that HBV infection induced an increased steady state of ATR protein and increased phosphorylation of multiple downstream targets including Chk1, p53 and H2AX. In contrast to ATR and its target, the phosphorylated form of ATM at Ser-1981 and its downstream substrate Chk2 phosphorylation at Thr-68 did not visibly increase upon infection. However, the level of Mre11 and p21 were reduced beginning at 0.5 h after HBV-positive serum addition. Also, HBV infection led to transient cell cycle arrest in the S and the G2 phases without accompanying increased apoptosis. Research on cell survival changes upon radiation following HBV infection showed that survival of UV-treated host cells was greatly increased by HBV infection, owing to the reduced apoptosis. Meanwhile, survival of IR-treated host cells was reduced by HBV infection. HBV infection activates ATR DNA damage response to replication stress and abrogates the checkpoint signaling controlled by DNA damage response.

Reoxygenation following hypoxia activates DNA-damage checkpoint signaling pathways that suppress cell-cycle progression in cultured human lymphocytes

Cellular responses to DNA damage after hypoxia and reoxygenation (H/R) were examined in human lymphocytes. Cultured lymphocytes exposed to H/R showed a lower cytokinesis block proliferation index and a higher frequency of micronuclei in comparison to control cells. Western blots showed that H/R exposure induced p53 expression; however, p21 and Bax expression did not increase, indicating that H/R did not affect p53 transactivational activity. Phosphorylation of p53 (Ser15), Chk1 (Ser345), and Chk2 (Thr68) was also observed, suggesting that H/R activates p53 through checkpoint signals. In addition, H/R exposure caused the phosphorylation and negative regulation of Cdc2 and Cdc25C, proteins that are involved in cell-cycle arrest at the G2/M checkpoint. The S-phase checkpoint, regulated by the ATM-p95/NBS1–SMC1 pathway, was also triggered in H/R-exposed lymphocytes. These results demonstrate that H/R exposure triggers checkpoint signaling and induces cell-cycle arrest in cultured human lymphocytes.

Accumulation of hRad9 protein in the nuclei of nonsmall cell lung carcinoma cells

DNA damage sensor proteins have received much attention as upstream components of the DNA damage checkpoint signaling pathway that are required for cell cycle control and the induction of apoptosis. Deficiencies in these proteins are directly linked to the accumulation of gene mutations, which can induce cellular transformation and result in malignant disease. Using 48 sets of tumor tissue specimens and peripheral normal lung tissue specimens from 48 patients with nonsmall cell lung carcinoma (NSCLC) who underwent surgery, the authors investigated the expression of hRad9 protein, a member of the human DNA damage sensor family, using immunohistochemical and Western blot analyses. Immunohistochemical analysis detected the accumulation of hRad9 in the nuclei of tumor cells in 16 tumor tissue specimens, (33% of tumor tissue specimens examined). Western blot analysis also revealed elevated levels of phosphorylated hRad9 protein in NSCLC cells that was accompanied by the detection of phosphorylated Chk1, a protein kinase that regulates the downstream signaling of the DNA damage checkpoint pathway. Furthermore, strong expression of hRad9 was correlated with an increase in Ki-67 expression index in the tumor cells that were examined. The findings made in the current study suggest that Rad9 expression may play an important role in cell cycle control in NSCLC cells and may influence NSCLC cell phenotype.

Xenopus cell-free extracts to study the DNA damage response.

Cell-free systems derived from Xenopus eggs represent a powerful tool, intermediate between in vitro and in vivo model systems. Here, we describe protocols to prepare cell-free extracts recapitulating several aspects of the DNA damage response, including the DNA damage-dependent activation of ATM/ATR protein kinases and several DNA damage checkpoint signaling pathways that inhibit initiation of DNA replication. We provide protocols to prepare cell-free extracts, DNA templates, protein kinase substrates, and to perform checkpoint assays. In addition, we describe related methods that provide useful readouts of the DNA damage response.

DNA damage sensor protein hRad9, a novel molecular target for lung cancer treatment

DNA damage sensor proteins are recognized as upstream components of the DNA damage checkpoint signaling pathway and are required for cell cycle control and the induction of apoptosis. hRad9 plays an important role as an upstream regulator of checkpoint signaling. In our previous studies, we confirmed the significant accumulation of hRad9 in the nuclei of tumor cells in surgically-resected non-small cell lung cancer (NSCLC) specimens. We also found that the capacity to produce a functional hRad9 protein was intact in lung cancer cells, a finding which suggests that hRad9 would be a vital component in the pathways that lead to the survival and progression of NSCLC. Small interfering RNA targeting hRad9 was transfected into human lung adenocarcinoma A549 and PC3 cells. After the hRad9 knockdown, the cytotoxicity of the transfected cells was measured by a neutral red uptake test, and the G2-M arrest of irradiated cells was examined by flow cytometry. Significant cytotoxicity was observed in the cancer cells in which hRad9 expression was down-regulated. We also detected the inhibition of Chk1 phosphorylation by Western blot analysis. This suggested that hRad9 silencing leads to the impairment of the DNA damage checkpoint signaling pathway in tumor cells. Flow cytometry indicated a reduced population of cells in the G2-M phase, an observation consistent with the findings of several studies that indicated that hRad9 is necessary for G2-M arrest. In conclusion, the current study demonstrated that RNA interference targeting hRad9 in cancer cells leads to the impairment of the DNA damage checkpoint signaling pathway, which appears to be essential for maintaining tumor cell proliferation, and induces cell death. Therefore, hRad9 may be a novel molecular target for lung cancer treatment.