Levels Of γH2AX Research Articles

Protective effect of borneol on the cutaneous toxicity of gilteritinib.

To investigate the effect of borneol on cutaneous toxicity of gilteritinib and to explore possible compounds that can intervene with the cutaneous toxicity. C57BL/6J male mice were given gilteritinib by continuous gavage for 28 d and the damage to keratinocytes in the skin tissues was observed with hematoxylin and eosin (HE) staining, TUNEL assay and immunohistochemistry. Human keratinocytes HaCaT were treated with gilteritinib, and cell death and morphological changes were examined by SRB staining and microscopy; apoptosis of HaCaT cells was examined by Western blotting, flow cytometry with propidium iodide/AnnexinⅤ double staining and immunofluorescence; the accumulation of cellular reactive oxygen species (ROS) was examined by flow cytometry with DCFH-DA. Compounds that can effectively intervene the cutaneous toxicity of gilteritinib were screened from a natural compound library using SRB method, and the intervention effect of borneol on gilteritinib cutaneous toxicity was further investigated in HaCaT cells and C57BL/6J male mice. In vivo studies showed pathological changes in the skin with apoptosis of keratinocytes in the stratum spinosum and stratum granulosum in the modeling group. Invitro studies showed apoptosis of HaCaT cells, significant up-regulation of cleaved poly (ADP-ribose) polymerase (c-PARP) and gamma-H2A histone family member X (γ-H2AX) levels, and increased accumulation of ROS in gilteritinib-modeled skin keratinocytes compared with controls. Screening of the natural compound library revealed that borneol showed excellent intervention effects on the death of HaCaT cells. In vitro, cell apoptosis was significantly reduced in the borneol+gilteritinib group compared to the gilteritinib control group. The levels of c-PARP, γ-H2AX and ROS in cells were significantly decreased. In vivo, borneol alleviated gilteritinib-induced skin pathological changes and skin cell apoptosis in mice. Gilteritinib induces keratinocytes apoptosis by causing intracellular ROS accumulation, resulting in cutaneous toxicity. Borneol can ameliorate the cutaneous toxicity of gilteritinib by reducing the accumulation of ROS and apoptosis of keratinocytes in the skin tissue.

Arthrospira platensis Nanoparticles Mitigate Aging-Related Oxidative Injured Brain Induced by D-galactose inRats Through Antioxidants, Anti-Inflammatory, and MAPK Pathways.

Loss of normal function is an inevitable effect of aging. Several factors contribute to the aging process, including cellular senescence and oxidative stress. We investigate how Arthrospira platensis Nanoparticles (NSP) protect against aging injury induced by d-galactose (D-gal) in the rat. So, we subcutaneously (S/C) injected D-gal at 200 mg/kg BW to see if Arthrospira platensis Nanoparticles (NSP) might protect against the oxidative changes generated by D-gal. NSP (0.5 mg/kg body weight once daily by gastric gavage) was given to all groups apart from the control and D-gal groups. The d-gal + NSP group was supplemented with 200 mg of D-gal per kg BW once a day and NSP 0.5 mg/kg BW given orally for 45 days. Biochemical, mRNA expression, and histological investigations of brain tissues were used to evaluate the oxidative alterations caused by d-gal and the protective role of NSP. Our data demonstrated that d-gal was causing significant reductions in relativebrain and body weight with increased malondialdehyde (MDA) and redox oxygen species (ROS) levels and increases in serum creatine phosphokinase (CPK) and creatine phosphokinase isoenzyme BB (CPK-BB) with marked decreases in the level of antioxidant enzyme activity in the brain and acetylcholinesterase activity augmented with a phosphorylated H2A histone family member X (γ-H2AX) level increased. The D-gal group had considerably higher phosphorylated p38 mitogen-activated protein kinases (P38MAPK) and C-Jun N-terminal (JNK) kinases. The d-gal administration stimulates the apoptotic gene expression by downregulating the brain superoxide dismutase (SOD), catalase (CAT), and nuclear factor erythroid 2-related factor 2 (Nrf2). The NSP administration saved these parameters in the direction of the control. The brain histopathologic and immunohistochemistry analysis findings support our findings on NSP's protective role. The NSP may be a promising natural protective compound that can prevent aging and preserve health.

EYA4 promotes breast cancer progression and metastasis through its role in replication stress avoidance

The Eyes Absent (EYA) family of proteins is an atypical group of four dual-functioning protein phosphatases (PP), which have been linked to many vital cellular processes and organogenesis pathways. The four family members of this PP family possess transcriptional activation and phosphatase functions, with serine/threonine and tyrosine phosphatase domains. EYA4 has been associated with several human cancers, with tumor-suppressing and tumor-promoting roles. However, EYA4 is the least well-characterized member of this unique family of PP, with its biological functions and molecular mechanisms in cancer progression, particularly in breast cancer, still largely unknown. In the present study, we found that the over-expression of EYA4 in breast tissue leads to an aggressive and invasive breast cancer phenotype, while the inhibition of EYA4 reduced tumorigenic properties of breast cancer cells in vitro and in vivo. Cellular changes downstream of EYA4, including cell proliferation and migration, may explain the increased metastatic power of breast cancer cells over-expressing EYA4. Mechanistically, EYA4 prevents genome instability by inhibiting the accumulation of replication-associated DNA damage. Its depletion results in polyploidy as a consequence of endoreplication, a phenomenon that can occur in response to stress. The absence of EYA4 leads to spontaneous replication stress characterized by the activation of the ATR pathway, sensitivity to hydroxyurea, and accumulation of endogenous DNA damage as indicated by increased γH2AX levels. In addition, we show that EYA4, specifically its serine/threonine phosphatase domain, plays an important and so far, unexpected role in replication fork progression. This phosphatase activity is essential for breast cancer progression and metastasis. Taken together, our data indicate that EYA4 is a novel potential breast cancer oncogene that supports primary tumor growth and metastasis. Developing therapeutics aimed at the serine/threonine phosphatase activity of EYA4 represents a robust strategy for killing breast cancer cells, to limit metastasis and overcome chemotherapy resistance caused by endoreplication and genomic rearrangements.

Erythritol Can Inhibit the Expression of Senescence Molecules in Mouse Gingival Tissues and Human Gingival Fibroblasts.

Oral aging causes conditions including periodontal disease. We investigated how the sugar alcohol erythritol, which has anti-caries effects, impacts aging periodontal tissues and gingival fibroblasts in mice and humans in vivo and in vitro. Mice were classified into three groups: control groups of six-week-old (YC) and eighteen-month-old mice (AC) and a group receiving 5% w/w erythritol water for 6 months (AE). After rearing, RNA was extracted from the gingiva, and the levels of aging-related molecules were measured using PCR. Immunostaining was performed for the aging markers p21, γH2AX, and NF-κB p65. p16, p21, γH2AX, IL-1β, and TNFα mRNA expression levels were higher in the gingiva of the AC group than in the YC group, while this enhanced expression was significantly suppressed in AE gingiva. NF-κB p65 expression was high in the AC group but was strongly suppressed in the AE group. We induced senescence in cultured human gingival fibroblasts using H2O2 and lipopolysaccharide before erythritol treatment, which reduced elevated senescence-related marker (p16, p21, SA-β-gal, IL-1β, and TNFα) expression levels. Knockdown of PFK or PGAM promoted p16 and p21 mRNA expression, but erythritol subsequently rescued pyruvate production. Overall, intraoral erythritol administration may prevent age-related oral mucosal diseases.

γ-H2AX and phospho-ATM enzyme-linked immunosorbent assays as biodosimetry methods for radiation exposure assessment: a pilot study.

In the event of a radiological incident, a fast and accurate biological dosimetry (biodosimetry) method for evaluating people who have been potentially exposed to ionising radiation is crucial. Among the many biodosimetry methods available, the immunodetection of phosphorylated H2AX (γ-H2AX) stands as a promising method to be used in the triage of patients exposed to radiation. Currently, the most common way to measure γ-H2AX levels is through fluorescence microscopy. In this pilot study, we assessed the feasibility of using an enzyme-linked immunosorbent assay (ELISA) for quantifying γ-H2AX for biodosimetry purposes. Moreover, the usefulness of measuring phosphorylated ATM (pATM) levels through ELISA for biodosimetry was also evaluated. Blood samples were obtained from three male donors (38y) and were irradiated with 60Co (0, 1, 2 and 6Gy). Peripheral blood mononuclear cells (PBMCs) were isolated and lysed before measuring γ-H2AX, total H2AX protein and pATM using ELISA kits. The dicentric chromosome assay (DCA) using whole blood was also performed for comparison. Data from all donors at each dose were pooled before statistical analysis. The ratio of γ-H2AX/total H2AX and pATM levels increased in a radiation-dose-dependent manner. The average γ-H2AX/total H2AX ratios were 0.816±0.219, 0.830±0.685, 1.276±1.151 and 1.606±1.098, whereas the average levels of pATM were 59.359±3.740, 63.366±0.840, 66.273±2.603 and 69.936±4.439, in PBMCs exposed to 0, 1, 2 and 6Gy, respectively. The linear-quadratic dose-response calibration curve for DCA was Y=0.0017 (±0.0010)+0.0251 (±0.0142)×D+0.0342 (±0.0039)×D2 $\boldsymbol{Y}=\mathbf{0.0017}\left(\pm \mathbf{0.0010}\right)+\mathbf{0.0208}\left(\pm \mathbf{0.0218}\right)\times \boldsymbol{D}+\mathbf{0.0350}\left(\pm \mathbf{0.0050}\right)\times{\boldsymbol{D}}^{\mathbf{2}}$. Overall, despite a large variability in the ratio of γ-H2AX/total H2AX among donors, the present study revealed the suitability of using the ratio of γ-H2AX/total H2AX and pATM for biodosimetry. Still, more research with a larger group of subjects is necessary to construct a reliable calibration curve for the ratio of γ-H2AX/total H2AX and pATM levels for biodosimetry.

Astaxanthin attenuated cigarette smoke extract-induced apoptosis via decreasing oxidative DNA damage in airway epithelium

Chronic obstructive pulmonary disease (COPD) is a lung inflammatory disease that is associated with environmental allergic component exposure. Cigarette smoke is an environmental toxicant that induces lung malfunction leading to various pulmonary diseases. Astaxanthin (AST) is a carotenoid that shows antioxidant and anti-inflammatory activities which might be a promising candidate for COPD therapy. In this study, we released that AST could attenuate cigarette smoke-induced DNA damage and apoptosis in vivo and in vitro. AST administration ameliorated cigarette smoke extract (CSE)-induced activation of Caspase-3 and apoptosis. Pretreated mice with AST significantly decrease CSE-induced DNA damage which shows lower nuclear γ-H2AX level. AST treatment also dramatically reduces the production of intracellular reactive oxygen species (ROS) by suppressing the expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme 4 (NOX4) and dual oxidase 1 (DUOX1). Taken together, this study suggested that AST can decrease CSE-induced DNA damage and apoptosis by inhibiting NOX4/DUOX1 expression that promotes ROS generation. AST may be a potential protective agent against CSE-associated lung disease that is worth in-depth investigation.

OCT1-dependent uptake of structurally diverse pyrrolizidine alkaloids in human liver cells is crucial for their genotoxic and cytotoxic effects

Pyrrolizidine alkaloids (PAs) are important plant hepatotoxins, which occur as contaminants in plant-based foods, feeds and phytomedicines. Numerous studies demonstrated that the genotoxicity and cytotoxicity of PAs depend on their chemical structure, allowing for potency ranking and grouping. Organic cation transporter-1 (OCT1) was previously shown to be involved in the cellular uptake of the cyclic PA diesters monocrotaline, retrorsine and senescionine. However, little is known about the structure-dependent transport of PAs. Therefore, we investigated the impact of OCT1 on the uptake and toxicity of three structurally diverse PAs (heliotrine, lasiocarpine and riddelliine) differing in their degree and type of esterification in metabolically competent human liver cell models and hamster fibroblasts. Human HepG2-CYP3A4 liver cells were exposed to the respective PA in the presence or absence of the OCT1-inhibitors d-THP and quinidine, revealing a strongly attenuated cytotoxicity upon OCT1 inhibition. The same experiments were repeated in V79-CYP3A4 hamster fibroblasts, confirming that OCT1 inhibition prevents the cytotoxic effects of all tested PAs. Interestingly, OCT1 protein levels were much lower in V79-CYP3A4 than in HepG2-CYP3A4 cells, which correlated with their lower susceptibility to PA-induced cytotoxicity. The cytoprotective effect of OCT1 inhibiton was also demonstrated in primary human hepatocytes following PA exposure. Our experiments further showed that the genotoxic effects triggered by the three PAs are blocked by OCT1 inhibition as evidenced by strongly reduced γH2AX and p53 levels. Consistently, inhibition of OCT1-mediated uptake suppressed the activation of the DNA damage response (DDR) as revealed by decreased phosphorylation of checkpoint kinases upon PA treatment. In conclusion, we demonstrated that PAs, independent of their degree of esterification, are substrates for OCT1-mediated uptake into human liver cells. We further provided evidence that OCT1 inhibition prevents PA-triggered genotoxicity, DDR activation and subsequent cytotoxicity. These findings highlight the crucial role of OCT1 together with CYP3A4-dependent metabolic activation for PA toxicity.

Multiparametric in vitro genotoxicity assessment of different variants of amorphous silica nanomaterials in rat alveolar epithelial cells

The hazard posed to human health by inhaled amorphous silica nanomaterials (aSiO2 NM) remains uncertain. Herein, we assessed the cyto- and genotoxicity of aSiO2 NM variants covering different sizes (7, 15, and 40 nm) and surface modifications (unmodified, phosphonate-, amino- and trimethylsilyl-modified) on rat alveolar epithelial (RLE-6TN) cells. Cytotoxicity was evaluated at 24 h after exposure to the aSiO2 NM variants by the lactate dehydrogenase (LDH) release and WST-1 reduction assays, while genotoxicity was assessed using different endpoints: DNA damage (single- and double-strand breaks [SSB and DSB]) by the comet assay for all aSiO2 NM variants; cell cycle progression and γ-H2AX levels (DSB) by flow cytometry for those variants that presented higher cytotoxic and DNA damaging potential. The variants with higher surface area demonstrated a higher cytotoxic potential (SiO2_7, SiO2_15_Unmod, SiO2_15_Amino, and SiO2_15_Phospho). SiO2_40 was the only variant that induced significant DNA damage on RLE-6TN cells. On the other hand, all tested variants (SiO2_7, SiO2_15_Unmod, SiO2_15_Amino, and SiO2_40) significantly increased total γ-H2AX levels. At high concentrations (28 µg/cm2), a decrease in G0/G1 subpopulation was accompanied by a significant increase in S and G2/M sub-populations after exposure to all tested materials except for SiO2_40 which did not affect cell cycle progression. Based on the obtained data, the tested variants can be ranked for its genotoxic DNA damage potential as follows: SiO2_7 = SiO2_40 = SiO2_15_Unmod > SiO2_15_Amino. Our study supports the usefulness of multiparametric approaches to improve the understanding on NM mechanisms of action and hazard prediction.

Early Detection of Genotoxic Hepatocarcinogens in Rats Using γH2AX and Ki-67: Prediction by Machine Learning.

Direct DNA double-strand breaks result in phosphorylation of H2AX, a variant of the histone H2 protein. Phosphorylated H2AX (γH2AX) may be a potential indicator in the evaluation of genotoxicity and hepatocarcinogenicity. In this study, γH2AX and Ki-67 were detected in the short-term responses (24 hours after chemical administration) to classify genotoxic hepatocarcinogens (GH) from non-GH chemicals. One hundred and thirty-five 6-week-old Crl: CD(SD) (SPF) male rats were treated with 22 chemicals including 11 GH and 11 non-GH, sacrificed 24 hours later, and immunostained with γH2AX and Ki-67. Positivity rates of these markers were measured in the 3 liver ZONEs 1-3; portal, lobular, and central venous regions. These values were input into three machine learning models -Naïve Bayes, Random Forest, and k-Nearest Neighbor to classify GH and non-GH using a 10-fold cross-validation method. All 11 and 10 out of 11 GH caused significant increase in γH2AX and Ki-67 levels, respectively (P < 0.05). Of the three machine learning models, Random Forest performed the best. GH were identified with 95.0% sensitivity (76/80 GH treated rats), 90.9% specificity (50/55 non-GH treated rats), and 90.0% overall correct response rate using γH2AX staining, and 96.2% sensitivity (77/80), 81.8% specificity (45/55), and 90.4% overall correct response rate using Ki-67 labeling. Random Forest model using γH2AX and Ki-67 could independently predict GH in the early stage with high accuracy.

The effects of PKI-402 on breast tumor models’ radiosensitivity via dual inhibition of PI3K/mTOR

Purpose PI3K/Akt/mTOR pathway activation causes relapse and resistance after radiotherapy in breast cancer (BC). We aimed to radiosensitize BC cell lines to irradiation (IR) by PKI-402, a dual PI3K/mTOR inhibitor. Methods We performed cytotoxicity, clonogenicity, hanging drop, apoptosis and double-strand break detection, and phosphorylation of 16 essential proteins involved in the PI3K/mTOR pathway. Results Our findings showed that PKI-402 has cytotoxic efficiency in all cell lines. Clonogenic assay results showed that PKI-402 plus IR inhibited the colony formation ability of MCF-7 and breast cancer stem cell lines. Results showed that PKI-402 plus IR causes more apoptotic cell death than IR alone in the MCF-7 cells but did not cause significant changes in the MDA-MB-231. γ-H2AX levels were increased in MDA-MB-231 in PKI-402 plus IR groups, whereas we did not observe any apoptotic and γ-H2AX induction in BCSCs and MCF-10A cells in all treatment groups. Some pivotal phosphorylated proteins of the PI3K/AKT pathway decreased, several proteins increased and others did not change. Conclusion In conclusion, if the combined use of PKI-402 with radiation is supported by in vivo studies, it can contribute to the treatment options and the course of the disease.

Methodological and Cellular Factors Affecting the Magnitude of Breast Cancer and Normal Cell Radiosensitization Using Gold Nanoparticles.

Breast cancer (BC) is the most common malignant tumor in women, which most often originates from the epithelial tissue of the breast gland. One of the most recommended kinds of treatment is radiotherapy (RT), but irradiation (IR) can affect not only the cancer tumor but also the healthy tissue around it. Au nanoparticles (AuNPs) were proposed as a radiosensitizing agent for RT which would allow for lower radiation doses, reducing the negative radiation effects on healthy tissues. The main objective of the study is to assess the dependence on the radiosensitivity of BC (MDA-MB-231) and normal mammary gland epithelial cells (MCF12A) to ionizing radiation, caused by functionalized AuNPs under diverse conditions. The viability, uptake, reactive oxygen species induction, and mitochondrial membrane potential in cells were analyzed applying a time and concentration-dependent manner. After different incubation times with AuNPs, cells were exposed to 2 Gy. The determination of radiation effect in combination with AuNPs was investigated using the clonogenic assay, p53, and γH2AX level, as well as, Annexin V staining. Our results highlighted the strong need for assessing the experimental conditions' optimization before the AuNPs will be implemented with IR. Moreover, results indicated that AuNPs did not act universally in cells. AuNPs could be a promising tool as a radiotherapy sensitizing agent, but it should be specified and deeply investigated under what conditions it will be applied taking into consideration not only AuNPs modifications but also the model and experimental conditions.

Detection of DNA Double-Stranded Breaks in Mouse Oocytes

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain arrested at the prophase of meiosis I. The quiescent state may last for years until the oocytes receive a stimulus to grow and obtain the competency to resume meiosis. This protracted state of arrest makes them extremely susceptible to accumulating DNA-damaging insults, which affect the genetic integrity of the female gametes and, therefore, the genetic integrity of the future embryo. Consequently, the development of an accurate method to detect DNA damage, which is the first step for the establishment of DNA damage response mechanisms, is of vital importance. This paper describes a common protocol to test the presence and progress of DNA damage in prophase-arrested oocytes during a period of 20 h. Specifically, we dissect mouse ovaries, retrieve the cumulus-oocyte complexes (COCs), remove the cumulus cells from the COCs, and culture the oocytes in Μ2 medium containing 3-isobutyl-1-methylxanthine to maintain the state of arrest. Thereafter, the oocytes are treated with the cytotoxic, antineoplasmic drug, etoposide, to engender double-strand breaks (DSBs). By using immunofluorescence and confocal microscopy, we detect and quantify the levels of the core protein γH2AX, which is the phosphorylated form of the histone H2AX. H2AX becomes phosphorylated at the sites of DSBs after DNA damage. The inability to restore DNA integrity following DNA damage in oocytes can lead to infertility, birth defects, and increased rates of spontaneous abortions. Therefore, the understanding of DNA damage response mechanisms and, at the same time, the establishment of an intact method for studying these mechanisms are essential for reproductive biology research.

Enhancer-driven transcription of MCM8 by E2F4 promotes ATR pathway activation and glioma stem cell characteristics

BackgroundGlioma stem cells (GSCs) are responsible for glioma recurrence and drug resistance, yet the mechanisms underlying their maintenance remains unclear. This study aimed to identify enhancer-controlled genes involved in GSCs maintenance and elucidate the mechanisms underlying their regulation.MethodsWe analyzed RNA-seq data and H3K27ac ChIP-seq data from GSE119776 to identify differentially expressed genes and enhancers, respectively. Gene Ontology analysis was performed for functional enrichment. Transcription factors were predicted using the Toolkit for Cistrome Data Browser. Prognostic analysis and gene expression correlation was conducted using the Chinese Glioma Genome Atlas (CGGA) data. Two GSC cell lines, GSC-A172 and GSC-U138MG, were isolated from A172 and U138MG cell lines. qRT-PCR was used to detect gene transcription levels. ChIP-qPCR was used to detect H3K27ac of enhancers, and binding of E2F4 to target gene enhancers. Western blot was used to analyze protein levels of p-ATR and γH2AX. Sphere formation, limiting dilution and cell growth assays were used to analyze GSCs growth and self-renewal.ResultsWe found that upregulated genes in GSCs were associated with ataxia-telangiectasia-mutated-and-Rad3-related kinase (ATR) pathway activation, and that seven enhancer-controlled genes related to ATR pathway activation (LIN9, MCM8, CEP72, POLA1, DBF4, NDE1, and CDKN2C) were identified. Expression of these genes corresponded to poor prognosis in glioma patients. E2F4 was identified as a transcription factor that regulates enhancer-controlled genes related to the ATR pathway activation, with MCM8 having the highest hazard ratio among genes positively correlated with E2F4 expression. E2F4 bound to MCM8 enhancers to promote its transcription. Overexpression of MCM8 partially restored the inhibition of GSCs self-renewal, cell growth, and the ATR pathway activation caused by E2F4 knockdown.ConclusionOur study demonstrated that E2F4-mediated enhancer activation of MCM8 promotes the ATR pathway activation and GSCs characteristics. These findings offer promising targets for the development of new therapies for gliomas.

Role of Nrf2 in 1,2-dichloropropane-induced cell proliferation and DNA damage in the mouse liver.

1,2-Dichloropropane (1,2-DCP) is recognized as the causative chemical of occupational cholangiocarcinoma in printing workers in Japan. However, the cellular and molecular mechanisms of 1,2-DCP-induced carcinogenesis remains elusive. The present study investigated cellular proliferation, DNA damage, apoptosis, and expression of antioxidant and proinflammatory genes in the liver of mice exposed daily to 1,2-DCP for 5 weeks, and the role of nuclear factor erythroid 2-related factor 2 (Nrf2) in these responses. Wild-type and Nrf2-knockout (Nrf2-/-) mice were administered 1,2-DCP by gastric gavage, and then the livers were collected for analysis. Immunohistochemistry for BrdU or Ki67 and TUNEL assay revealed that exposure to 1,2-DCP dose-dependently increased proliferative cholangiocytes, whereas decreased apoptotic cholangiocytes in wild-type mice but not in Nrf2-/- mice. Western blot and quantitative real-time PCR showed that exposure to 1,2-DCP increased the levels of DNA double-strand break marker γ-H2AX and mRNA expression levels of NQO1, xCT, GSTM1, and G6PD in the livers of wild-type mice in a dose-dependent manner, but no such changes were noted in Nrf2-/- mice. 1,2-DCP increased glutathione levels in the liver of both the wild-type and Nrf2-/- mice, suggesting that an Nrf2-independent mechanism contributes to 1,2-DCP-induced increase in glutathione level. In conclusion, the study demonstrated that exposure to 1,2-DCP induced proliferation but reduced apoptosis in cholangiocytes, and induced double-strand DNA breaks and upregulation of antioxidant genes in the liver in an Nrf2-dependent manner. The study suggests a role of Nrf2 in 1,2-DCP-induced cell proliferation, antiapoptotic effect, and DNA damage, which are recognized as key characteristics of carcinogens.

Nerve Injury-Induced γH2AX Reduction in Primary Sensory Neurons Is Involved in Neuropathic Pain Processing.

Phosphorylation of the serine 139 of the histone variant H2AX (γH2AX) is a DNA damage marker that regulates DNA damage response and various diseases. However, whether γH2AX is involved in neuropathic pain is still unclear. We found the expression of γH2AX and H2AX decreased in mice dorsal root ganglion (DRG) after spared nerve injury (SNI). Ataxia telangiectasia mutated (ATM), which promotes γH2AX, was also down-regulated in DRG after peripheral nerve injury. ATM inhibitor KU55933 decreased the level of γH2AX in ND7/23 cells. The intrathecal injection of KU55933 down-regulated DRG γH2AX expression and significantly induced mechanical allodynia and thermal hyperalgesia in a dose-dependent manner. The inhibition of ATM by siRNA could also decrease the pain threshold. The inhibition of dephosphorylation of γH2AX by protein phosphatase 2A (PP2A) siRNA partially suppressed the down-regulation of γH2AX after SNI and relieved pain behavior. Further exploration of the mechanism revealed that inhibiting ATM by KU55933 up-regulated extracellular-signal regulated kinase (ERK) phosphorylation and down-regulated potassium ion channel genes, such as potassium voltage-gated channel subfamily Q member 2 (Kcnq2) and potassium voltage-gated channel subfamily D member 2 (Kcnd2) in vivo, and KU559333 enhanced sensory neuron excitability in vitro. These preliminary findings imply that the down-regulation of γH2AX may contribute to neuropathic pain.

Determination of the effects of fusaric acid, a mycotoxin, on cytotoxicity, gamma-H2AX, 8-hydroxy-2 deoxyguanosine and DNA repair gene expressions in pancreatic cancer cells

Pancreatic cancer has a poor prognosis and is an important public health problem for developing countries. Oxidative stress plays an important role in cancer initiation, progression, proliferation, invasion, angiogenesis and metastasis. For this reason, one of the important strategic targets of new cancer therapeutics is to drive cancer cells into apoptosis through oxidative stress. In nuclear and mitochondrial DNA, 8-hydroxy-2′-deoxyguanosine and gamma-H2AX (γ-H2AX) are used as important oxidative stress biomarkers. Fusaric acid (FA) is a mycotoxin that mediates toxicity produced by Fusarium species and exhibits anticancer effects in various cancers via inducing apoptosis, cell cycle arrest, or other cellular mechanisms. The aim of this study was to determine the effects of fusaric acid on cytotoxic and oxidative damage in MIA PaCa-2 and PANC-1 cell lines. In this context, dose and time dependent cytotoxic effect of fusaric acid was determined by XTT method, mRNA expression levels of genes related to DNA repair were determined by RT-PCR, and its effect on 8-hydroxy-2′-deoxyguanosine and γ-H2AX levels was revealed by ELISA assay. According to XTT results, fusaric acid inhibits cell proliferation in MIA PaCa-2 and Panc-1 cells in a dose- and time-dependent manner. IC50 doses were determined as 187.74 μM at 48 h in MIA PaCa-2 cells and 134.83 μM at 48 h in PANC-1 cells, respectively. γ-H2AX and 8-OHdG changes were not found significant in pancreatic cancer cells. The mRNA expression levels of DNA repair-related genes NEIL1, OGG1, XRCC and Apex-1 change with exposure to fusaric acid. This study contributes to the therapeutic approaches to be developed for pancreatic cancer and demonstrates the potential of fusaric acid as an anticancer agent.

SIRT1 regulates DNA damage signaling through the PP4 phosphatase complex.

The Sirtuin family of NAD+-dependent enzymes plays an important role in maintaining genome stability upon stress. Several mammalian Sirtuins have been linked directly or indirectly to the regulation of DNA damage during replication through Homologous recombination (HR). The role of one of them, SIRT1, is intriguing as it seems to have a general regulatory role in the DNA damage response (DDR) that has not yet been addressed. SIRT1-deficient cells show impaired DDR reflected in a decrease in repair capacity, increased genome instability and decreased levels of γH2AX. Here we unveil a close functional antagonism between SIRT1 and the PP4 phosphatase multiprotein complex in the regulation of the DDR. Upon DNA damage, SIRT1 interacts specifically with the catalytical subunit PP4c and promotes its inhibition by deacetylating the WH1 domain of the regulatory subunits PP4R3α/β. This in turn regulates γH2AX and RPA2 phosphorylation, two key events in the signaling of DNA damage and repair by HR. We propose a mechanism whereby during stress, SIRT1 signaling ensures a global control of DNA damage signaling through PP4.

Ten-Eleven Translocation 1 and 2 Enzymes Affect Human Skin Fibroblasts in an Age-Related Manner.

Ten-eleven translocation (TET) enzymes catalyze the oxidation of 5-methylcytosine (5mC), first to 5-hydroxymethylcytosine (5hmC), then to 5-formylcytosine (5fC), and finally to 5-carboxycytosine (5caC). Evidence suggests that changes in TET expression may impact cell function and the phenotype of aging. Proliferation, apoptosis, markers of autophagy and double-strand DNA break repair, and the expression of Fibulin 5 were assessed by flow cytometry in TET1 and TET2-overexpressing fibroblasts isolated from sun-unexposed skin of young (23-35 years) and age-advanced (75-94 years) individuals. In cells derived from young individuals, TET1 overexpression resulted in the inhibition of proliferation and apoptosis by 37% (p = 0.03) and 24% (p = 0.05), respectively, while the overexpression of TET2 caused a decrease in proliferation by 46% (p = 0.01). Notably, in cells obtained from age-advanced individuals, TETs exhibited different effects. Specifically, TET1 inhibited proliferation and expression of autophagy marker Beclin 1 by 45% (p = 0.05) and 28% (p = 0.048), respectively, while increasing the level of γH2AX, a marker of double-strand DNA breaks necessary for initiating the repair process, by 19% (p = 0.04). TET2 inhibited proliferation by 64% (p = 0.053) and increased the level of γH2AX and Fibulin 5 by 46% (p = 0.007) and 29% (p = 0.04), respectively. These patterns of TET1 and TET2 effects suggest their involvement in regulating various fibroblast functions and that some of their biological actions depend on the donor's age.

Abstract B076: IGFBP3 promotes resistance to olaparib via modulating EGFR signaling in advanced prostate cancer

Abstract Background: Castration-resistant prostate cancer (CRPC) is an incurable disease and a leading cause of cancer death in men worldwide. Olaparib (Lynparza) was among the first PARP inhibitors (PARPi) approved for the treatment of CRPC tumors harboring DNA repair defects. However, clinical resistance to PARPi’s has been documented. The mechanisms underlying resistance to PARPi’s remain elusive. To study acquired resistance, we developed olaparib-resistant LN-OlapR and 2B-OlapR cell lines generated through chronic olaparib treatment of the olaparib-sensitive cell lines LNCaP and C4-2B, respectively. RNA-seq revealed IGFBP3 is overexpressed in both OlapR cell lines. IGFBP3 overexpression is correlated with poor clinical outcome and is thought to participate in DNA repair pathways. IGFBP3 plays a key role in nonhomologous end joining (NHEJ) repair through a ternary complex with EGFR and DNA-PKcs. The IGFBP3/EGFR signaling axis is thought to modulate NHEJ repair and could have implications for PARPi sensitivity. We hypothesize that increased IGFBP3 expression promotes PARPi resistance by enhancing DNA repair capacity. Methods: RNA-sequencing and gene set enrichment analysis were used to determine the expression profile changes in resistant cells compared to parental cells. Real time PCR and western blots confirmed the expression of DNA damage repair genes such as γH2AX, EGFR, and DNA-PKcs. ELISA was used to determine IGFBP3 secretion. RNAi was used to inhibit IGFBP3 and EGFR expression. Gefitinib was used to inhibit EGFR activity. Cell viability assays were used to assess cell growth. Results: Transcriptomic profiling revealed that IGFBP3 is highly expressed in resistant models. We verified increased levels of IGFBP3 RNA and protein in both OlapR models. We found that RNAi inhibition of IGFBP3 increases γH2AX and cleaved-PARP protein levels in the resistant models, which suggests accumulation of DNA double strand breaks (DSBs) leading to genomic instability and cell death. We discovered increased phosphorylation of EGFR and DNA-PKcs in the resistant cells. Furthermore, silencing/inhibiting IGFBP3 and EGFR reduces OlapR cell viability and resensitizes resistant cells to treatment. Conclusions: Our findings demonstrated that inhibiting IGFBP3 and EGFR aids in PARPi sensitivity in the resistant setting. Future work will utilize OlapR models to study how the IGFBP3/EGFR/DNA-PKcs protein complex promotes the development of resistance. Understanding the role of IGFBP3 in PARPi resistance will enhance our ability to re-sensitize resistant CRPC to PARPi therapeutics. Citation Format: Amy R. Leslie, Allen C. Gao, Alan P. Lombard, Shu Ning. IGFBP3 promotes resistance to olaparib via modulating EGFR signaling in advanced prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B076.

Overexpressed FOXM1 collaborates with MMB to increase WEE1 inhibitor sensitivity in NSCLC.

Non-small cell lung cancer (NSCLC) is a common lung tumor with high mortality. The complex formed by MYB-MuvB complex (MMB) and forkhead box M1 (FOXM1) (MMB-FOXM1) plays a vital role in cell cycle progression to affect the progression of diseases. The role of the FOXM1-MMB complex in Wee1-like protein kinase (WEE1) inhibitor sensitivity in NSCLC keeps unclear. The reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed to measure the mRNA levels of FOXM1, LIN54, Replication Protein A (RPA), gammaH2AX (γH2AX) and Cyclin B (CCNB). The western blot was performed to examine the corresponding protein expressions. The Cell Counting Kit-8 (CCK-8) assay was performed to test cell survival. It was demonstrated that after AZD-1775 treatment, the decrease in cell survival mediated by FOXM1 overexpression (P<0.001) could be reversed by LIN54 knockdown (P<0.01) and that cell survival in the control group did not differ obviously from that in the pcDNA3.1-FOXM1+siLIN54 group, indicating that the FOXM1-MMB complex was necessary for WEE1 inhibitor sensitivity. Moreover, the mRNA and protein expression levels of RPA and γH2AX were increased after AZD-1775 treatment and FOXM1 overexpression (P<0.01), suggesting that FOXM1 upregulation enhanced DNA replication stress and DNA damage. Finally, we found that the increases in the mRNA and protein expression levels of CCNB mediated by FOXM1 (P<0.01) could be rescued by silencing LIN54 (P<0.001) and that CCNB expression in the control group did not differ obviously from that in the pcDNA3.1-FOXM1+siLIN54 group. These findings revealed that the FOXM1-MMB complex activated G2/M checkpoints. In our work, it was discovered that FOXM1 overexpression increased DNA replication stress, which increased DNA replication and pressure on the WEE1 checkpoint. On the other hand, FOXM1 can enhance CCNB expression, increase the threshold content of the CCNB/CDK1 complex, facilitate mitosis, and promote WEE1 dephosphorylation. Under these two conditions, sensitivity to the WEE1 inhibitor AZD-1775 is increased, which leads to the accumulation of DNA damage and drives the activation of apoptosis. Overexpressed FOXM1 collaborates with MMB to increase WEE1 inhibitor sensitivity in NSCLC. This discovery might highlight the regulatory function of FOXM1/MMB in the treatment of NSCLC patients.