Affect DNA Damage Research Articles

C-MYC-Induced AP4 Attenuates DREAM-Mediated Repression by p53

The deregulated expression of the c-MYC oncogene activates p53, which is presumably mediated by ARF/INK4, as well as replication-stress-induced DNA damage. Here, we aimed to determine whether the c-MYC-inducible AP4 transcription factor plays a role in this context using a genetic approach. We used a CRISPR/Cas9 approach to generate AP4- and/or p53-deficient derivatives of MCF-7 breast cancer cells harboring an ectopic, inducible c-MYC allele. Cell proliferation, senescence, DNA damage, and comprehensive RNA expression profiles were determined after activation of c-MYC. In addition, we analyzed the expression data from primary breast cancer samples. Loss of AP4 resulted in elevated levels of both spontaneous and c-MYC-induced DNA damage, senescence, and diminished cell proliferation. Deletion of p53 in AP4-deficient cells reverted senescence and proliferation defects without affecting DNA damage levels. RNA-Seq analyses showed that loss of AP4 enhanced repression of DREAM and E2F target genes after p53 activation by c-MYC. Depletion of p21 or the DREAM complex component LIN37 abrogated this effect. These p53-dependent effects were conserved on the level of clinical and gene expression associations found in primary breast cancer tumors. Our results establish AP4 as a pivotal factor at the crossroads of c-MYC, E2F, and p53 target gene regulation.

RNA G-Quadruplex within the 5′-UTR of FEN1 Regulates mRNA Stability under Oxidative Stress

Reactive oxygen species (ROS) are a group of highly oxidative molecules that induce DNA damage, affecting DNA damage response (DDR) and gene expression. It is now recognized that DNA base excision repair (BER) is one of the important pathways responsible for sensing oxidative stress to eliminate DNA damage, in which FEN1 plays an important role in this process. However, the regulation of FEN1 under oxidative stress is still unclear. Here, we identified a novel RNA G-quadruplex (rG4) sequence in the 5'untranslated region (5'UTR) of FEN1 mRNA. Under oxidative stress, the G bases in the G4-forming sequence can be oxidized by ROS, resulting in structural disruption of the G-quadruplex. ROS or TMPyP4, a G4-structural ligand, disrupted the formation of G4 structure and affected the expression of FEN1. Furthermore, pull-down experiments identified a novel FEN1 rG4-binding protein, heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), and cellular studies have shown that hnRNPA1 plays an important role in regulating FEN1 expression. This work demonstrates that rG4 acts as a ROS sensor in the 5'UTR of FEN1 mRNA. Taken together, these results suggest a novel role for rG4 in translational control under oxidative stress.

Adaptive Response in Rat Retinal Cell Cultures Irradiated with γ-rays.

Retina can receive incidental γ-ray exposure from various sources. For example, although radiation therapy is a crucial tool for managing head and neck tumors, patients may develop ocular complications as collateral damage from accidental irradiation. Recently, there has been concern that retinal irradiation during space flight may compromise mission goals and long-term quality of life after space travel. Previously, in our in vitro model, we proved that immature retinal cells are more vulnerable to γ-radiation than differentiated neurons. Here, we investigate if a low-dose pre-irradiation (0.025 Gy), known to have a protective effect in various contexts, can affect DNA damage and oxidative stress in cells exposed to a high dose of γ-rays (2 Gy). Our results reveal that pre-irradiation reduces 2 Gy effects in apoptotic cell number, H2AX phosphorylation and oxidative stress. These defensive effects are also evident in glial cells (reduction in GFAP and ED1 levels) and antioxidant enzymes (catalase and CuZnSOD). Overall, our results confirm that rat retinal cultures can be an exciting tool to study γ-irradiation toxic effects on retinal tissue and speculate that low irradiation may enhance the skill of retinal cells to reduce damage induced by higher doses.

Abstract C075: The loss of ATRX induces a sex-specific progression of pancreatic ductal adenocarcinoma in the presence of oncogenic KRASG12D

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is currently the third leading cause of cancer-related deaths primarily due to a lack of effective early screening and the tumor microenvironment increasing resistance to chemotherapy. Somatic mutation in oncogenic KRASG12D appears in 97% of PDAC patients, however, the addition of environmental and epigenetic stressors are required to promote PDAC initiation and progression from acinar cells. We are elucidating the mechanism by which a loss of α-thalassemia, mental-retardation, X-linked (ATRX), a SWI/SNF chromatin remodeling protein, potentiates acinar cells to develop neoplastic lesions in females. Young et al. have confirmed that female mice with a loss of Atrx, combined with KRASG12D, have shown increased acinar-to-duct cell metaplasia (ADM), increased fibrosis, inflammation, and pancreatic intraepithelial neoplasias (PanINs) lesions relative to mice expressing KRASG12D and Atrx. I hypothesize that a loss of ATRX function increases sensitivity to KRASG12D in PDAC by affecting DNA damage and repair pathways and altering gene expression in acinar cells in a sex-specific fashion. Methods: Mice with an acinar-specific Atrx deletion and oncogenic KRASG12D expression were generated by mating to mice targeting an inducible creERT to the Ptf11α gene (PtflacreERT). Mice were gavaged with tamoxifen and pancreatic injury was induced in these mice (Ptf11αcreERT KRASLSLG12DAtrxfl/fl) with cerulein 10 days later. Mice are monitored for two and five weeks following cerulein treatment and pancreatic tissue was collected for molecular, biochemical, and histological analysis. In addition, pre-neoplastic pancreatic organoid cultures +/- ATRX expression were generated to assess differences in growth rates and chemotherapy resistance. Results: Preliminary results have shown increased sensitivity to chronic pancreatitis is predicted to be observed specifically in KRASG12D Atrx-/- female pancreatic tissue and derived organoids. Conversely, a protective effect stimulated by a loss of ATRX is observed in KRASG12D Atrx-/- male mice, indicated by less ADM/PanIN lesions and greater acinar cells. Discussion: A healthier prognosis in Atrx-/-, KRAS activated male mice suggests that the interaction between ATRX and various scaffold proteins (PRC2, DAXX, EZH2, etc.) alters chromosomal architecture, possibly altering gene expression via X chromosome inactivation or reactivation. Sex-specific differences in pancreatic tissue phenotype of Atrx-/- mice are predicted to arise from differential effects of ATRX in triggering acinar cell genomic instability in female mice to prevent the resolution of dsDNA breaks via non-homologous end joining to sensitize acinar cells to oncogenic KRAS. Future experiments will focus on establishing pre-neoplastic organoid lines derived from inflamed murine pancreata to elucidate the signalling pathway of ATRX that likely stimulates a differential gene and protein expression involved in a better prognosis in KRASG12D activated, Atrx-/- male mice. Citation Format: Aswin Sureshkumar, Phyo Win, Christopher Pin. The loss of ATRX induces a sex-specific progression of pancreatic ductal adenocarcinoma in the presence of oncogenic KRASG12D [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C075.

High Expression of BCAT1 Affects DNA Damage Response By Inhibiting α-KG-Dependent Histone Demethylase

Branched-chain amino acids (BCAA) are essential for tumor growth and proliferation. Branched-chain aminotransferase 1(BCAT1) correlates with AML aggression, not only that, it has been reported that overexpression of BCAT1 in leukemia cells decreases intracellular αKG and impairs the activities of αKG-dependent dioxygenase. In this study, we firstly analyzed the prognostic significance of BCAT1 expression in Cytogenetically normal AML(CN-AML) patients. We found that patients with high levels of BCAT1 had poor prognosis, the median overall survival time is shorter in BCAT1 high group (356 days versus 570 days,p=0.004)(Figure A).Next, we performed gene knockout in THP-1 and MV4-11 cells and gene overexpressing in OCI-AML3 cells. In summary, BCAT1 suppression of BCAT1 markedly limited cell proliferation and colony-forming ability by blocking cell circle at G0/G1 phase(Figure B,C),whereas overexpression contributed to colony-forming and cell growth(Figure D,E). In vivo experiments confirmed that BCAT1 knockout can reduce the tumor burden of xenograft mice, while BCAT1 overexpression remarkably increased tumor burden (Figure F).Survival analysis showed that the BCAT1 OE group significantly shortened survival time(22.5 days vs 26.0 days), and the median survival time of the knockout group was 2.5 days longer than control(26.0 days vs 28.5 day ).(Figure G). To further clarify the mechanism by which BCAT1 affects the proliferation of AML cells, we collected specimens from 87 AML patients and performed RNA-Seq analysis. Through Gene Set Enrichment Analysis, we found that the DNA Genes down-regulated in response to ultraviolet (UV) radiation pathway was enriched in the high expression group of BCAT1(Figure H). Therefore, we further examined the effect of BCAT1 expression level on DNA damage repair in vitro.DNA damage foci studies were performed in control and BCAT1 OE cells. More γ-H2AX focis in BCAT1 overexpressed cells were observed. About 48.20% of BCAT1 OE cell were obeseved ＞5 γ-H2AX foci, which was much higher than the control group(13.6%). And significant regression of γ-H2AX levels in BCAT1 overexpressing cells after the addition of DM-αKG(21%)(Figure I).We used western blot to detect DSB repair-related proteins and found that ATM was also down-regulated in BCAT1 overexpressing cells, along with an upregulation γ-H2AX and the changes of these proteins can also be rescued by exogenous supplementation of DM-aKG(Figure J). High expression of BCAT1 reduces the level of intracellular α-KG and inhibits the activity of α-KG-dependent dioxygenases. Histone demethylase KDM2-7 are αKG-dependent dioxygenase, so we speculated that the decrease in a-KG caused by high BCAT1 expression would limit the activity of histone demethylase, thereby affecting the cellular histone methylation levels. We detected several classic histone methylation markers after BCAT1 overexpression, results showed that the levels of H3K9me3 were significantly up-regulated after BCAT1 overexpression, which could be reversed by supplementation of DM-α-KG(Figure K). Therefore, we believe that the regulation of cellular histone methylation levels by BCAT1 is affected by α-KG levels. H3K9me3 is an epigenetic suppressive marker. IDH1mut cells inhibited ATM expression by upregulating histone H3K9 methylation levels and impede cellular DNA damage repair. The elevated DNA damage level in BCAT1 overexpression condition may be therapeutically exploited using therapies that induce or augment DNA damage, such as chemotherapy and PARP inhibitor, Next, we tried to compare the responses of BCAT1 overexpression and control AML cells to the PARP inhibitor BMN673(Talazoparib). After treatment with BMN673, the survival fraction of BCAT1 overexpressing AML cells was lower than that of negative control in MTS assay. Vice versa, The IC50 of BMN673 in BCAT1 knockout AML cells is much larger than that of the control(Figure L). In summary, this study verified BCAT1 high expression is an independent prognostic predictor of CN-AML.BCAT1 reduces intracellular αKG levels, inhibits αKG-dependent histone demethylation enzymes to inhibit H3K9me3 demethylation, thereby suppressing ATM expression and inhibiting DNA damage response in AML cells. Therefore, BCAT1 high expressing AML cells have higher sensitivity to PARP inhibitors. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Targeting integrin α2 as potential strategy for radiochemosensitization of glioblastoma.

Glioblastoma (GBM) is a fast-growing primary brain tumor characterized by high invasiveness and resistance. This results in poor patient survival. Resistance is caused by many factors, including cell-extracellular matrix (ECM) interactions. Here, we addressed the role of adhesion protein integrin α2, which we identified in a high-throughput screen for novel potential targets in GBM cells treated with standard therapy consisting of temozolomide (TMZ) and radiation. In our study, we used a range of primary/stem-like and established GBM cell models in vitro and in vivo. To identify regulatory mechanisms, we employed high-throughput kinome profiling, Western blotting, immunofluorescence staining, reporter, and activity assays. Our data showed that integrin α2 is overexpressed in GBM compared to normal brain and, that its deletion causes radiochemosensitization. Similarly, invasion and adhesion were significantly reduced in TMZ-irradiated GBM cell models. Furthermore, we found that integrin α2-knockdown impairs the proliferation of GBM cells without affecting DNA damage repair. At the mechanistic level, we found that integrin α2 affects the activity of activating transcription factor 1 (ATF1) and modulates the expression of extracellular signal-regulated kinase 1 (ERK1) regulated by extracellular signals. Finally, we demonstrated that integrin α2-deficiency inhibits tumor growth and thereby prolongs the survival of mice with orthotopically growing GBM xenografts. Taken together our data suggest that integrin α2 may be a promising target to overcome GBM resistance to radio- and chemotherapy. Thus, it would be worth evaluating how efficient and safe the adjuvant use of integrin α2 inhibitors is to standard radio(chemo)therapy in GBM.

Oxidant stress-sensitive circRNA Mdc1 controls cardiomyocyte chromosome stability and cell cycle re-entry during heart regeneration

Targeting cardiomyocyte plasticity has emerged as a new strategy for promoting heart repair after myocardial infarction. However, the precise mechanistic network underlying heart regeneration is not completely understood. As noncoding RNAs, circular RNAs (circRNAs) play essential roles in regulating cardiac physiology and pathology. The present study aimed to investigate the potential roles of circMdc1 in cardiac repair after injury and elucidate its underlying mechanisms. Here, we identified that circMdc1 levels were upregulated in postnatal mouse hearts but downregulated in the regenerative myocardium. The expression of circMdc1 in cardiomyocytes is sensitive to oxidative stress, which was attenuated by N-acetyl-cysteine. Enforced circMdc1 expression inhibited cardiomyocyte proliferation, while circMdc1 silencing led to cardiomyocyte cell cycle re-entry. In vivo, the cardiac-specific adeno-associated virus-mediated knockdown of circMdc1 promoted cardiac regeneration and heart repair accompanied by improved heart function. Conversely, circMdc1 overexpression blunted the regenerative capacity of neonatal hearts after apex resection. Moreover, circMdc1 was able to block the translation of its host gene Mdc1 specifically by binding to PABP, affecting DNA damage and the chromosome stability of cardiomyocytes. Furthermore, overexpression of Mdc1 caused damaged mouse hearts to regenerate and repair after myocardial infarction in vivo. Oxidative stress-sensitive circMdc1 plays an important role in cardiac regeneration and heart repair after injury by regulating DNA damage and chromosome stability in cardiomyocytes by blocking the translation of the host gene Mdc1.

RAD21 Confers Poor Prognosis and Affects Ovarian Cancer Sensitivity to Poly(ADP-Ribose)Polymerase Inhibitors Through DNA Damage Repair.

BackgroundPoly(ADP-ribose)polymerase (PARP) inhibitors are a class of molecular-targeted cancer drugs. Synthetic lethality is a phenomenon that renders homologous recombination repair defective cells more sensitive to PARP inhibitors. As a component of the cohesin complex, RAD21 regulates DNA damage repair. However, the biological roles of RAD21 in ovarian cancer and their underlying mechanisms remain unclear.MethodsAn immunohistochemical assay was used to validate the expression of RAD21 in ovarian cancer and its correlation with prognosis. The effects of RAD21 were evaluated through Cell Counting Kit-8 (CCK8), wound-healing, and invasion assays in vitro and the tumor growth in vivo. Furthermore, CCK8 assay and immunofluorescence assay were used to detect the effect of RAD21 on cell sensitivity to PARP inhibitors and their mechanism. The pathway changes were detected by Western blotting.ResultsRAD21 was markedly upregulated in ovarian cancer samples. High RAD21 expression was correlated with poor differentiation and poor prognosis in patients with ovarian cancer. Functionally, RAD21 overexpression promoted cancer cell proliferation, migration, and invasion. Moreover, RAD21 knockdown increased the sensitivity of ovarian cancer cells to three kinds of PARP inhibitors by affecting DNA damage repair. In vivo experiments indicated that RAD21 promoted tumor growth. Mechanistically, the overexpression of RAD21 led to increased phosphorylation levels of Akt and mTOR. Blocking the Akt/mTOR signaling pathway reversed RAD21 overexpression-induced cancer progression and drug resistance.ConclusionsRAD21 can serve as a valuable prognostic marker for ovarian cancer and has the potential as a therapeutic target that can expand the utility of PARP inhibitors.

Co-occurrence of germline pathogenic variants in Chinese patients with solid tumors.

10579 Background: Germline mutations play an important role in cancer risk and susceptibility. With the growing implementation of next generation sequence of multiple gene panel, an increasing number of cases that carry two or more inherited cancer-predisposing alleles in the same individual are being described. However, understanding the impact of these combined mutations on the patient's phenotype can be particularly challenging. To further understand these combined mutations, we analyze the genetic susceptibility mutations of Chinese solid tumor patients to explore the characteristics of multiple germline variants. Methods: Genetic mutations were reviewed in cancer patients who underwent hybridization capture based next-generation sequencing (NGS). The pathogenicity of germline mutations was categorized based on American College of Medical Genetics and Genomics (ACMG) guidelines, and only pathogenic and likely pathogenic variants were included in this study. Results: Germline variants were identified in 1267 individuals, including 1244 patients harbored one germline mutations and 23 patients carried ≥2 germline mutations, and there was no age difference in the two groups (57 vs 55, p = 0.62). In the ≥2 germline mutations group, genes most frequently involved were BRCA1/2 (12/47, 25.5%), CHEK2 (4/47, 5,1%), TP53(4/47, 5,1%) and MUTYH (4/47, 5,1%). Most of these variants affected DNA damage repair pathways [57% homologous recombination repair (HR), 15% mismatch repair (MMR)]. Besides, double variants in HR and HR pathway were found in 8 patients, HR and MMR pathway were found in 5 patients. Surprisingly, the 50-year-old man with colorectal cancer with three pathogenic variants of the [ FH - BRCA2- FANCD2] genes showed a high aggregation of tumors associated with leiomyomatosis and renal cell cancer and HBOC. Conclusions: We found over 56.5% patients with multiple pathogenic variants in DNA damage repair pathways, which may have important implications for the treatment strategy for these patients. Next-generation sequencing technologies now provide the opportunity to perform simultaneous parallel testing of large numbers of inherited cancer genes, which could guide the clinician as to what the effect of each combination of mutations might be.

8-methoxypsoralen Potentiates Solar UV-Induced DNA Damage of Skin Melanocytes

ObjectivesThere is little research on how diet may influence skin cancer. 8-methoxypsoralen (8-MOP) is a photocarcinogenic dietary furocoumarin that exists in parsley, parsnip, and celery. Phototherapy that uses 8-MOP and ultraviolet radiation (UVR) exposure to treat skin disorders is known to induce skin cancer. However, the biological mechanism of its carcinogenicity is not understood. We aimed to determine whether 8-MOP affects the extent of UVA-induced DNA damage in human epidermal melanocytes, the origin of melanoma, the most fatal type of skin cancer. MethodsOxidative DNA damage leads to the phosphorylation of H2AX (a member of the histone H2A family), generating γ-H2AX. We used γ-H2AX as a marker for DNA damage in human epidermal melanocytes untreated or treated with 100 μM 8-MOP for 12 hours and exposed or not to 3J/cm2 UVA. Each of the 4 groups were analyzed by western blots using H2AX and γ-H2AX antibodies. We used ImageJ/FIJI software for densitometric analysis to quantify the band intensities. The intensities of γ-H2AX bands were divided by that of H2AX bands (γ-H2AX/total H2AX) to measure the amount of DNA damage. To measure how 8-MOP interacts with UV to affect DNA damage, the UV: Control ratios for untreated and 8-MOP treated cells were calculated as the ratio of γ-H2AX/total H2AX in cells exposed to UV to that of cells without UV. T-tests were performed to calculate the significances of differences between γ-H2AX/total H2AX and between UV: Control ratios of the 4 groups. ResultsThe mean UV: Control ratio for 8-MOP treated cells was borderline significantly higher than the mean UV: Control ratio for untreated cells (p = 0.07), suggesting that 8-MOP interacts with UV to enhance DNA damage. When cells were treated with 8-MOP and exposed to UV, γ-H2AX/total H2AX increased compared to untreated cells exposed to UV, although the increase was not statistically significant (p = 0.20). Conclusions8-MOP potentiates UVA-induced DNA damage in melanocytes, which could be one of the mechanisms how 8-MOP and UV exposure induce skin cancer. Other dietary furocoumarins in fruits and vegetables should be evaluated in terms of the effect on DNA damage. Funding SourcesBrown Cancer Center.

Ionizing radiation-induced DNA damage responses affect cell compressibility.

Radiotherapy is an important treatment modality for cancer patients. Ionizing radiation kills cancer cells by inducing DNA damage. However, it remains unclear how this process affects the biomechanical properties of cells and how cellular mechanics affect DNA damage and repair. In this study, the effects of ionizing radiation on cell mechanics were investigated by precisely measuring the compressibility of cells in suspension using an in-house developed microfluidic device. We found that cell compressibility significantly increased after irradiation, depending on cell type and radiation dose. Radiation-induced DNA damage response (DDR) is an important process that alters cell mechanics, and this association was confirmed experimentally by inhibiting the DDR process. Furthermore, the mechanisms involved in changes in cell compressibility were investigated from the perspective of the nucleus and cytoplasm. Experiments showed that radiation reduced H3K9me3 staining intensity but had no effect on F-actin. This suggested that chromatin decondensation caused by radiation-induced DDR is the primary cause of changes in cell compressibility after radiation. The effects of cell mechanics on radiation-induced DNA damage were also investigated. The addition of blebbistatin reduced cytoskeletal forces on the nucleus, which resulted in a reduction in radiation-induced DNA damage. Collectively, this study elucidated reciprocal mechanisms of radiation-induced DNA damage and cell mechanics.

The Roles of Non-Coding RNAs in Radiotherapy of Gastrointestinal Carcinoma.

Radiotherapy (RT), or radiation therapy, has been widely used in clinical practice for the treatment of local advanced gastrointestinal carcinoma. RT causes DNA double-strand breaks leading to cell cytotoxicity and indirectly damages tumor cells by activating downstream genes. Non-coding RNA (including microRNAs, long non-coding RNAs (ncRNAs), and circular RNAs) is a type of RNA that does not encode a protein. As the field of ncRNAs increasingly expands, new complex roles have gradually emerged for ncRNAs in RT. It has been shown that ncRNAs can act as radiosensitivity regulators in gastrointestinal carcinoma by affecting DNA damage repair, cell cycle arrest, irradiation-induced apoptosis, cell autophagy, stemness, EMT, and cell pyroptosis. Here, we review the complex roles of ncRNAs in RT and gastrointestinal carcinoma. We also discuss the potential clinical significance and predictive value of ncRNAs in response to RT for guiding the individualized treatment of patients. This review can serve as a guide for the application of ncRNAs as radiosensitivity enhancers, radioresistance inducers, and predictors of response in RT of gastrointestinal carcinoma.

High Melanin Content in Melanoma Cells Contributes to Enhanced DNA Damage after Rose Bengal Photosensitization.

Melanoma is a type of tumor that originates from melanocytes. Irradiation of melanin with UVA and visible light can produce reactive oxygen species (ROS) such as singlet molecular oxygen (1 O2 ). The objective of this study was to examine DNA damage in melanoma cells (B16-F10) with different melanin contents, subjected to 1 O2 generation. To this end, we used the photosensitizer Rose Bengal acetate (RBAc) and irradiation with visible light (526 nm) (RBAc-PDT). We used the modified comet assay with the repair enzymes hOGG1 and T4 endonuclease V to detect the DNA damage associated with 8-oxo-7,8-dihydro-2'-deoxyguanosine and cyclobutane pyrimidine dimers lesions, respectively. We observed increased formation of hOGG1- and T4endoV-sensitive DNA lesions after light exposure (with or without RBAc). Furthermore, 18 h after irradiation, hOGG1-sensitive DNA lesions increased compared to that at the initial time point (0 h), which shows that a high melanin content contributes to post-irradiation formation of them, mainly via sustained oxidative stress, as confirmed by the measurement of ROS levels and activity of antioxidant enzymes. Contrastingly, the number of T4endoV-sensitive DNA lesions decreased over time (18 h). Our data indicate that in melanoma cells, a higher amount of melanin may affect DNA damage levels when subjected to RBAc-PDT.

Do 50/60 Hz magnetic fields influence oxidative or DNA damage responses in human SH-SY5Y neuroblastoma cells?

Purpose We investigated the possible effects of 50 and 60 Hz magnetic fields (MFs) on reactive oxygen species (ROS) production, DNA damage, DNA damage repair rate, as well as gene expression related to oxidative stress and DNA damage signaling. Materials and methods Human SH-SY5Y neuroblastoma cells were sham-exposed or exposed to 100 µTRMS MFs for 24 h, then assayed or further treated with 100 µM menadione for 1 h before the assay. The levels of ROS and cytosolic superoxide anion (O2 •–) were assayed fluorometrically. DNA damage and gene expression were assayed by comet assay and RT-qPCR, respectively. To examine whether MFs affected DNA damage repair rate, cells were allowed to repair their DNA for 1 or 2 h after menadione treatment and then assayed for DNA damage. Results There was suggestive evidence of a general low-magnitude increase in the expression of ROS-related genes (primarily genes with antioxidant activity) when quantified immediately after MF exposure, suggesting a response to a small increase in ROS level. The possible upregulation of ROS-related genes is supported by the finding that the level of menadione-induced ROS was consistently decreased by 50 Hz MFs (not significantly by 60 Hz MFs) in several measurements 30–60 min after MF exposure. MF exposures did not affect cytosolic O2 •– levels, DNA damage, or its repair rate. Changes in the expression of DNA damage-signaling genes in the MF-exposed cells did not exceed the expected rate of false-positive findings. No firm evidence was found for differential effects from 50 vs. 60 Hz MFs. Conclusions While only weak effects were found on the endpoints measured, the results are consistent with MF effects on ROS signaling.

Clinical significance and biomarker potential of MGMT protein measurement in colorectal cancer.

136 Background: O6-methylguanine DNA methyltransferase (MGMT) is the principal repair mechanism of DNA damage created by alkylating agents. Approximately 40% of colorectal cancers (CRC) exhibit MGMT promoter hypermethylation, expected to result in gene silencing. The role of MGMT as a biomarker is poorly understood, and quantitative measurement of MGMT protein has not been performed in CRC. We hypothesized that altered tumor MGMT would affect DNA damage response, modulate adaptive anti-tumor immune responses, and influence survival in CRC. Methods: We used multiplexed quantitative immunofluorescence (QIF) to study 4 clinically annotated retrospective patient cohorts treated from 2000 to 2017 at Yale, consisting of 400 formalin-fixed paraffin-embedded CRC cases represented in tissue microarray format. These included paired tumors and adjacent non-tumor colonic mucosa (n = 112 pairs in Cohort 1), paired primary CRC and lymph node metastases (n = 31 pairs in Cohort 2), and 2 cohorts with primary stage I-IV tumors (n = 250 in Cohort 3; n = 150 in Cohort 4). We established a QIF panel for simultaneous, localized measurement of DAPI (all cells), cytokeratin (CK; tumor epithelial cells), MGMT, γH2AX (DNA damage response), and CD8. The measurement of fluorescent signals in CK-positive tumor cells or CK-negative stromal cells was achieved using co-localization strategies and the AQUA QIF platform. Results: We identified lower MGMT protein levels in CRC cells than in non-tumor colonic epithelium (p < 0.0001), and in lymph node metastases compared to paired primary CRC tumors (p = 0.0411). Using the visual detection threshold, tumor selective MGMT protein downregulation was identified in 20% of cases in Cohorts 3 and 4. Microsatellite instability-high (MSI-H) cases showed decreased tumoral MGMT protein compared to microsatellite stable (MSS) cases (p = 0.002). In Cohorts 3 and 4, low tumoral MGMT was consistently associated with increased CD8+ tumor infiltrating lymphocytes (p = 0.0472; p = 0.0002). The association between MGMT and γH2AX was inconsistent. The association between MGMT and CD8 was significant only in MSS cases (p = 0.0001) but not in MSI-H cases (p = 0.0979), supporting that the effect is not driven by MSI-H status. Tumor-selective MGMT downregulation evidenced by a low tumor-to-stroma ratio was associated with improved progression-free survival and overall survival in both Cohorts 3 and 4, but statistical significance was only seen in Cohort 3. Conclusions: MGMT protein downregulation occurs in 20% of CRCs and is associated with increased adaptive anti-tumor immune responses, mismatch repair (MMR) deficiency and better prognosis. MGMT deficiency may alter DNA repair in tumor cells and mediate the accumulation of antigenic mutations or neopeptides, independent from MMR status. Our results support a biomarker role of MGMT protein and suggest a role for immunotherapy combinations in MGMT deficient tumors.

Effect of Cold Atmospheric Plasma on Epigenetic Changes, DNA Damage, and Possibilities for Its Use in Synergistic Cancer Therapy.

Cold atmospheric plasma has great potential for use in modern medicine. It has been used in the clinical treatment of skin diseases and chronic wounds, and in laboratory settings it has shown effects on selective decrease in tumour-cell viability, reduced tumour mass in animal models and stem-cell proliferation. Many researchers are currently focusing on its application to internal structures and the use of plasma-activated liquids in tolerated and effective human treatment. There has also been analysis of plasma’s beneficial synergy with standard pharmaceuticals to enhance their effect. Cold atmospheric plasma triggers various responses in tumour cells, and this can result in epigenetic changes in both DNA methylation levels and histone modification. The expression and activity of non-coding RNAs with their many important cell regulatory functions can also be altered by cold atmospheric plasma action. Finally, there is ongoing debate whether plasma-produced radicals can directly affect DNA damage in the nucleus or only initiate apoptosis or other forms of cell death. This article therefore summarises accepted knowledge of cold atmospheric plasma’s influence on epigenetic changes, the expression and activity of non-coding RNAs, and DNA damage and its effect in synergistic treatment with routinely used pharmaceuticals.

The Dual Effect of the HDAC Inhibitor Romidepsin on Androgen Receptor Signaling and DNA Damage Repair in Prostate Cancer.

Aberrant epigenetic modifications play an important role in prostate cancer development and progression. Histone deacetylase inhibitors (HDACi) are a novel class of small-molecular compounds that have been shown to have activity in several tumor types. Some HDACi's induce a stress response that affects DNA damage repair (radiosensitization) and androgen receptor (AR) signaling (increased sensitivity to androgen blockage). We hypothesized that the HDACi, romidepsin, would increase prostate cancer cell killing through dual effects on AR signaling and radiation DNA damage response.The prostate cancer cell lines LNCaP (androgen sensitive) and 22Rv1 (androgen insensitive) were treated with romidepsin (10 nM) plus or minus 2 Gy radiation. The added effect of enzalutamide (10 mM) was also tested. Cell viability was performed by Real-time Cell assay (RTCA), whole-transcriptome analysis by RNA sequencing, western blot analyses, immunofluorescent quantification of γ-H2AX double strand break foci, and colonogenic survival. Western blot analysis was used to measure the expression of (i) AR signaling pathway proteins that included wild-type AR and its AR-V7 splice variant and (ii) DNA repair genes that included Rad51, DNA-PK and γ-H2AX.Romidepsin reduced cell viability by RTCA of both cell lines in a dose dependent manner. The addition of enzalutamide further reduced cell viability and clonogenic survival. Gene ontology analysis from RNA-Seq data in both LNCaP and 22Rv1 cells revealed that romidepsin suppressed pathways related to prostate cancer aggressiveness including the AR, MYC, DNA damage response, and cell cycle progression pathways. By western blot analysis, romidepsin suppressed AR (LNCaP) and AR-V7 (22Rv1) expression and reduced DNA repair genes including RAD51, DNA-PK and phospho-ATM levels. After 2 Gy radiation, we found that treatment with 10nM romidepsin for 24 hours resulted in an increase in γ-H2AX foci in 35.2% in LNCaP and 27% in 22Rv1 cells respectively. By colonogenic assay, 10 nM romidepsin for 24 hours prior to 2 Gy to 8 Gy radiation resulted in a significant reduction in the surviving fractions. The addition of enzalutamide resulted in the greatest reduction in colonogenic survival.Our study demonstrated for the first time that HDACi romidepsin exerted dual effects on AR signaling and DNA damage response genes in androgen sensitive and insensitive prostate cancer cell lines, and that there is an added suppressive effect with the addition of enzalutamide to HDAC inhibition plus radiation. The use of HDACi's have promise clinically for the treatment of high-risk prostate cancer, for which androgen deprivation plus radiotherapy are the standard treatments.

Dynamically monitoring cellular γ-H2AX reveals the potential of carcinogenicity evaluation for genotoxic compounds.

Amongst all toxicological endpoints, carcinogenicity might pose the greatest concern. Genetic damage has been considered an important underlying mechanism for the carcinogenicity of chemical substances. The demand for in vitro genotoxic tests as alternative approaches is growing rapidly with the implementation of new regulations for compounds. However, currently available in vitro genotoxicity tests are often limited by relatively high false positive rates. Moreover, few studies have explored carcinogenicity potential by in vitro genotoxicity testing due to the shortage of suitable toxicological biomarkers to link gene damage with cancer risk. γ-H2AX is a recently acknowledged attractive endpoint (biomarker) for evaluating DNA damage and can simultaneously reflect the DNA damage response and repair of cells. We previously reported an ultrasensitive and reliable method, namely stable-isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS), for detecting cellular γ-H2AX and evaluating genotoxic chemicals. More importantly, our method can dynamically monitor the specific processes of genotoxic compounds affecting DNA damage and repair reflected by the amount of γ-H2AX. To clarify the possibility of using this method to assess the potential carcinogenicity of genotoxic chemicals, we applied it to a set of 69 model compounds recommended by the European Center for the Validation of Alternative Methods (ECVAM), with already-characterized genotoxic potential. Compared to conventional in vitro genotoxicity assays, including the Ames test, the γ-H2AX assay by MS has high accuracy (94-96%) due to high sensitivity and specificity (88% and 100%, respectively). The dynamic profiles of model compounds after exposure in HepG2 cells were explored, and a mathematical approach was employed to simulate and quantitatively model the DNA repair kinetics of genotoxic carcinogens (GCs) based on γ-H2AX time-effect curves up to 8h. Two crucial parameters, i.e., k (rate of γ-H2AX decay) and t50 (time required for γ-H2AX from maximum decrease to half) estimated by the least squares method, were achieved. An open web server to help researchers calculate these two key parameters and profile simulated curves of the tested compound is available online ( http://ccb1.bmi.ac.cn:81/shiny-server/sample-apps/prediction1/ ). We detected a positive association between carcinogenic levels and k and t50 values of γ-H2AX in tested GCs, validating the potential of using this MS-based γ-H2AX in vitro assay to help preliminarily evaluate carcinogenicity and assess genotoxicity. This approach may be used alone or integrated into an existing battery of in vitro genetic toxicity tests.

A combination of Class-I fumarases and metabolites (α-ketoglutarate and fumarate) signal the DNA damage response in Escherichia coli

Class-II fumarases (fumarate hydratase, FH) are dual-targeted enzymes occurring in the mitochondria and cytosol of all eukaryotes. They are essential components in the DNA damage response (DDR) and, more specifically, protect cells from DNA double-strand breaks. Similarly, the gram-positive bacterium Bacillus subtilis class-II fumarase, in addition to its role in the tricarboxylic acid cycle, participates in the DDR. Escherichia coli harbors three fumarase genes: class-I fumA and fumB and class-II fumC Notably, class-I fumarases show no sequence similarity to class-II fumarases and are of different evolutionary origin. Strikingly, here we show that E. coli fumarase functions are distributed between class-I fumarases, which participate in the DDR, and the class-II fumarase, which participates in respiration. In E. coli, we discover that the signaling molecule, alpha-ketoglutarate (α-KG), has a function, complementing DNA damage sensitivity of fum-null mutants. Excitingly, we identify the E. coli α-KG-dependent DNA repair enzyme AlkB as the target of this interplay of metabolite signaling. In addition to α-KG, fumarate (fumaric acid) is shown to affect DNA damage repair on two different levels, first by directly inhibiting the DNA damage repair enzyme AlkB demethylase activity, both in vitro and in vivo (countering α-KG). The second is a more global effect on transcription, because fum-null mutants exhibit a decrease in transcription of key DNA damage repair genes. Together, these results show evolutionary adaptable metabolic signaling of the DDR, in which fumarases and different metabolites are recruited regardless of the evolutionary enzyme class performing the function.

Effective DNA damage response after acute but not chronic immune challenge: SARS-CoV-2 vaccine versus Systemic Lupus Erythematosus

Whether and how an acute immune challenge may affect DNA Damage Response (DDR) is unknown. By studying vaccinations against Influenza and SARS-CoV-2 (mRNA-based) we found acute increases of type-I interferon-inducible gene expression, oxidative stress and DNA damage accumulation in blood mononuclear cells of 9 healthy controls, coupled with effective anti-SARS-CoV-2 neutralizing antibody production in all. Increased DNA damage after SARS-CoV-2 vaccine, partly due to increased oxidative stress, was transient, whereas the inherent DNA repair capacity was found intact. In contrast, in 26 patients with Systemic Lupus Erythematosus, who served as controls in the context of chronic immune activation, we validated increased DNA damage accumulation, increased type-I interferon-inducible gene expression and induction of oxidative stress, however aberrant DDR was associated with deficiencies in nucleotide excision repair pathways. These results indicate that acute immune challenge can indeed activate DDR pathways, whereas, contrary to chronic immune challenge, successful repair of DNA lesions occurs.