DNA Damage Signaling Pathways Research Articles

Decoding molecular features of bovine oocyte fate during antral follicle growth via single-cell multi-omics analysis.

Antral follicle size is a useful predictive marker of the competency of enclosed oocytes for yielding an embryo following in vitro maturation and fertilization. However, the molecular mechanisms underpinning oocyte developmental potential during bovine antral follicle growth are still unclear. Here, we used a modified single-cell multi-omics approach to analyze the transcriptome, DNA methylome and chromatin accessibility in parallel for oocytes and cumulus cells collected from bovine antral follicles of different sizes. Transcriptome profiling identified three types of oocytes (Small, Medium and Large) that underwent different developmental trajectories, with Large oocytes exhibiting the largest average follicle size and characteristics resembling metaphase-II oocytes. Differential expression analysis and real-time PCR assay showed that most replication-dependent histone genes were highly expressed in Large oocytes. The joint analysis of multi-omics data revealed that the transcription of 20 differentially expressed genes in Large oocytes was associated with both DNA methylation and chromatin accessibility. In addition, oocyte-cumulus interaction analysis showed that inflammation, DNA damage, and p53 signaling pathways were active in Small oocytes, which had the smallest average follicle sizes. We further confirmed that p53 pathway inhibition in in vitro maturation experiments using oocytes obtained from small antral follicles could improve the quality of oocytes and increased the blastocyte rate after in vitro fertilization and culture. Our work provides new insights into the intricate orchestration of bovine oocyte fate determination during antral folliculogenesis, which is instrumental for optimizing in vitro maturation techniques to optimize oocyte quality.

Combined in vitro and in silico mechanistic approach to explore the potential of Alternaria mycotoxins alternariol and altertoxin II to hamper γH2AX formation in DNA damage signaling pathways

Risk assessment of food and environmental contaminants is faced by substantial data gaps and novel strategies are needed to support science-based regulatory actions. The Alternaria mycotoxins alternariol (AOH) and altertoxin II (ATXII) have garnered attention for their possible genotoxic effects. Nevertheless, data currently available are rather scattered, hindering a comprehensive hazard characterization. This study combined in vitro/in silico approaches to elucidate the potential of AOH and ATXII to induce double-strand breaks (DSBs) in HepG2 cells. Furthermore, it examines the impact of co-exposure to AOH and the DSB-inducing drug doxorubicin (Doxo) on γH2AX expression. AOH slightly increased γH2AX expression, whereas ATXII did not elicit this response. Interestingly, AOH suppressed Doxo-induced γH2AX expression, despite evidence of increased DNA damage in the comet assay. Building on these observations, AOH was postulated to inhibit γH2AX-forming kinases. Along this line, in silico analysis supported AOH potential interaction with the ATP-binding sites of these kinases and immunofluorescence experiments showed decreased intracellular phosphorylation events. Similarly, in silico results suggested that ATXII might also interact with these kinases. This study emphasizes the importance of understanding the implications of AOH-induced γH2AX expression inhibition on DNA repair processes and underscores the need for caution when interpreting γH2AX assay results.

Abstract A003: HPV infection causes dependence on alternative DNA damage response pathways providing cancer specific targets for radiosensitization

Abstract Targeted radiosensitization is an attractive approach to improving the efficacy of radiotherapy in cancer treatment. However, drugs targeting ubiquitous DNA damage response (DDR) pathways are not cancer specific and can increase radiation related side effects. Thus, there is an unmet clinical need for tumor specific molecular targets. To address this need we sought to identify DNA damage signaling pathways that are unique to cancer cells. We hypothesized that by expressing its genome in host cells high-risk Human Papilloma Virus (HPV) can rewire DNA damage signaling making HPV-positive tumor cells dependent on alternative pathways to survive radiation, which can be exploited for targeted radiosensitization of HPV-induced cancers. Using a CRISPR/Cas9 screen in cells that selectively express HPV16 proteins E6 or E7 we uncovered genes that cells rely on to survive radiation-induced DNA damage in the context of E6 or E7 expression. We demonstrate that targeting these newly identified genes sensitizes cancer cells to radiation specifically in the presence of E6 or E7. We provide mechanistic insights showing that E6 and E7 alter the cellular epigenome by inhibiting histone lysine acetylation and tri-methylation, respectively. In this setting, a member of a centromeric protein complex identified in the screen is critical for mitotic progression and survival in E6-expressing cells following radiation exposure. Deletion of this factor resulted in delayed mitotic progression, chromosomal missegregation and rapid post-mitotic death in irradiated HPV-positive cells, a phenotype that was mitigated by ablation of E6 expression. Furthermore, we identified an E3 ubiquitin ligase that promotes survival in E7-expressing cells following radiation exposure by alleviating perturbations during DNA replication. Together, we have uncovered DDR pathways that are critical for cell survival after radiation exposure specifically in HPV-positive cells. Our findings suggest that expression of viral proteins E6 and E7 rewires DNA damage signaling networks causing dependence on alternative response pathways. We propose that these pathways can be targeted for tumor-specific radiosensitization of HPV-induced cancers. Citation Format: Michael Goldstein, Akhil Kotwal, Nishanth Gabriel, William Buchser, Monica Sentmanat, Stephanie Markovina, Julie Schwarz, Xia Cui. HPV infection causes dependence on alternative DNA damage response pathways providing cancer specific targets for radiosensitization [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A003.

The MRE11–ATM–SOG1 DNA damage signaling pathway confers rice immunity to Xanthomonas oryzae

Plants are constantly exposed to microbial pathogens in the environment. One branch of innate plant immunity is mediated by cell-membrane-localized receptors, but less is known about associations between DNA damage and plant immune responses. Here, we show that rice (Oryza sativa) mesophyll cells are prone to DNA double-stranded breaks (DSBs) in response to ZJ173, a strain of Xanthomonas oryzae pv. oryzae (Xoo). The DSB signal transducer ataxia telangiectasia mutated (ATM), but not the ATM and Rad3-related branch, confers resistance against Xoo. Mechanistically, the MRE11–ATM module phosphorylates suppressor of gamma response 1 (SOG1), which activates several phenylpropanoid pathway genes and prompts downstream phytoalexin biosynthesis during Xoo infection. Intriguingly, overexpression of the topoisomerase gene TOP6A3 causes a switch from the classic non-homologous end joining (NHEJ) pathway to the alternative NHEJ and homologous recombination pathways at Xoo-induced DSBs. The enhanced ATM signaling of the alternative NHEJ pathway strengthens the SOG1-regulated phenylpropanoid pathway and thereby boosts Xoo-induced phytoalexin biosynthesis in TOP6A3-OE1 overexpression lines. Overall, the MRE11–ATM–SOG1 pathway serves as a prime example of plant–pathogen interactions that occur via host non-specific recognition. The function of TOP6-facilitated ATM signaling in the defense response makes it a promising target for breeding of rice germplasm that exhibits resistance to bacterial blight disease without a growth penalty.

Pan-cancer analyses of senescence-related genes in extracellular matrix characterization in cancer

PurposeThe aged microenvironment plays a crucial role in tumor onset and progression. However, it remains unclear whether and how the aging of the extracellular matrix (ECM) influences cancer onset and progression. Furthermore, the mechanisms and implications of extracellular matrix senescence-related genes (ECM-SRGs) in pan-cancer have not been investigated.MethodsWe collected profiling data from over 10,000 individuals, covering 33 cancer types, 750 small molecule drugs, and 24 immune cell types, for a thorough and systematic analysis of ECM-SRGs in cancer.ResultsWe observed a significant correlation between immune cell infiltrates and Gene Set Variation Analysis enrichment scores of ECM-SRGs in 33 cancer types. Moreover, our results revealed significant differences in immune cell infiltration among patients with copy number variations (CNV) and single nucleotide variations (SNV) in ECM-SRGs across various malignancies. Aberrant hypomethylation led to increased ECM-SRGs expression, and in specific malignancies, a connection between ECM-SRGs hypomethylation and adverse patient survival was established. The frequency of CNV and SNV in ECM-SRGs was elevated. We observed a positive correlation between CNV, SNV, and ECM-SRGs expression. Furthermore, a correlation was found between the high frequency of CNV and SNV in ECM-SRGs and poor patient survival in several cancer types. Additionally, the results demonstrated that ECM-SRGs expression could serve as a predictor of patient survival in diverse cancers. Pathway analysis unveiled the role of ECM-SRGs in activating EMT, apoptosis, and the RAS/MAPK signaling pathway while suppressing the cell cycle, hormone AR, and the response to DNA damage signaling pathway. Finally, we conducted searches in the “Genomics of Drug Sensitivity in Cancer” and “Genomics of Therapeutics Response Portal” databases, identifying several drugs that target ECM-SRGs.ConclusionsWe conducted a comprehensive evaluation of the genomes and immunogenomics of ECM-SRGs, along with their clinical features in 33 solid tumors. This may provide insights into the relationship between ECM-SRGs and tumorigenesis. Consequently, targeting these ECM-SRGs holds promise as a clinical approach for cancer treatment.

HPV Infection Causes Dependence on Alternative DNA Damage Response Pathways Providing Cancer Specific Targets for Radiosensitization

Cancer-specific radiosensitization is an attractive approach to improving the efficacy of radiotherapy. However, drugs targeting ubiquitous DNA damage response pathways are not cancer specific and can increase radiation related side effects. Thus, there is an unmet need for tumor specific molecular targets. This approach requires an identification of DNA damage signaling pathways that are unique to cancer cells. We hypothesized that by expressing its genome in the host cells HPV infection can rewire DNA damage signaling making HPV-positive tumor cells dependent on alternative pathways to survive radiation, which can be exploited for radiosensitization of HPV-induced cancers. We have performed a CRISPR/Cas9 screen in HPV-positive SiHa cells that either express HPV16 proteins E6 and E7 or contain a selective knock-out of E6 or E7. We used next-generation sequencing to determine the abundance of gRNA in cells that were mock-treated or irradiated. We identified genes that were required for survival of radiation specifically in the context of E6 or E7 expression. Results of the screen were validated by generating knock-outs of the discovered genes using CRISPR/Cas9. By applying an array of molecular tools to analyze cell survival, cell cycle progression and mitotic progression we assessed the role of these genes in radiation response in HPV-positive cancer cells. We have identified genes that are required for survival of radiation-induced DNA damage in the context of either E6 or E7 expression. We demonstrate that targeting these genes results in hypersensitization of cervical cancer cells to radiation specifically in the presence of E6 or E7. We show that the gene product required for survival in E6-expressing cells is critical for mitotic progression after radiation exposure serving as a member of a protein complex stabilizing the attachment of mitotic spindle to centromeres. We have uncovered specific genes that are critical for DNA damage response and cell survival after radiation exposure in HPV-positive cells. Our findings suggest that expression of the HPV proteins E6 and E7 rewires DNA damage signaling causing dependence on alternative response pathways. We propose that these pathways can be targeted for a tumor-specific radiosensitization of HPV-induced cancers.

DDB2 and MDM2 genes are promising markers for radiation diagnosis and estimation of radiation dose independent of trauma and burns.

In the field of biodosimetry, the current accepted method for evaluating radiation dose fails to meet the need of rapid, large-scale screening, and most RNA marker-related studies of biodosimetry are concentrating on a single type of ray, while some other potential factors, such as trauma and burns, have not been covered. Microarray datasets that contain the data of human peripheral blood samples exposed to X-ray, neutron, and γ-ray radiation were obtained from the GEO database. Totally, 33 multi-type ray co-induced genes were obtained at first from the differentially expressed genes (DEGs) and key genes identified by weighted gene co-expression network analysis (WGCNA), and these genes were mainly enriched in DNA damage, cellular apoptosis, and p53 signaling pathway. Following transcriptome sequencing of blood samples from 11 healthy volunteers, 13 patients with severe burns, and 37 patients with severe trauma, 6635 trauma-related DEGs and 7703 burn-related DEGs were obtained. Through the exclusion method, a total of 12 radiation-specific genes independent of trauma and burns were identified. ROC curve analysis revealed that the DDB2 gene performed the best in diagnosis of all three types of ray radiation, while correlation analysis showed that the MDM2 gene was the best in assessment of radiation dose. The results of multiple-linear regression analysis indicated that such analysis could improve the accuracy in assessment of radiation dose. Moreover, the DDB2 and MDM2 genes remained effective in radiation diagnosis and assessment of radiation dose in an external dataset. In general, the study brings new insights into radiation biodosimetry.

A Dihydroartemisinin-Loaded Nanoreactor Motivates Anti-Cancer Immunotherapy by Synergy-Induced Ferroptosis to Activate Cgas/STING for Reprogramming of Macrophage.

Infiltration of tumor-associated macrophages (TAM) characterized by an M2 phenotype is an overriding feature in malignant tumors. Reprogramming TAM is the most cutting-edge strategy for cancer therapy. In the present study, an iron-based metal framework (MOF) nanoreactor loaded with dihydroartemisinin (DHA) was developed, which provided high uptake by TAM and retains their viability, thus effectively addressing the inefficiency of the DHA at low concentrations. Impressively, DHA@MIL-101 could selectively accumulate in tumor tissues and remodel TAM to the M1 phenotype. The results of RNA sequencing further suggested that this nanoreactor may regulate ferroptosis, a DNA damage signaling pathway in TAM. Indeed, the outcomes confirmed that DHA@MIL-101 triggered ferroptosis in TAM. In addition, the findings revealed that DNA damage induced by DHA nanoreactors activated the intracellular cGAS sensor, resulting in the binding of STING to IRF3 and thereby up-regulating the immunogenicity. In contrast, blocking ferroptosis impaired DHA@MIL-101-induced activation of STING signaling and phenotypic remodeling. Finally, we showed that DHA nanoreactors deployed anti-tumor immunotherapy through ferroptosis-mediated TAM reprogramming. Taken together, we have achieved immune efficacy through TAM's remodeling by delivering DHA and iron ions into TAM using nanoreactors, providing a novel approach for combining phytopharmaceuticals with nanocarriers to regulate the immune microenvironment. This article is protected by copyright. All rights reserved.

BRCA1 heterozygosity promotes DNA damage-induced senescence in a sex-specific manner following repeated mild traumatic brain injury.

Emerging evidence suggests cellular senescence, as a consequence of excess DNA damage and deficient repair, to be a driver of brain dysfunction following repeated mild traumatic brain injury (rmTBI). This study aimed to further investigate the role of deficient DNA repair, specifically BRCA1-related repair, on DNA damage-induced senescence. BRCA1, a repair protein involved in maintaining genomic integrity with multiple roles in the central nervous system, was previously reported to be significantly downregulated in post-mortem brains with a history of rmTBI. Here we examined the effects of impaired BRCA1-related repair on DNA damage-induced senescence and outcomes 1-week post-rmTBI using mice with a heterozygous knockout for BRCA1 in a sex-segregated manner. Altered BRCA1 repair with rmTBI resulted in altered anxiety-related behaviours in males and females using elevated zero maze and contextual fear conditioning. Evaluating molecular markers associated with DNA damage signalling and senescence-related pathways revealed sex-specific differences attributed to BRCA1, where females exhibited elevated DNA damage, impaired DNA damage signalling, and dampened senescence onset compared to males. Overall, the results from this study highlight sex-specific consequences of aberrant DNA repair on outcomes post-injury, and further support a need to develop sex-specific treatments following rmTBI.

FACS-based genome-wide CRISPR screens define key regulators of DNA damage signaling pathways

DNA damage-activated signaling pathways are critical for coordinating multiple cellular processes, which must be tightly regulated to maintain genome stability. To provide a comprehensive and unbiased perspective of DNA damage response (DDR) signaling pathways, we performed 30 fluorescence-activated cell sorting (FACS)-based genome-wide CRISPR screens in human cell lines with antibodies recognizing distinct endogenous DNA damage signaling proteins to identify critical regulators involved in DDR. We discovered that proteasome-mediated processing is an early and prerequisite event for cells to trigger camptothecin- and etoposide-induced DDR signaling. Furthermore, we identified PRMT1 and PRMT5 as modulators that regulate ATM protein level. Moreover, we discovered that GNB1L is a key regulator of DDR signaling via its role as a co-chaperone specifically regulating PIKK proteins. Collectively, these screens offer a rich resource for further investigation of DDR, which may provide insight into strategies of targeting these DDR pathways to improve therapeutic outcomes.

Rad1 attenuates DNA double-strand breaks and cell cycle arrest in type II alveolar epithelial cells of rats with bronchopulmonary dysplasia

BackgroundBronchopulmonary dysplasia (BPD) is the most common and serious chronic lung disease in preterm infants with pathological characteristics of arrested lung development. DNA double-strand breaks (DSBs) are a serious manifestation of oxidative stress damage, but little is known about the role of DSBs in BPD. The current study set out to detect DSB accumulation and cell cycle arrest in BPD and study the expression of genes related to DNA damage and repair in BPD through DNA damage signaling pathway-based PCR array to determine a suitable target to improve arrested lung development associated with BPD.MethodsDSB accumulation and cell cycle arrest were detected in a BPD animal model and primary cells, then a DNA damage signaling pathway-based PCR array was used to identify the target of DSB repair in BPD.ResultsDSB accumulation and cell cycle arrest were shown in BPD animal model, primary type II alveolar epithelial cells (AECII) and cultured cells after exposure to hyperoxia. Of the 84 genes in the DNA damage-signaling pathway PCR array, eight genes were overexpressed and 11 genes were repressed. Rad1, an important protein for DSB repair, was repressed in the model group. Real-time PCR and western blots were used to verify the microarray results. Next, we confirmed that silencing Rad1 expression aggravated the accumulation of DSBs and cell cycle arrest in AECII cells, whereas its overexpression alleviated DSB accumulation and cell cycle arrest.ConclusionsThe accumulation of DSBs in AECII might be an important cause of alveolar growth arrest associated with BPD. Rad1 could be an effective target for intervention to improve this arrest in lung development associated with BPD.

Cancer researchers' perceptions of the importance of the sex of cell lines, animals, and human samples for cancer biology research.

Sex differences in cancer risk and outcome are currently a topic of major interest in clinical oncology. It is however unknown to what extent cancer researchers consider sex as a biological variable for their research. We conducted an international survey among 1243 academic cancer researchers and collected both quantitative and qualitative data. Although most of the participants indicated that they were familiar with the concept of studying sex differences in cancer biology, they did not think it was important to investigate sex differences in every context of cancer research nor in all tumor types. This is in stark contrast to the current recommendations and guidelines and illustrates the need for increased awareness among cancer researchers regarding the potential impact of the sex of cell lines, animals, and human samples in their studies.

A virus-borne DNA damage signaling pathway controls the lysogeny-induction switch in a group of temperate pleolipoviruses.

Many prokaryotic viruses are temperate and their reactivation is tightly regulated. However, except for a few bacterial model systems, the regulatory circuits underlying the exit from lysogeny are poorly understood, especially in archaea. Here, we report a three-gene module which regulates the switch between lysogeny and replicative cycle in a haloarchaeal virus SNJ2 (family Pleolipoviridae). The SNJ2 orf4 encodes a winged helix-turn-helix DNA binding protein which maintains lysogeny through repressing the expression of the viral integrase gene intSNJ2. To switch to the induced state, two other SNJ2-encoded proteins, Orf7 and Orf8, are required. Orf8 is a homolog of cellular AAA+ATPase Orc1/Cdc6, which is activated upon mitomycin C-induced DNA damage, possibly through posttranslational modification. Activated Orf8 initiates the expression of Orf7 which, in turn, antagonizes the function of Orf4, leading to the transcription of intSNJ2, thereby switching SNJ2 to the induced state. Comparative genomics analysis revealed that the SNJ2-like Orc1/Cdc6-centered three-gene module is common in haloarchaeal genomes, always present in the context of integrated proviruses. Collectively, our results uncover the first DNA damage signaling pathway encoded by a temperate archaeal virus and reveal an unexpected role of the widely distributed virus-encoded Orc1/Cdc6 homologs.

Identification and Analysis of Hub Genes and Immune Cells Associated with the Formation of Acute Aortic Dissection.

Acute type A aortic dissection (AAD) is a catastrophic disease with high mortality, but the pathogenesis has not been fully elucidated. This study is aimed at identifying hub genes and immune cells associated with the pathogenesis of AAD. The datasets were downloaded from Gene Expression Omnibus (GEO). Gene Set Enrichment Analysis (GSEA), gene set variation analysis (GSVA), and differential analysis were performed. The differentially expressed genes (DEGs) were intersected with specific genes collected from MSigDB. The gene function and pathway enrichment analysis were also performed on intersecting genes. The key modules were selected by weighted gene coexpression network analysis (WGCNA). Hub genes were identified by least absolute shrinkage and selection operator (LASSO) analysis and were verified in the metadataset. The immune cell infiltration was analyzed by CIBERSORT, and the relationship between hub genes and immune cells was performed by Pearson's correlation analysis. The single-cell RNA sequencing (scRNA-seq) dataset was used to verify the differences in DNA damage and repair signaling pathways and hub genes in different cell types. The results of GSEA and GSVA indicated that DNA damage and repair processes were activated in the occurrence of AAD. The gene function and pathway enrichment analysis on differentially expressed DNA damage- and repair-related genes showed that these genes were mainly involved in the regulation of the cell cycle process, cellular response to DNA damage stimulus, response to wounding, p53 signaling pathway, and cellular senescence. Three key modules were identified by WGCNA. Five genes were screened as hub genes, including CDK2, EIF4A1, GLRX, NNMT, and SLCO2A1. Naive B cells and Gamma delta T cells (γδ T cells) were decreased in AAD, but monocytes and M0 macrophages were increased. scRNA-seq analysis included that DNA damage and repair processes were activated in smooth muscle cells (SMCs), tissue stem cells, and monocytes in the aortic wall of patients with AAD. Our results suggested that DNA damage- and repair-related genes may be involved in the occurrence of AAD by regulating many biological processes. The hub genes and immune cells reported in this study also increase the understanding of AAD.

Combined inhibition of Wee1 and Chk1 as a therapeutic strategy in multiple myeloma.

Multiple myeloma (MM) is a hematological malignancy characterized by an abnormal clonal proliferation of malignant plasma cells. Despite the introduction of novel agents that have significantly improved clinical outcome, most patients relapse and develop drug resistance. MM is characterized by genomic instability and a high level of replicative stress. In response to replicative and DNA damage stress, MM cells activate various DNA damage signaling pathways. In this study, we reported that high CHK1 and WEE1 expression is associated with poor outcome in independent cohorts of MM patients treated with high dose melphalan chemotherapy or anti-CD38 immunotherapy. Combined targeting of Chk1 and Wee1 demonstrates synergistic toxicities on MM cells and was associated with higher DNA double-strand break induction, as evidenced by an increased percentage of γH2AX positive cells subsequently leading to apoptosis. The therapeutic interest of Chk1/Wee1 inhibitors' combination was validated on primary MM cells of patients. The toxicity was specific of MM cells since normal bone marrow cells were not significantly affected. Using deconvolution approach, MM patients with high CHK1 expression exhibited a significant lower percentage of NK cells whereas patients with high WEE1 expression displayed a significant higher percentage of regulatory T cells in the bone marrow. These data emphasize that MM cell adaptation to replicative stress through Wee1 and Chk1 upregulation may decrease the activation of the cell-intrinsic innate immune response. Our study suggests that association of Chk1 and Wee1 inhibitors may represent a promising therapeutic approach in high-risk MM patients characterized by high CHK1 and WEE1 expression.

Effects of elevated temperature on 8-OHdG expression in the American oyster (Crassostrea virginica): Induction of oxidative stress biomarkers, cellular apoptosis, DNA damage and γH2AX signaling pathways

Global temperature is increasing due to anthropogenic activities and the effects of elevated temperature on DNA lesions are not well documented in marine organisms. The American oyster (Crassostrea virginica, an edible and commercially important marine mollusk) is an ideal shellfish species to study oxidative DNA lesions during heat stress. In this study, we examined the effects of elevated temperatures (24, 28, and 32 °C for one-week exposure) on heat shock protein-70 (HSP70, a biomarker of heat stress), 8‑hydroxy-2′-deoxyguanosine (8-OHdG, a biomarker of pro-mutagenic DNA lesion), double-stranded DNA (dsDNA), γ-histone family member X (γH2AX, a molecular biomarker of DNA damage), caspase-3 (CAS-3, a key enzyme of apoptotic pathway) and Bcl-2-associated X (BAX, an apoptosis regulator) protein and/or mRNA expressions in the gills of American oysters. Immunohistochemical and qRT-PCR results showed that HSP70, 8-OHdG, dsDNA, and γH2AX expressions in gills were significantly increased at high temperatures (28 and 32 °C) compared with control (24°C). In situ TUNEL analysis showed that the apoptotic cells in gill tissues were increased in heat-exposed oysters. Interestingly, the enhanced apoptotic cells were associated with increased CAS-3 and BAX mRNA and/or protein expressions, along with 8-OHdG levels in gills after heat exposure. Moreover, the extrapallial (EP) fluid (i.e., extracellular body fluid) protein concentrations were lower; however, the EP glucose levels were higher in heat-exposed oysters. Taken together, these results suggest that heat shock-driven oxidative stress alters extracellular body fluid conditions and induces cellular apoptosis and DNA damage, which may lead to increased 8-OHdG levels in cells/tissues in oysters.

Radiation-induced PD-L1 expression in tumor and its microenvironment facilitates cancer-immune escape: a narrative review.

Radiotherapy (RT) is one of the fundamental anti-cancer regimens by means of inducing in situ tumor vaccination and driving a systemic anti-tumor immune response. It can affect the tumor microenvironment (TME) components consisting of blood vessels, immunocytes, fibroblasts, and extracellular matrix (ECM), and might subsequently suppress anti-tumor immunity through expression of molecules such as programmed death ligand-1 (PD-L1). Immune checkpoint inhibitors (ICIs), especially anti-programmed cell death 1 (PD-1)/PD-L1 therapies, have been regarded as effective in the reinvigoration of the immune system and another major cancer treatment. Experimentally, combination of RT and ICIs therapy shows a greater synergistic effect than either therapy alone. We performed a narrative review of the literature in the PubMed database. The research string comprised various combinations of "radiotherapy", "programmed death-ligand 1", "microenvironment", "exosome", "myeloid cell", "tumor cell", "tumor immunity". The database was searched independently by two authors. A third reviewer mediated any discordance of the results of the two screeners. RT upregulates PD-L1 expression in tumor cells, tumor-derived exosomes (TEXs), myeloid-derived suppressor cells (MDSCs), and macrophages. The signaling pathways correlated to PD-L1 expression in tumor cells include the DNA damage signaling pathway, epidermal growth factor receptor (EGFR) pathway, interferon gamma (IFN-γ) pathway, cGAS-STING pathway, and JAK/STATs pathway. PD-L1 upregulation post-RT is found not only in tumor cells but also in the TME and is one of the mechanisms of tumor evasion. Therefore, further studies are necessary to fully comprehend this biological process. Meanwhile, combination of therapies has been shown to be effective, and novel approaches are to be developed as adjuvant to RT and ICIs therapy.

Organophosphorus Flame Retardant TCPP Induces Cellular Senescence in Normal Human Skin Keratinocytes: Implication for Skin Aging.

Tris (1-chloro-2-propyl) phosphate (TCPP) is one of the most frequently detected organophosphorus flames in the environment. Continuous daily exposure to TCPP may harm human skin. However, little is known about the adverse effects of TCPP on human skin. In this study, we first evaluated the detrimental effects and tried to uncover the underlying mechanisms of TCPP on human skin keratinocytes (HaCaT) after 24 h exposure. We found that TCPP caused a concentration-dependent decrease in HaCaT cell viability after exposure to 1.56-400 μg/mL for 24 h, with an IC50 of 275 μg/mL. TCPP also promoted the generation of intracellular reactive oxygen species (ROS) and triggered DNA damage, evidenced by an increase of phosphorylated histone H2A.X (γH2A.X) in the nucleus. Furthermore, the cell cycle was arrested at the G1 phase at 100 μg/mL by upregulation of the mRNA expression of p53 and p21 and downregulation of cyclin D1 and CDK4 expression. Additionally, both the senescence-associated-β-galactosidase activity and related proinflammatory cytokine IL-1β and IL-6 were elevated, indicating that TCPP exposure caused cellular senescence may be through the p53-dependent DNA damage signal pathway in HaCaT cells. Taken together, our data suggest that flame-retardant exposure may be a key precipitating factor for human skin aging.

Differential Gene Expression Profile in DNA Damage Signaling Pathways, in De Novo Acute Myeloid Leukemia Patients before Induction Chemotherapy

Introduction: Acute Myeloid Leukemia (AML) is a life-threatening myeloid malignancy with poor prognosis. Response to induction treatment determines long-term prognosis; however, mechanisms underlying response have not been thoroughly investigated. DNA damage and repair mechanisms influence not only the genetic predisposition to leukemia but are also important for refractoriness to treatment. Aim: The aim of this study is to investigate the possible alterations in the gene expression profile of DNA damage signaling pathways in two leukemic cell lines following their exposure to chemotherapeutic agents, namely, idarubicin and cytarabine, and to verify the findings in AML patient samples at diagnosis before the onset of chemotherapy. Methods: Cell lines Kasumi-1 with the t(8;21) and MV4-11 (biphenotypic B-myelomonocytic leukemia) were exposed either to idarubicin (0.1μΜ) for 6h or cytarabine (1μΜ) for 48h. Dead cells were eliminated from drug-treated cells using an appropriate commercial kit. Gene expression profiling through PCR arrays analysis (RT2 Profiler, Qiagen) was performed in triplicate after RNA extraction from untreated, chemotherapy-treated and live (chemo resistant) cells following chemotherapy exposure. Human DNA damage signaling pathway-related gene expression and statistical analysis was performed through the RT2 Profiler PCR Array data analysis tool. Moreover, mononuclear cells were isolated from bone marrow of 11 lymphoma patients, used as controls, and from 64 AML patients at diagnosis who subsequently received chemotherapy with the 7+3 regimen. Response to first-line chemotherapy was recorded and patients were assigned in two groups: Responders (n=53 patients) and non-Responders (n=11 patients). Total RNA was isolated (RNeasy plus Mini Kit, Qiagen), quantified and cDNA was synthesized. Relative expression of genes of interest was quantified using SYBR® Greenbased real-time RT-PCR (QuantiTect® Primer Assay, Qiagen) based on the ddCt method (Housekeeping gene GADPH). All samples were obtained from patients after informed consent and treated in accordance with the Declaration of Helsinki. Statistical analysis was performed using R Statistical Software (version 4.2.1). Results: Five genes were found significantly [p < 0.05, Students t-test] up-regulated in live cells from either leukemic cell lines after treatment with both agents and had a FoldChange > 2. More specifically PPP1R15A, CDKN1A and GADD45G genes were up-regulated in both live cell lines, GADD45A in live MV4-11 cells and EXO1 in live Kasumi cells (Figure 1A). The aforementioned genes were selected and their expression levels were quantified in human bone marrow samples. CDKN1A and GADD45G were found down-regulated in Responders [p=0.032 and p=0.001 respectively; Welch t-test] while PPP1R15A was significantly up-regulated [over 8-fold change, p= 2.08E-09; Welch t-test] compared to controls. Likewise, GADD45G and PPP1R15A were also significantly down and up regulated respectively, in non-Responders [p=0.08 and p=1.51E-05, Welch t-test]. Interestingly, PPP1R15A presented a 1.7-fold overexpression trend in non-Responders compared to Responders [p=0.18, Welch t-test] (Figure 1B). Conclusion: The results of our study suggest a dysregulation of the DNA damage and repair pathways in AML. Upregulation of GADD45G is known to dramatically induce apoptosis, differentiation, and growth arrest while increasing sensitivity of AML cells to chemotherapeutic drugs (Guo, Dan et al., Blood 2021). CDKN1A is involved in p53/TP53 mediated inhibition of cellular proliferation in response to DNA damage. Both were down-regulated in AML patients and are likely to play an important role in the pathogenesis of AML. PPP1R15A participates in DNA damage response facilitating the recovery of cells from stress. The up-regulation of PPP1R15A both in live cells treated with Ara-C and Cytarabine, as well as the 1.7-fold overexpression trend in non-Responders compared to Responders, supports the notion that PPP1R15A could be associated with resistance to induction treatment, which may have significant prognostic and therapeutic implications. A greater sample of AML patients both responding and not responding to chemotherapy, would be essential to delineate the role of genes involved in DNA damage and repair pathways in resistance to induction treatment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Targeting Sumoylation with Subasumstat (TAK-981), a Selective Inhibitor of SUMO-Activating Enzyme (SAE), Induces Mitochondrial Dysfunction in Preclinical Models of Non-Hodgkin Lymphoma (NHL)

SUMOylation is a post-translational modification which leads to covalent attachment of small ubiquitin-like modifiers (SUMO) proteins to lysine residues of a target protein. SUMOylation is essential for the regulation of genomic integrity, gene expression and intracellular signaling. Many tumors exhibit deregulation of SUMOylation pathway. Subasumstat, (TAK-981), a novel selective inhibitor of SUMO-activating enzyme (SAEi), has entered early phase II clinical trials. Here we studied SAEi in pre-clinical models of B-cell non-Hodgkin's lymphoma (NHL). TAK-981 was provided by Takeda Development Center Americas, Inc. (Lexington, MA). Diffuse large B-cell lymphoma (DLBCL) cell lines (OCI-LY3, VAL, U-2932, SU-DHL10), and primary mantle cell lymphoma (MCL) cells were assayed for survival, mitochondrial function, and metabolic phenotype. In vivo experiments were performed using DLBCL cell line xenografts and MCL PDX models. Treatment with TAK-981 restricted growth of DLBCL cell lines with an IC50~10 nM. SAEi led to rapid (within 2 h) protein desumoylation in a dose dependent manner, prominently in the ~90 kDa region. TAK-981 induced pronounced double strand DNA breaks in DLBCL cells (comet assay) and cell cycle arrest (propidium iodine). RNA-Seq and ATAC-Seq analysis of TAK-981-treated DLBCL cells revealed upregulation of the DNA damage, NF-κB, and OXPHOS signaling pathways. We noted that in addition to cytoplasmic proteins, mitochondrial proteins were heavily de-SUMOylated upon treatment of NHL cells with TAK-981. In vitro exposure to TAK-981 resulted in mitochondrial membrane depolarization, loss of mitochondrial integrity (by electron microscopy) and a change of mitochondrial dynamics as assessed by expression of mitochondrial proteins (eg, Drp1). Seahorse respirometry analysis revealed dramatic downmodulation of OXPHOS following SAEi. Furthermore, metabolomic profiling of DLBCL cells treated with TAK-981 showed downmodulation of TCA substrates, while glycolysis substrates remained unchanged. The mitochondrial disruption observed following TAK-981 treatment was accompanied by rapid accumulation of reactive oxygen species, a mechanism that has been previously shown to lead to DNA damage in cancer cells. Consistent with cell line data, exposure of primary MCL cells to TAK-981 in stromal conditions (CD40L- or BAFF expressing stroma) induced apoptosis and reduced OXPHOS. Next, we conducted genome-wide CRISPR-Cas9 loss-of-function library screens and identified that loss of genes in the NFκB, TP53, DNA damage and centromere/telomere gene pathways contributed to resistance to SAEi in DLBCL cells. By means of genetic knockout we demonstrated that TP53 and its transcriptional target BAX contributed to TAK-981-induced G2 arrest and apoptosis, further implicating mitochondrial dysfunction in sensitivity to SAEi. For in vivo experiments, we used DLBCL xenografts. OCI-LY3 and U-2932 cells were inoculated subcutaneously in NSG mice. Once tumors reached 100 mm3, mice received 10 twice-weekly IV doses of 7.5 mg/kg TAK-981 (over 5 weeks), leading to resolution of flank tumors and improved survival compared with control. Finally, we used an MCL PDX model where MCL cells were inoculated intravenously. Once cells became detectable in the blood, mice were treated as above. Treatment with TAK-981 resulted in delayed tumor expansion in blood and spleen and extended animal survival. TAK-981-exposed splenocytes exhibited a reduction of maximal respiration by Seahorse analysis. Conclusions. Pharmacologic SAEi with TAK-981 demonstrated pre-clinical activity in NHL models in vitro and in vivo, accompanied by DNA damage, mitochondrial dysfunction, and metabolic reprogramming.