Phosphorylated Histone H2AX Research Articles

Murine Myoblasts Exposed to SYUIQ-5 Acquire Senescence Phenotype and Differentiate into Sarcopenic-Like Myotubes, an In Vitro Study.

The musculoskeletal system is one of the most affected organs by aging that correlates well with an accumulation of senescent cells as for other multiple age-related pathologies. The molecular mechanisms underpinning muscle impairment because of senescent cells are still elusive. The availability of in vitro model of skeletal muscle senescence is limited and restricted to a small panel of phenotypic features of these senescent cells in vivo. Here, we developed a new in vitro model of senescent C2C12 mouse myoblasts that, when subjected to differentiation, the resulting myotubes showed sarcopenic features. To induce senescence, we used SYUIQ-5, a quindoline derivative molecule inhibitor of telomerase activity, leading to the expression of several senescent hallmarks in treated myoblasts. They had increased levels of p21 protein accordingly with the observed cell cycle arrest. Furthermore, they had enhanced SA-βgalactosidase enzyme activity and phosphorylation of p53 and histone H2AX. SYUIQ-5 senescent myoblasts had impaired differentiation potential and the resulting myotubes showed increased levels of ATROGIN-1 and MURF1, ubiquitin ligases components responsible for protein degradation, and decreased mitochondria content, typical features of sarcopenic muscles. Myotubes differentiated from senescent myoblasts cultures release increased levels of MYOSTATIN that could affect skeletal muscle cell growth. Overall, our data suggest that a greater burden of senescent muscle cells could contribute to sarcopenia. This study presents a well-defined in vitro model of muscle cell senescence useful for deeper investigation in the aging research field to discover new putative therapeutic targets and senescence biomarkers associated with the aged musculoskeletal system.

Abstract B022: C11ORF68 is essential for γH2AX formation after replication stress

Abstract Introduction: Unresolved replication stress (RS) often leads to genomic instability – a feature of many diseases including cancer. RS can activate downstream DNA damage response (DDR) phenotypes such as phosphorylation of histone H2AX (γH2AX). Therefore, understanding the molecular foundation of DDR is crucial for identifying potential biomarkers or therapeutic targets. Our lab has identified an uncharacterized protein that is symmetrically dimethylated by PRMT5 under hydroxyurea (HU) induced RS. Therefore, we hypothesize that C11ORF68 is involved in the RS and DNA damage response. Materials and methods: RS and DNA damaging agents including HU and etoposide were used to induce RS or DNA damage in various cell lines. C11ORF68 was depleted using siRNA and dicer siRNA (dsiRNA) systems. Immunofluorescence and immunoblotting were used to evaluate DDR phenotypes. Results: One of the earliest phenomenon in DDR is phosphorylation of histone H2AX (γH2AX). Depletion of C11ORF68 under HU-induced RS reduced γH2AX foci formation as compared to cells treated with control siRNA or dsiRNA. Since HU affects cells entering S-phase, we evaluated if this is a cell cycle-related effect by using other DNA damaging agents that target cells in all phases of the cell cycle. Similarly, depletion of C11ORF68 under DNA damaging conditions showed reduced γH2AX foci formation. This may be due to a lack of upstream PIKK kinases activation as knockdown of C11ORF68 also attenuated phosphorylation of PIKKs important for H2AX phosphorylation after treatment with DNA damaging agents and RS-inducing agents. Conclusions: Our data suggests that C11ORF68 it appears to modulate early DDR even before H2AX phosphorylation and upstream PIKK activation. We are currently investigating if C11ORF68 is involved in initial events after DNA damage or RS such as PARylation events or MRN sensing of DNA breaks. Citation Format: Clarissa Chin Min Toh, Mai Phuong Hoang, Anand Jeyasekharan. C11ORF68 is essential for γH2AX formation after replication stress [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 B022.

The Genotoxicity of Organic Extracts from Particulate Emissions Produced by Neat Gasoline (E0) and a Gasoline-Ethanol Blend (E15) in BEAS-2B Cells.

Emissions from modern gasoline engines represent an environmental and health risk. In this study, we aimed to compare the toxicity of organic compound mixtures extracted from particulate matter (PM extracts) produced by neat gasoline (E0) and a blend containing 15% ethanol (E15), which is offered as an alternative to non-renewable fossil fuels. Human lung BEAS-2B cells were exposed to PM extracts, and biomarkers of genotoxicity, such as DNA damage evaluated by comet assay, micronuclei formation, levels of phosphorylated histone H2AX, the expression of genes relevant to the DNA damage response, and exposure to polycyclic aromatic hydrocarbons (PAHs), were determined. Results showed that both PM extracts significantly increased the level of oxidized DNA lesions. The E0 extract exhibited a more pronounced effect, possibly due to the higher content of nitrated PAHs. Other endpoints were not substantially affected by any of the PM extracts. Gene expression analysis revealed mild but coordinated induction of genes related to DNA damage response, and a strong induction of PAH-inducible genes, indicating activation of the aryl hydrocarbon receptor (AhR). Our data suggest that the addition of ethanol into the gasoline diminished the oxidative DNA damage, but no effect on other genotoxicity biomarkers was observed. Activated AhR may play an important role in the toxicity of gasoline PM emissions.

Abstract C146: VIC-1911, a selective Aurora kinase A inhibitor, synergizes with sacituzumab govitecan in triple-negative breast cancer

Abstract BACKGROUND: Triple-negative breast cancer (TNBC) accounts for 15-20% of breast cancer cases and 30-40% of U.S. breast cancer deaths. Although current precision treatments improve TNBC patient outcomes, TNBC heterogeneity has led to therapeutic targeting challenges. Also, the resistance of TNBC to conventional treatment remains a significant clinical problem. Therefore, informed evaluation of new targets can improve the treatment of TNBC, representing an unmet need. Dysregulation of the cell cycle is a hallmark of cancer for unlimited proliferation. Abnormal mitosis and cell-cycle checkpoints are crucial in leading to aneuploidy and genetic instability. Aurora kinase A (AURKA), a serine/threonine kinase, regulates mitosis in the early stages (G2/M phase) by regulating centrosome maturation and disjunction, thereby establishing a bipolar mitotic spindle. VIC-1911 (previously known as TAS-119), a novel, orally active, highly selective inhibitor of AURKA with a low toxicity profile, suppresses the growth of various cancer cell lines in vitro and in vivo. As AURKA can modulate DNA damage response, we hypothesized that VIC-1911 combined with sacituzumab govitecan (SG), which has a topoisomerase 1 inhibitor as a payload, has a synergistic antitumor effect on TNBC. MATERIALS AND METHODS: To evaluate the short-term in vitro efficacy of VIC-1911 alone and combination with SG, a sulforhodamine B cell proliferation assay was performed using 10 TNBC cell lines. For long-term treatment conditions, a colony formation assay was conducted. Western blot and immunofluorescent analyses were conducted to confirm treatment-mediated DNA damage and apoptosis. Human TNBC cell line–derived mammary fat pad xenograft models were used to evaluate the antitumor effect of VIC-1911 and SG. RESULTS: In vitro proliferation, data demonstrated that single-agent VIC-1911 had a nanomolar to micromolar range of half-maximal inhibitory concentrations (2.69 nM to 10.6 mM) in the tested TNBC cell lines. The half-maximal inhibitory concentration was correlated with c-Myc expression (R2=0.4876, p=0.0247). Exposure to the combination of VIC-1911 and SG led to significant death of VIC-1911–sensitive TNBC cell lines (p<0.05). Western blot and immunofluorescent analysis revealed that VIC-1911 and SG combination caused TNBC cell death by inducing expression of phosphorylated histone H2AX (DNA damage marker) and cleaved poly (ADP-ribose) polymerase (apoptosis marker). Furthermore, the combination of VIC-1911 and SG had significantly greater antitumor efficacy than did either agent alone in SUM149 TNBC xenograft models (tumor growth inhibition rate (TGI): VIC-1911, 61.4%; SG, 68.3%; combination, 82.9%; p<0.01) and HCC1806 TNBC xenograft models (TGI: VIC-1911, 49.3%; SG, 66.3%; combination, 85.9%; p<0.01). CONCLUSION: The AURKA inhibitor VIC-1911 has a synergistic antitumor effect with SG by inducing DNA damage in TNBC cells. PDX and additional mechanistic studies are planned to further evaluate VIC-1911 and SG as novel combination therapy for TNBC. Citation Format: Jangsoon Lee, YoungJin Gi, Thomas Myers, Linda Paradiso, Debu Tripathy, Naoto T. Ueno. VIC-1911, a selective Aurora kinase A inhibitor, synergizes with sacituzumab govitecan in triple-negative breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C146.

DNA Damage as an Early Marker of Senescence in Tauopathies

AbstractBackgroundHyperphosphorylated tau accumulation in neurons and astrocytes is a pathological hallmark of a class of neurodegenerative disorders known as “tauopathies”, such as progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Alzheimer’s disease (AD) and chronic traumatic encephalopathy (CTE). Previous research investigating the toxic effects of astrocytic tau pathology identified the DNA damage marker p16 in astrocytes from post‐mortem human cases of AD and FTD. DNA damage is one of many markers of cellular senescence, a state in which cells undergo irreversible cell cycle arrest. Senescence initiation can also be identified via the expression of phosphorylated histone H2AX (gamma‐H2AX) and cyclin‐dependent kinase inhibitor p21. Senescence in astrocytes leads to reduced functional support for neurons. While recognized, the role of senescent astrocytes in tauopathies is not well understood. Here, we sought to determine how expression of other DNA damage markers, such as gamma‐H2AX and p21, and the DAMP protein HMGB1 varies across different tauopathies.MethodImmunohistochemistry (IHC) was performed on postmortem human frontal cortex tissue of CTE (n = 3), AD (N = 3), PSP (n = 3), CBD (n = 3), Healthy (n = 1) and glioblastoma (GBM) (n = 1) cases with antibodies against markers of early senescence gamma H2A.x (yH2A.x) and p21, late senescence, p16, and a senescence‐associated secretory phenotype, HMGB1. Positively stained nuclei were quantified. Immunofluorescence (IF) was employed on postmortem cases using either glial fibrillary acidic protein (GFAP) or Neurofilament H (NF‐H) antibodies with yH2A.x, p16, p21 and HMGB1 markers in order to qualitatively and quantitatively measure the prevalence of senescent astrocytes and neurons in tauopathies.ResultImmunohistochemistry revealed yH2Ax expression was observed in AD, PSP, and CBD cases. Immunofluorescence experiments employing yH2AX co‐stained with astrocytic and neuronal markers revealed expression in AD, PSP and CBD cases. Minimal expression of yH2Ax was revealed in the CTE and healthy control cases. Results from p21 stains were inconclusive. Preliminary evidence reveals HMGB1 expression in CBD and PSP cases utilizing IF techniques, and p16 expression in cells employing IHC.ConclusionOverall, results indicate that DNA damage may be broadly associated with astrocytes in tauopathies. Utilizing alternative approaches to recognize and evaluate astrocyte senescence in post‐mortem human tissue will provide further insight into these findings.

γH2AX in mouse embryonic stem cells: Distribution during differentiation and following γ-irradiation

Phosphorylated histone H2AX (γH2AX) represents a sensitive molecular marker of DNA double-strand breaks (DSBs) and is implicated in stem cell biology. We established a model of mouse embryonic stem cell (mESC) differentiation and examined the dynamics of γH2AX foci during the process. Our results revealed high numbers of γH2AX foci in undifferentiated mESCs, decreasing as the cells differentiated towards the endothelial cell lineage. Notably, we observed two distinct patterns of γH2AX foci: the typical discrete γH2AX foci, which colocalize with the transcriptionally permissive chromatin mark H3K4me3, and the less well-characterized clustered γH2AX regions, which were only observed in intermediate progenitor cells. Next, we explored responses of mESCs to γ-radiation (137Cs). Following exposure to γ-radiation, mESCs showed a reduction in cell viability and increased γH2AX foci, indicative of radiosensitivity. Despite irradiation, surviving mESCs retained their differentiation potential. To further exemplify our findings, we investigated neural stem progenitor cells (NSPCs). Similar to mESCs, NSPCs displayed clustered γH2AX foci associated with progenitor cells and discrete γH2AX foci indicative of embryonic stem cells or differentiated cells. In conclusion, our findings demonstrate that γH2AX serves as a versatile marker of DSBs and may have a role as a biomarker in stem cell differentiation. The distinct patterns of γH2AX foci in differentiating mESCs and NSPCs provide valuable insights into DNA repair dynamics during differentiation, shedding light on the intricate balance between genomic integrity and cellular plasticity in stem cells. Finally, the clustered γH2AX foci observed in intermediate progenitor cells is an intriguing feature, requiring further exploration.

Venetoclax Synergizes with IMGN632, a Novel CD123-Targeting Antibody Conjugated to a DNA Alkylating Payload, By Suppressing DNA Damage Response and Potentiating Apoptosis in Acute Myeloid Leukemia in Vitro Models

CD123, a component of interleukin-3 receptor, is overexpressed on the surface of acute myeloid leukemia (AML) blasts and leukemic stem cells compared with normal hematopoietic progenitors, which makes this antigen an attractive therapeutic target. IMGN632 is a novel antibody-drug-conjugate comprising a high affinity CD123-targeting antibody and DNA alkylating cytotoxic payload. In preclinical studies, IMGN632 demonstrated high potency and selective cytotoxicity against AML leukemic cells vs normal myeloid progenitors, suggesting reduced likelihood of myelosuppression (Kovtun, 2018). Importantly, we previously showed high synergy of IMGN632 combination with BCL-2 inhibitor venetoclax (VEN) and DNA hypomethylating azacytidine (AZA) in AML cell lines and xenograft models (ASH 2020, #617). IMGN632 demonstrated encouraging single agent activity and favorable tolerability in relapsed/refractory AML patients (ASH 2019 #734, ASCO 2020 TPS7563, NCT03386513) and is currently tested in combination with VEN and/ or AZA in relapsed and frontline CD123-positive AML patients (NCT04086264). In this study, we provide new insight into molecular mechanisms underlying the synergistic efficacy of these combinations using AML models in vitro. AML cells with FLT3-ITD mutations expressed higher levels of CD123, as previously reported (Han 2013). FLT3-ITD cell lines were particularly sensitive to IMGN632 with IC 50 values at low ng/ml range. Correspondingly, as evidenced by large scale drug combination screen using BLISS independence model, triple IMGN632/VEN/AZA treatment was synergistic at multiple dosage levels and ratios, suggesting a favorable potency of triple combination in FLT3-mutated subtypes of AML associated with poor prognosis. The efficacy of IMGN632/VEN/AZA combination was however decreased in cells lacking functional TP53. shRNA-mediated knockdown of TP53 in FLT3-ITD MOLM13 cells resulted in increased cell viability, reduced apoptosis and downregulation of cleaved caspase-3 and PARP. While both treated wild-type and TP53 KD cells showed robust overexpression of phosphorylated histone H2AX (p-H2AX), TP53 KD cells expressed higher levels of anti-apoptotic MCL-1 and lower levels of pro-apoptotic BAX, indicative of their intrinsic inability to fully execute apoptosis and resulting in suppressed response to triple combination. Importantly, in sensitive AML cells, IMGN632 induced profound DNA damage, G 1 (MOLM13 and MOLM14 cells) or G 2/M (MV4-11 cells) phase cell cycle arrest and decline in S-phase population, all above augmented by VEN/AZA co-treatment. Observed increase in p-H2AX was accompanied by apoptotic caspase-3 and PARP cleavage and was fully prevented by QVD-OPh caspase inhibitor, suggesting that double-stranded DNA damage and p-H2AX triggered by triple combination were secondary events associated with apoptosis but not the initial cause of cell cycle arrest. Thus, given that cytotoxic payload of IMGN632 is a DNA alkylator, we next tested its impact on mediators of the DNA damage response (DDR). IMGN632 alone induced phosphorylation of p53 at Ser15, a step required for p53 transcriptional activity, and triggered activating phosphorylation of checkpoint kinase Chk1 (Ser345), known to facilitate cell cycle arrest and DNA damage repair, thus preventing treated cells from progressing through S-phase but also increasing the potential of repairing IMGN632-induced DNA damage (Fig. 1A). Upon combined IMGN632+VEN treatment, p53 phosphorylation was sustained, but p-ChK1 decreased, suggesting that VEN may impair protective DDR triggered by IMGN632, resulting in greater cell death. To support this hypothesis, we showed that IMGN632 treatment prior to VEN produced significant synergistic activity (Fig. 1B). Together, these results suggest that VEN, apart from its canonical inhibitory effect on anti-apoptotic BCL-2, exerts previously unrecognized ability to suppress DDR program in AML and augments activity of DNA damaging IMGN632. Failure of cells to sustain DDR in the presence of VEN constitutes a key aspect of high efficacy of IMGN/VEN/AZA combination in AML.

Evaluating a novel protective agent against radiation-induced acute intestinal injury

ObjectiveTo develop and synthesize a novel derivative of ethyl pyruvate, named TZC02, and investigate its radioprotective effects against ionizing radiation (IR)-induced intestinal injury in mice. MethodsMale C57BL/6J mice weighing (20 ​± ​2) g in the survival experiment were randomly divided into five groups (n ​= ​10 in each): control group, IR group, IR ​+ ​TZC02 (50 ​mg/kg) group, IR ​+ ​TZC02 (100 ​mg/kg) group, and IR ​+ ​TZC02 (200 ​mg/kg) group. Mice's survival rates were monitored for 7 ​d. In other experiments, the male mice were randomly divided into three groups (n ​= ​5 per group): control group, IR group, and IR ​+ ​TZC02 (100 ​mg/kg) group. TZC02 was intragastrically administered 1 ​h before 12 ​Gy abdominal γ-ray irradiation (ABI) and 24 h, 48 h after irradiation, respectively. Three days after IR exposure, small intestinal tissues were collected and the number of small intestinal crypts was determined using hematoxylin & eosin (H&E) staining. Immunohistochemical analysis was used to assess the regenerative capacity of the small intestine (SI) and radiation-induced damage, stemness markers or DNA repair surrogates, including Ki67, lysozyme, and villus. The expressions of histone H2AX phosphorylation (γH2AX) and caspase-3 were evaluated through immunofluorescence analyses. Additionally, in vitro cultured small intestinal organoids were employed to investigate the effects of TZC02 on SI regeneration after irradiation. ResultsThe administration of TZC02 significantly improved the 7 ​d- survival rate of mice exposed to 12 ​Gy ABI (P ​< ​0.05). Compared to the IR group, TZC02 treatment attenuated the decrease of SI Ki67-positive cells [(59.60 ​± ​6.33) vs. (37.70 ​± ​7.82), t ​= ​11.89, P ​< ​0.0001) and Paneth cells [(9.90 ​± ​1.37) vs.(5.50 ​± ​1.71) , t ​= ​6.02, P ​< ​0.001) in five crypts, and reduced structural damage to the SI [villus height, (349.49 ​± ​60.17) μm vs. (294.72 ​± ​40.09) ​μm; t ​= ​3.39; P < ​0.05]. TZC02 also significantly decreased the crypt apoptosis detected by caspase-3 [(10.75 ​± ​1.26) vs. ​(29.83 ​± ​2.56), t ​= ​13.39, P ​< ​0.0001) and DNA damage detected by gH2AX [(10.40 ​± ​1.14) vs. ​(29.60 ​± ​2.70), t ​= ​10.13, P ​< ​0.0001)]. The organoid survival 7 ​d post-irradiation further confirmed the protective effects of TZC02 (area of organoids, (0.119 ​± ​0.081) ​mm2vs. ​(0.080 ​± ​0.037) ​mm2; t ​= ​2.30; P ​< ​0.05). ConclusionsThis study demonstrate that TZC02 can offer effective protection against IR-induced intestinal injury, suggesting its potential as a promising protective compound for patients treated with radiotherapy.

Prediction of late adverse events in pelvic cancer patients receiving definitive radiotherapy using radiation-induced gamma-H2AX foci assay.

Radiation can induce DNA double-stranded breaks, which are typically detected by the fluorescence of phosphorylated histone H2AX. In this study, we examined the usefulness of the dynamics of radiation-induced gamma-H2AX foci of peripheral blood lymphocytes (PBLs), as a marker of DNA repair ability, in predicting late adverse events from radiotherapy. A total of 46 patients with cervical, vaginal and anal canal cancers treated with radical radiotherapy between 2014 and 2019 were included in this analysis. Concurrent chemotherapy was administered in 36 cases (78.3%). Peripheral blood was obtained before treatment, and then irradiated ex vivo with 1Gy X-ray. The ratio of radiation-induced gamma-H2AX foci in PBLs measured at 30min and at 4h was defined as the foci decay ratio (FDR). With a median follow-up of 54months, 9 patients (19.6%) were observed to have late genitourinary or gastrointestinal (GU/GI) toxicity. The FDR ranged from 0.51 to 0.74 (median 0.59), with a significantly higher incidence of Grade 1 or higher late adverse events in the FDR ≥ 0.59 group. In multivariate analysis, FDR ≥ 0.59 and hypertension also emerged as significant factors associated with the development of late toxicities. Overall, our results suggest that measurement of radiation-induced gamma-H2AX foci in PBLs may predict the risk of late GU/GI toxicities from chemoradiotherapy, which can enable tailoring the radiation dose to minimize adverse effects.

Pro-Apoptotic Activity of MCL-1 Inhibitor in Trametinib-Resistant Melanoma Cells Depends on Their Phenotypes and Is Modulated by Reversible Alterations Induced by Trametinib Withdrawal.

Although BRAFV600/MEK inhibitors improved the treatment of melanoma patients, resistance is acquired almost inevitably. Trametinib withdrawal/rechallenge and MCL-1 inhibition in trametinib-resistance models displaying distinct p-ERK1/2 levels were investigated. Trametinib withdrawal/rechallenge caused reversible changes in ERK1/2 activity impacting the balance between pro-survival and pro-apoptotic proteins. Reversible alterations were found in MCL-1 levels and MCL-1 inhibitors, BIM and NOXA. Taking advantage of melanoma cell dependency on MCL-1 for survival, we used S63845. While it was designed to inhibit MCL-1 activity, we showed that it also significantly reduced NOXA levels. S63845-induced apoptosis was detected as the enhancement of Annexin V-positivity, caspase-3/7 activation and histone H2AX phosphorylation. Percentages of Annexin V-positive cells were increased most efficiently in trametinib-resistant melanoma cells displaying the p-ERK1/2low/MCL-1low/BIMhigh/NOXAlow phenotype with EC50 values at concentrations as low as 0.1 μM. Higher ERK1/2 activity associated with increased MCL-1 level and reduced BIM level limited pro-apoptotic activity of S63845 further influenced by a NOXA level. Our study supports the notion that the efficiency of an agent designed to target a single protein can largely depend on the phenotype of cancer cells. Thus, it is important to define appropriate phenotype determinants to stratify the patients for the novel therapy.

Evaluation of guide-free Cas9-induced genomic damage and transcriptome changes in pig embryos

Cas9 protein without sgRNAs can induce genomic damage at the cellular level invitro. However, whether the detrimental effects occur in embryos after Cas9 treatment remains unknown. Here, using pig embryos as subjects, we observed that Cas9 protein transcribed from injected Cas9 mRNA can persist until at least the blastocyst stage. Cas9 protein alone can induce genome damage in preimplantation embryos, represented by the increased number of phosphorylated histone H2AX foci on the chromatin fiber, which led to apoptosis and decreased cell number of blastocysts. In addition, single-blastocyst RNA sequencing confirmed that Cas9 protein without sgRNAs can cause changes in the blastocyst transcriptome, depressing embryo development signal pathways, such as cell cycle, metabolism, and cellular communication-related signal pathways, while activating apoptosis and necroptosis signal pathways, which together resulted in impaired preimplantation embryonic development. These results indicated that attention should be given to the detrimental effects caused by the Cas9protein when using CRISPR-Cas9 for germline genome editing, especially for the targeted correction of human pathological mutations using germline gene therapy.

The disulphide cleavage derivative (C42-4) of 11′-deoxyverticillin A (C42) fails to induce apoptosis and genomic instability in HeLa cells

ABSTRACT Our previous study revealed 11’-deoxyverticillin A (C42), a natural product isolated from the Ophiocordyceps-associated fungus Clonostachys rogersoniana and a member of the epipolythiodioxopiperazines (ETPs), induced both apoptosis and autophagy in HCT116 cells; however, the role of disulphide/polysulphide bridges of C42 in the regulation of autophagy remains unexplored. Here, we revealed that C42 activated both caspase-dependent apoptosis and autophagy in HeLa cells, whereas its disulphide cleavage derivative C42-4 failed to induce the cleavage of both caspase-3 and PARP-1. In contrast, both C42 and C42-4 increased the formation of autophagosomes, punctate staining of LC3, and the ratio of LC3-II to actin, suggesting that disulphide/polysulphide bridges are dispensable for the induction of the autophagic process. Moreover, we found that C42 but not C42-4 led to nuclear instability by increasing the formation of micronuclei and expression of phosphorylated histone H2AX (γ-H2AX), a widely used marker for DNA double strand breaks (DSBs), while Rad51, a protein pivotal for DNA repair, was decreased upon challenge with C42. These results demonstrate that the disulphide bonds in ETPs play an essential role in the induction of caspase-dependent apoptosis and nuclear stability.

Oxidative and DNA damage in obese patients undergoing bariatric surgery: A one-year follow-up study

The pathogenesis of obesity and related comorbidities has long been associated with oxidative stress. The excess of adipose tissue contributes to the production of free radicals that sustain both a local and a systemic chronic inflammatory state, whereas its reduction can bring to an improvement in inflammation and oxidative stress. In our work, using the fluorescent lipid probe BODIPY® 581/591 C11 and the γH2AX foci assay, a well-known marker of DNA double strand breaks (DSB), we evaluated the extent of cell membrane oxidation and DNA damage in peripheral blood lymphocytes of normal weight (NW) controls and obese patients sampled before and after bariatric surgery. Compared to NW controls, we observed a marked increase in both the frequencies of oxidized cells or nuclei exhibiting phosphorylation of histone H2AX in preoperatory obese patients. After bariatric surgery, obese patients, resampled over one-year follow-up, improved oxidative damage and reduced the presence of DSB. In conclusion, the present study highlights the importance for obese patients undergoing bariatric surgery to also monitor these molecular markers during their postoperative follow-up.

Insights into DNA Damage in Regenerating Young and Aged Mouse Livers

Abstract Background The maintenance of genome stability is a key process to slow aging. One of the mechanisms ensuring this stability is the correct coordination of origins of replication (ORI), resulting in the successful transmission of DNA. Previously, we mapped and compared ORI firing between young and aged mice in vivo, using a regenerating liver mouse model. We confirmed a decreased hepatocyte ORI efficiency in aged mice, known to have impaired liver regeneration. ORI firing proved to be fully rescued when blocking the aged mice's ATR serine/threonine-protein kinase, suggesting that the DNA replication checkpoint actively mediates ORI firing impairment upon DNA damage detection. Aims To explore DNA damage differences between young and aged mice regenerating livers. Methods To induce proliferation, mice were subjected to partial hepatectomy (PH) and liver sections were harvested at different timepoints. Immunohistochemistry staining (IHC) were used to address proliferation and DNA damage, using Ki67 and serine 139 phosphorylated histone H2AX (g-H2AX) as markers, respectively. Results We confirmed a lack of Ki67 signal in young and aged mice prior to PH. The signal takes off at 24-28h post-PH and reaches its peak at 36-48h, which is significantly lower in aged mice. After 48h post-PH, young hepatocytes’ Ki67 reaches its basal level 120h post-PH. However, aged hepatocytes’ Ki67 is maintained at low levels overtime. Next, we compared DNA damage kinetics between young and aged mice livers. Both mouse groups present an increase of g-H2AX upon PH, higher in young mice. The g-H2AX signal decreases in young hepatocytes after 48h post-PH until disappearing 120h post PH. Aged mice hepatocytes maintain the g-H2AX rates overtime after 48h post-PH. Conclusions Our data suggests that hepatocytes develop DNA damage upon proliferation, which is able to be resolved in young mice hepatocytes but remains present in aged livers, ultimately leading to impaired liver regeneration.

Resistance of Leukemia Cells to 5-Azacytidine: Different Responses to the Same Induction Protocol.

Three AML cell variants (M/A, M/A* from MOLM-13 and S/A from SKM-1) were established for resistance by the same protocol using 5-azacytidine (AZA) as a selection agent. These AZA-resistant variants differ in their responses to other cytosine nucleoside analogs, including 5-aza-2'-deoxycytidine (DAC), as well as in some molecular features. Differences in global DNA methylation, protein levels of DNA methyltransferases, and phosphorylation of histone H2AX were observed in response to AZA and DAC treatment in these cell variants. This could be due to changes in the expression of uridine-cytidine kinases 1 and 2 (UCK1 and UCK2) demonstrated in our cell variants. In the M/A variant that retained sensitivity to DAC, we detected a homozygous point mutation in UCK2 resulting in an amino acid substitution (L220R) that is likely responsible for AZA resistance. Cells administered AZA treatment can switch to de novo synthesis of pyrimidine nucleotides, which could be blocked by inhibition of dihydroorotate dehydrogenase by teriflunomide (TFN). This is shown by the synergistic effect of AZA and TFN in those variants that were cross-resistant to DAC and did not have a mutation in UCK2.

Association of encapsulation of CDK12 inhibitor with anti-tumour activity in colon cancer cell lines.

e15077 Background: Colorectal cancer (CRC) is the third most common cancer worldwide. Unfortunately, despite the incorporation of new therapeutic options, the metastatic setting is still an incurable disease. Cyclin-dependent kinases (CDKs) are a large family of proteins involved in cell cycle and transcriptional regulation, and are highly expressed in colon cancer, such as CDK12. Here, we explore the antitumor activity of a novel CDK12 inhibitor (CDK12-IN-E9) in preclinical models of colon cancer, and its liposomal formulation (LP-CDK12-IN-E9). Methods: Expression of CDK12 at a genomic level was assessed using web tools such as the Colonomics database. CDK12-IN-E9 was encapsulated in a liposomal formulation to evaluate differences between the free and the liposome form. The effect of compounds on cell viability was assessed with MTT assays. Matrigel assays were performed to study invasiveness and wound assays for migration. In addition, cell cycle and apoptosis studies were carried out by flow cytometry with 7-AAD, annexin V staining, and western-blot for specific protein expression. DNA damage and caspase degradation assays were performed by immunofluorescence. Results: Genomic mapping of CDK12 showed a higher expression in CRC than in normal tissue ( p−value = 1.424e−43). The CDK12 inhibitor was formulated in liposomes LP-CDK12-IN-E9 with the following characteristics: a polydispersity index of 0.11 ±0.02, a hydrodynamic radius of 62.98 ±10.29 nm and a zeta potential of -27.12±1.65 mV. CDK12-IN-E9 induced an antiproliferative effect in SW620, SW480 and HCT116 colon cancer cell lines. LP-CDK12-IN-E9 displayed an EC50 two times higher compared to CDK12-IN-E9 and a higher reduction of invasiveness between free inhibitor and its liposomal formulation mainly in HCT116 ( p-value &lt; 0.001). Cell cycle studies showed no significant differences between CDK12-IN-E9 and LP-CDK12-IN-E9, but there was a profound induction of cell death at the highest doses through apoptosis mediated largely by caspases. In parallel to the effect on cell death, an increase in histone H2AX phosphorylation and an increase in cleaved caspase-3 was observed, which also implies a large increase in DNA damage in tumor cells. In vivo studies are ongoing. Conclusions: In summary, LP- CDK12-IN-E9 showed more antitumoral activity than its free formulation in CRC cell lines mainly through a caspase-dependent induction of apoptosis. This data suggests that liposomal formulation of novel chemical entities can augment its antitumoral properties.

The Function of MAP Kinases in Induced Histone H2AX Phosphorylation in Transformed Cells

The Function of MAP Kinases in Induced Histone H2AX Phosphorylation in Transformed Cells

Astaxanthin protected against the adverse effects induced by diesel exhaust particulate matter via improving membrane stability and anti-oxidative property

Diesel exhaust particulate matter (DPM), which has been clarified as a Group I carcinogenic agent, is still challenging in its detoxification due to the complex composition and toxic mechanisms. Astaxanthin (AST) is a pleiotropic small biological molecule widely used in medical and healthcare with surprising effects and applications. The present study aimed to investigate the protective effects of AST on DPM-induced injury and the underlying mechanism. Our results indicated that AST significantly suppressed the generation of phosphorylated histone H2AX (γ-H2AX, marker of DNA damage) and inflammation caused by DPM both in vitro and in vivo. Mechanistically, AST prevented the endocytosis and intracellular accumulation of DPM via regulating the stability and fluidity of plasma membranes. Moreover, the oxidative stress elicited by DPM in cells could also be effectively inhibited by AST, together with protecting the structure and function of mitochondria. These investigations provided clear evidence that AST notably reduced DPM invasion and intracellular accumulation by modulating the membrane-endocytotic pathway, which eventually reduced intracellular oxidative stress caused by DPM. Our data might provide a novel clue for curing and treating the harmful effects of particulate matter.

Observation of Histone H2AX Phosphorylation by Radiation-Induced Bystander Response Using Titanium Characteristic X-ray Microbeam.

Radiation-induced bystander response (RIBR) is a response induced in non-irradiated cells that receive bystander signals from directly irradiated cells. X-ray microbeams are useful tools for elucidating the mechanisms underlying RIBR. However, previous X-ray microbeams used low-energy soft X-rays with higher biological effects, such as aluminum characteristic X-rays, and the difference from conventional X-rays and γ-rays has often been discussed. The microbeam X-ray cell irradiation system at the Central Research Institute of Electric Power Industry has been upgraded to generate higher energy titanium characteristic X-rays (TiK X-rays), which have a longer penetration distance sufficient to irradiate 3D cultured tissues. Using this system, we irradiated the nuclei of HeLa cells with high precision and found that the pan-nuclear induction of phosphorylated histone H2AX on serine 139 (γ-H2AX) in the non-irradiated cells increased 180 and 360 min after irradiation. We established a new method to quantitatively evaluate bystander cells, using the fluorescence intensity of γ-H2AX as an indicator. The percentage of bystander cells increased significantly to 23.2% ± 3.2% and 29.3% ± 3.5% at 180 and 360 min after irradiation, respectively. Our irradiation system and the obtained results may be useful for studies of cell competition as well as non-targeted effects.

Petite Integration Factor 1 knockdown enhances gemcitabine sensitivity in pancreatic cancer cells via increasing DNA damage.

Chemoresistance is still a vital obstacle in various tumors chemotherapy. This study aimed to explore the role of Petite Integration Factor 1 (PIF1) in the sensitivity of gemcitabine response to pancreatic cancer cells. Gene Expression Profiling Interactive Analysis (GEPIA) database was employed for evaluating the level of PIF1 in pancreatic cancer tissues and normal tissues. The mRNA level of PIF1 was detected via reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. The relative protein expression of PIF1, cleaved caspase-3, and phosphorylated histone H2Ax (γH2Ax) was assessed through western blot. Cell viability and apoptosis were assessed via Cell Counting Kit-8 (CCK-8) assay and flow cytometry, respectively. Moreover, lactate dehydrogenase (LDH) release and caspase-3 activity were determined via the corresponding LDH Cytotoxicity Assay Kit and caspase-3 colorimetric assay kit. PIF1 expression was upregulated in pancreatic cancer tissues and cells. Knockdown of PIF1 exhibited the repressive impact on the viability of AsPC-1 and PANC-1 cells. PIF1 knockdown enhanced LDH release and apoptosis in both AsPC-1 and PANC-1 cells. PIF1 downregulation could augment the sensitivity of gemcitabine in pancreatic cancer cells, as evidenced by lower cell viability and higher LDH release and apoptosis rate after knocking down PIF1 in gemcitabine-treated pancreatic cancer cells relative to pancreatic cancer cells treated with gemcitabine alone. Moreover, PIF1 knockdown increased γH2Ax protein expression and DNA damage, and gemcitabine treatment-induced DNA damage in AsPC-1 and PANC-1 cells was exacerbated by PIF1 silencing. Furthermore, gemcitabine treatment-caused increase of DNA damage was alleviated by PIF1 overexpression; whereas, this effect of PIF1 upregulation was reversed by thymidine, a DNA synthesis inhibitor. In addition, the decreased gemcitabine sensitivity response to pancreatic cancer cells caused by PIF1 upregulation was also hindered by thymidine treatment. In conclusion, PIF1 silencing enhanced gemcitabine sensitivity response to pancreatic cancer cells through aggrandizing DNA damage.