Breaks In Lines Research Articles

A New Source and Large Quantity of Resveratrol in Cratoxylum Species and Their Activities on Normal Human and Cancer Cells.

Cratoxylum formosum ssp. formosum (Cff), C. formosum ssp. pruniflorum (Cfp), and C. sumatranum (Cs) were investigated for phytochemical analysis. Toxicity testing, programmed cell death, and cell cycle arrest were tested on CHL-1, HCT-116, and HepG2 cancer cell lines, and human normal PBMCs. The results are revealed in the following order. The phytochemical percentages varied in each species, the quantity and concentration of α-amyrin and resveratrol were 0.038 mg/g and 0.955 mg/mL, and 0.064 mg/g and 0.640 mg/mL. The most studied Cratoxylum extracts showed IC50 values in PBMCs and cancer cell lines except for the hexane Cff and ethanol Cfp extracts. All studied extracts did not induce DNA breaks in PBMCs but caused significant DNA breaks in the cancer cell lines. All studied extracts induced both apoptosis and necrosis in cancer cell lines, and the DNA quantity in the S and G2-M phases decreased significantly but did not induce apoptosis and necrosis in PBMCs. Except for the ethanolic extracts of Cff and Cfp that induced PBMCs apoptosis and necrosis, these data confirmed that the three studied Cratoxylum samples have inhibiting properties for the growth of cancer cells and low toxicity to PBMCs. Cs showed more toxicity to cancer cell lines than Cf and cisplatin.

Relationship between key offensive performance indicators and match running performance in the FIFA Women’s World Cup 2023

ABSTRACT Football teams usually adopt different offensive playing styles to increase the probability of success. However, the strategy adopted can influence match-running performance. This study aimed to verify the relationship between the key indicators of offensive performance and match running performance in FIFA Women´s World Cup 2023 (2023-WC). Thirteen key offensive performance indicators were collected from 64 matches (from 32 national teams) with a multi-camera optical tracking system (TRACAB Gen5, ChyronHego). Possession, line breaks completed, receptions in the final third and ball progressions were negatively related to distance covered. On the other hand, goals, total passes, and defensive line breaks were positively associated. K-means cluster analysis indicates that the team that outperformed the above variables was the one that was successful in the 2023-WC. Therefore, the superior performance of some variables, such as ball possession and progression, may prevent athletes from covering greater distances. Teams that make larger pass exchanges and make more breaks in defensive lines may be asked to run more. There are specific key offensive indicators that are related to match running performance. This information can be useful for coaches in choosing the strategy adopted.

Ideological Transformations of Family Discourses Produced in Popular Comedy Films in Turkey

The aim of this study is to discuss the ideological transformations of family discourses that becomes to visible in popular comedy films produced in Turkey. The discussions on family are shaped by three approaches that are categorized based on the narrative structures of the films. The first of these approaches is the corporatization of the family, which is discussed through the films Eltilerin Savaşı (Onur Bilgetay 2020) and Kocan Kadar Konuş 1-2 (Kıvanç Baruönü 2015, 2016). The second approach is the Oedipalization of the family, developed through the films Görümce (Kıvanç Baruönü 2016), Celal ile Ceren (Togan Gökbakar 2013) and Romantik Komedi 1-2 (Hakan Kırvavaç 2010 and Erol Özlevi 2013). As the last approach, the dissolution of the truth of the family and the emergence of the post-family discourse were discussed through the film Aile Arasında (Ozan Açıktan 2017).
 The findings obtained by the discourse analysis method used in the study, covering the corporatization of the family, Oedipalization, and the transformations of the family as a truth regime, show that popular comedy films are parallel to the breaks in today's popular discussion lines in Turkey.

DNA strand break levels in cryopreserved mononuclear blood cell lines measured by the alkaline comet assay: results from the hCOMET ring trial.

The comet assay is widely used in biomonitoring studies for the analysis of DNA damage in leukocytes and peripheral blood mononuclear cells. Rather than processing blood samples directly, it can be desirable to cryopreserve whole blood or isolated cells for later analysis by the comet assay. However, this creates concern about artificial accumulation of DNA damage during cryopreservation. In this study, 10 laboratories used standardized cryopreservation and thawing procedures of monocytic (THP-1) or lymphocytic (TK6) cells. Samples were cryopreserved in small aliquots in 50% foetal bovine serum, 40% cell culture medium, and 10% dimethyl sulphoxide. Subsequently, cryopreserved samples were analysed by the standard comet assay on three occasions over a 3-year period. Levels of DNA strand breaks in THP-1 cells were increased (four laboratories), unaltered (four laboratories), or decreased (two laboratories) by long-term storage. Pooled analysis indicates only a modest positive association between storage time and levels of DNA strand breaks in THP-1 cells (0.37% Tail DNA per year, 95% confidence interval: -0.05, 0.78). In contrast, DNA strand break levels were not increased by cryopreservation in TK6 cells. There was inter-laboratory variation in levels of DNA strand breaks in THP-1 cells (SD = 3.7% Tail DNA) and TK6 reference sample cells (SD = 9.4% Tail DNA), whereas the intra-laboratory residual variation was substantially smaller (i.e. SD = 0.4%-2.2% Tail DNA in laboratories with the smallest and largest variation). In conclusion, the study shows that accumulation of DNA strand breaks in cryopreserved mononuclear blood cell lines is not a matter of concern.

Peculiarities of transient recovering voltages at short circuit breaks in cable lines of high-voltage electric networks

Peculiarities of transient recovering voltages at short circuit breaks in cable lines of high-voltage electric networks

Exploiting the DNA Damaging Activity of Liposomal Low Dose Cytarabine for Cancer Immunotherapy.

Perhaps the greatest limitation for the continually advancing developments in cancer immunotherapy remains the immunosuppressive tumor microenvironment (TME). The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) axis is an emerging immunotherapy target, with the resulting type I interferons and transcription factors acting at several levels in both tumor and immune cells for the generation of adaptive T cell responses. The cGAS-STING axis activation by therapeutic agents that induce DNA damage, such as certain chemotherapies, continues to be reported, highlighting the importance of the interplay of this signaling pathway and the DNA damage response in cancer immunity/immunotherapy. We have developed a multi-targeted mannosylated cationic liposomal immunomodulatory system (DS) which contains low doses of the chemotherapeutic cytarabine (Ara-C). In this work, we show that entrapment of non-cytotoxic doses of Ara-C within the DS improves its ability to induce DNA double strand breaks in human ovarian and colorectal cancer cell lines, as well as in various immune cells. Importantly, for the first time we demonstrate that the DNA damage induced by Ara-C/DS translates into cGAS-STING axis activation. We further demonstrate that Ara-C/DS-mediated DNA damage leads to upregulation of surface expression of immune ligands on cancer cells, coinciding with priming of cytotoxic lymphocytes as assessed using an ex vivo model of peripheral blood mononuclear cells from colorectal cancer patients, as well as an in vitro NK cell model. Overall, the results highlight a broad immunotherapeutic potential for Ara-C/DS by enhancing tumor-directed inflammatory responses.

407 Glucose activates STAT3, promoting XRCC1 expression and increased DNA repair after exogenous challenge

OBJECTIVES/GOALS: Inflammation-promoting exposures, including hyperglycemia and inflammatory cytokines, are common in the development and progression of multiple cancers. Here, we determine if high glucose drives the dysregulation of XRCC1, changing BER/SSBR capacity, and increasing resistance to DNA damaging agents. METHODS/STUDY POPULATION: Here, we acutely exposed cell lines models with 30 mM glucose to mimic hyperglycemic changes and monitored the gene and protein expression of STAT3 and XRCC1. We selected the osteosarcoma cell line U2OS, with high STAT3 activation before glucose challenge, and the non-tumorigenic human embryonic kidney cell line HEK293T, with low STAT3 activation before glucose challenge, to dissect the role STAT3 plays in dysregulating DNA repair. We also examined changes in STAT3 occupancy at the XRCC1 promoter following glucose challenge using chromatin immunoprecipitation (ChIP). Finally, we measured changes in the sensitivity to the alkylating agent methyl methanesulfonate (MMS) induced by the glucose challenge using cell survival and DNA strand break analysis. RESULTS/ANTICIPATED RESULTS: High glucose challenge increases the phosphorylation and activation of STAT3 and subsequently increases XRCC1 gene and protein content in the cell. Acute high glucose activated STAT3, driving the subsequent expression of XRCC1 in U2OS and HEK293T cells through increased STAT3 occupancy at the XRCC1 promoter. High glucose also reduced sensitivity to MMS, increasing cell survival. The most significant increase in resistance to MMS occurred in the HEK293T, which also showed the largest increase in XRCC1 protein expression following the glucose challenge. Increased survival correlated with the faster resolution of DNA strand breaks in glucose-challenged cell lines. DISCUSSION/SIGNIFICANCE: This work has identified a novel regulatory mechanism by which high glucose drives the expression of XRCC1 through STAT3 activation, increasing DNA repair and resistance to the DNA damaging agent MMS. These data suggest dietary choices induce sustained XRCC1 expression and may contribute to chemoresistance and poor survival outcomes in cancers.

Abstract P1-07-10: Estrogen-stimulated DNA damage in mammary epithelium varies among strains of rodents and women

Abstract Background: Estrogens play an important role in breast development, but prolonged exposures and higher levels have been associated with increased risk of breast cancer. Recently, estrogens and environmental estrogenic chemicals have been shown to promote the formation of estrogen receptor-mediated co-transcriptional R-loops that lead to DNA double strand breaks in breast cancer cell lines. However, the DNA damage mediated by the estrogen receptors in the breast epithelium may vary among individuals. Experimental Design: Strains of mice and rats were used to investigate the genetic basis for variation in estrogen-induced DNA damage. The BALB/c and C57BL/6 strains of mice were selected as they differ in susceptibility to mammary tumors (Majhi et al., 2021). Similarly, the ACI and BN strains of rats differ in their susceptibility to mammary tumors induced by estrogen exposure. The mice and rats were treated with 17β-estradiol (E2) for up to 28 days, then tissues were analyzed for γH2AX foci to assess the presence of DNA double strand breaks. Normal breast tissues from average-risk and high-risk patients were maintained in culture (patient-derived explant cultures or PDEs) and exposed to either control media or supplemented with 10nM E2, then analyzed for γH2AX foci. Results: Proliferative responses were similar in both BALB/c and C57BL/6 mouse strains. However, DNA damage and R loops were significantly higher in the mammary epithelium of BALB/c mice whereas only background levels were detected in C57BL/6 mice. The highest level of γH2AX foci was found colocalized in nuclei of cells that were positive for estrogen receptor alpha (ERα+). The E2-stimulated γH2AX in BALB/c mice was not associated with differences in estrogen receptor (Esr1) level or transcriptional responses in target genes Pgr, Areg or expression of Apobec3. Similarly, DNA damage was increased significantly in the mammary epithelium of mammary tumor-susceptible ACI rats treated with E2, but not in the tumor-resistant BN strain. The effect of E2 was also examined in humans using breast explant cultures. Levels of E2-induced γH2AX were significantly higher in tissues from donors undergoing prophylactic mastectomy due to familial risk compared to tissues from average risk women undergoing reduction mammoplasty. Among average risk donors, significant increases in γH2AX by E2 treatment was observed in only 1 of five donors. Conclusions: These data demonstrate that E2-induced DNA damage varies among strains of rodents and women. The levels of E2-stimulated DNA damage can provide a valuable diagnostic tool to identify subsets of individuals for whom estrogen exposure may be pathogenic in breast epithelial cells due to impaired DNA double strand break repair due to inherited risk alleles or epigenetic alterations. Citation Format: Karen A. Dunphy, Aman Sharma, Prabin Majhi, Sallie S Schneider, Giovanna M Crisi, Grace Makari-Judson, D Joseph Jerry. Estrogen-stimulated DNA damage in mammary epithelium varies among strains of rodents and women [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-07-10.

The RNA‐binding protein La/SSB associates with radiation‐induced DNA double‐strand breaks in lung cancer cell lines

BackgroundPlatinum‐based chemotherapy and radiotherapy are standard treatments for non‐small cell lung cancer, which is the commonest, most lethal cancer worldwide. As a marker of treatment‐induced cancer cell death, we have developed a radiodiagnostic imaging antibody, which binds to La/SSB. La/SSB is an essential, ubiquitous ribonuclear protein, which is over expressed in cancer and plays a role in resistance to cancer therapies.AimIn this study, we examined radiation‐induced DNA double strand breaks (DSB) in lung cancer cell lines and examined whether La/SSB associated with these DSB.MethodThree lung cancer lines (A549, H460 and LL2) were irradiated with different X‐ray doses or X‐radiated with a 5 Gy dose and examined at different time‐points post‐irradiation for DNA DSB in the form of γ‐H2AX and Rad51 foci. Using fluorescence microscopy, we examined whether La/SSB and γ‐H2AX co‐localise and performed proximity ligation assay (PLA) and co‐immunoprecipitation to confirm the interaction of these proteins.ResultsWe found that the radio‐resistant A549 cell line compared to the radio‐sensitive H460 cell line showed faster resolution of radiation‐induced γ‐H2AX foci over time. Conversely, we found more co‐localised γ‐H2AX and La/SSB foci by PLA in irradiated A549 cells.ConclusionThe co‐localisation of La/SSB with radiation‐induced DNA breaks suggests a role of La/SSB in DNA repair, however further experimentation is required to validate this.

Molecular targeted drugs resistance impairs double-strand break repair and sensitizes ER-positive breast cancer to PARP inhibitors.

There are various treatments for estrogen-positive breast cancer, mainly hormone therapy and molecular-targeted drugs. Acquiring resistance to these drugs is a major clinical problem. Additionally, little is known about the effect of drug resistance on the DNA repair mechanism. Poly ADP ribose polymerase (PARP) inhibitors currently used for treating HER2-negative metastatic breast cancer with BRCA mutations have been shown to be effective in BRCA-deficient cells with impaired homologous recombination repair. Here, we investigated the effect of drug resistance acquisition on the DNA repair mechanism and the effect of PARP inhibitors on ER (estrogen receptor) -positive breast cancer. We investigated changes in the expression of DNA repair mechanism-related factors and repair ability of double-strand breaks (DSB) in various drug-resistant cell lines established in our laboratory. Additionally, PARP inhibitor susceptibility was investigated using olaparib. DSB repairs in MCF-7 and hormone therapy-resistant model cells were normal, and these cells demonstrated low sensitivity to olaparib. The resistant cell lines against CDK4/6 inhibitors, fulvestrant and mTOR/PI3K inhibitors showed decreased DSB repair ability and high olaparib sensitivity. They showed low sensitivity to CDK4/6 inhibitors, a close link between acquiring resistance to CDK4/6 inhibitors and hypersensitivity to olaparib. Our study suggests some cases of acquiring drug resistance impairs DSB repair ability and sensitizes ER-positive breast cancer to PARP inhibitors.

DNA Double-Strand Breaks in Murine Mammary Tumor Cells Induced by Combined Treatment with Doxorubicin and Controlled Stable Cavitation

Chemotherapeutic agents such as doxorubicin induce cell cytotoxicity through induction of DNA double-strand breaks. Recent studies have reported the occurrence of DNA double-strand breaks in different cell lines exposed to cavitational ultrasound. As ultrasound stable cavitation can potentiate the therapeutic effects of cytotoxic drugs, we hypothesized that combined treatment with unseeded stable cavitation and doxorubicin would lead to increased DNA damage and would reduce cell viability and proliferation in vitro. In this study, we describe how we determined, using 4T1 murine mammary carcinoma as a model cell line, that unseeded stable cavitation combined with doxorubicin leads to additive DNA double-strand break induction. Combined treatment with doxorubicin and unseeded stable cavitation significantly reduced cell viability and proliferation at 72 h. A mechanistic study of the potential mechanisms of action of the combined treatment identified the presence of cavitation necessary to increase early DNA double-strand break induction, likely mediated by a bystander effect with release of extracellular calcium.

Protective Activity of A\u03b2 on Cell Cultures (PC12 and THP-1 after Differentiation) Preincubated with Lipopolysaccharide (LPS)

Amyloid-β (Aβ), the influence of which is considered the pathomechanism of Alzheimer’s disease, is also present in healthy people. The microbiome’s impact is also taken into account, where bacterial lipopolysaccharide (LPS) activates inflammatory processes and stimulates microglia via TLRs. Molecules of bacterial origin can co-create senile plaques with Aβ. This study evaluated the activity of physiological Aβ concentrations on neuronal and microglial cells after preincubation with LPS. Two cell lines were used in the study: PC12 cells differentiated with NGF and THP-1 cells differentiated with phorbol 12-myristate 13-acetate (PMA). Cells were incubated with LPS at concentrations of 1–100 μM for 24 h and then with Aβ25–35 at a concentration of 0.001 μM or 1.0 μM for another 24 h. The viability of the culture and free oxygen radicals and the number of DNA strand breaks in both cell lines were evaluated. Additionally, for PC12 cells, neural features were assessed. Stimulation of repair processes in the presence of Aβ was observed for both studied cell lines. There was a decrease in free radical level and DNA damage number compared to control cultures (cells treated with LPS and without Aβ). The neurotrophic activity of Aβ was observed—the effect on neurites’ growth even after the preincubation of PC12 cells with LPS. At the lowest concentration of LPS used, the increase in neurite length was about 50% greater than in the negative control. At low concentrations, Aβ has a protective effect on neuron-like PC12 cells pretreated with LPS.

Fletcher’s Versification

The essay deals with Fletcher’s versification compared to his contemporaries and co-authors. Fletcher had the most feminine endings compared to other playwrights, more compound feminine endings, and more heavy (stressed) feminine endings. He had few run-on lines: run-on lines and compound feminine endings are in reverse proportion. The main features outlined in the paper are the distribution of stressing and strong syntactic breaks in the line, types of line endings, and the syntactic and semantic function of enclitic micro-phrases. Fletcher’s verse had undergone an evolution, from Bonduca and Valentinian (1610–13) to The Island Princess (1621–23), e. g. the changed place of strong syntactic breaks. Analysis of Massinger’s verse confirmed Oras’s impression that it was prose-like: Massinger often squeezed two syllables into the same metrical slot; he had little hemistich segmentation; and he often divided his lines syntactically; the second part of the divided line ran-over onto the next line, thus effacing the division of his verse into lines. Unlike Shakespeare’s enjambments, which are composed with masculine endings and with an unstressed monosyllabic grammatical word placed on position 10, Massinger’s syntactic breaks in midline and run-on lines occur with compound light feminine endings.

Construction of Radiation Surviving/Resistant Lung Cancer Cell Lines with Equidifferent Gradient Dose Irradiation.

Radiotherapy plays an increasingly crucial role in the treatment of non-small cell lung cancer (NSCLC). Local tumor recurrence and tumor progression caused by intratumoral heterogeneity induced radiotherapy resistance remain the primary causes of radiotherapy failure. However, the lack of a suitable cell line model has hampered the exploration of the dynamic mechanisms of radiation resistance. We established 3 groups of equidifferent gradient dose irradiation surviving/resistant human lung cancer cell lines based on A549, H520, and H460 cells with clinical conventional fractionated radiotherapy (CFRT) (2 Gy × 20 F, 2 Gy × 30 F, and 2 Gy × 40 F). The radiosensitivity of the cells was detected by clone formation assay, EDU cell proliferation assay, neutral comet assay, and γ-H2AX immunofluorescence staining. The radiosensitivity and proliferation viability were increased in a received dose-dependent manner. Compared with parental cells, DNA double-strand breaks (DSBs) in cell lines that received higher-dose irradiation were significantly reduced. We successfully constructed equidifferent gradient dose irradiation surviving/resistant NSCLC cell lines whose radiation surviving and resistant abilities were increased in a received dose-dependent manner. This preclinical cell model could be used to dynamically observe and detect the radiation surviving/resistant biomarkers during radiotherapy stress, elucidate the mechanism of radiation resistance.

Abstract 6267: Repurposing antiandrogens to overcome therapy resistance in androgen receptor-positive glioblastoma

Abstract New approaches are needed to overcome intrinsic therapy resistance in glioblastoma (GBM). Because GBMs exhibit sexual dimorphism and are reported to express steroid hormone receptors, we reasoned that signaling through the androgen receptor (AR) could mediate therapy resistance in GBM, as it does in AR-positive prostate and breast cancers. Using RNAseq, immunoblot and immunohistochemistry, we found that nearly half of GBM cell lines, patient-derived xenografts and human tumors express AR transcript and protein with levels that overlap those of primary prostate cancer. AR expression in GBM did not vary by sex, age or common molecular alterations. We identified two cell line models of GBM that expressed AR protein (LN18 and T98G: termed “AR positive”) and two that did not (8MGBA and AM38: termed “AR negative”). Seviteronel, a blood-brain barrier permeable CYP17 lyase inhibitor and antiandrogen slowed growth in AR positive GBM cell lines (GI50 3-4 µM) but not AR negative lines (GI50 > 500 µM) as measured by the colony formation assay. The antiandrogen enzalutamide, which also penetrates the blood brain barrier, similarly preferentially slowed growth in AR positive GBM cell lines. Seviteronel and enzalutamide sensitized AR positive GBM cell lines to radiation with enhancement ratios of 1.3-1.6 as measured by the clonogenic survival assay. Antiandrogens had no effect on the radiosensitivity of AR negative GBM cell lines. Seviteronel treatment did not affect the growth of AR positive T98G xenografts grown in vivo, but did sensitize these tumors to radiation (median time to tripling: 15 d with radiation alone and not reached with radiation combined with seviteronel). Enzalutamide similarly had modest single agent effects on an AR positive GBM patient-derived xenograft (GBM26 from the Mayo Clinic GBM PDX national resource) grown in vivo but sensitized these tumors to radiation (median time to tripling: 25.5 d with radiation alone and 39 d with radiation combined with enzalutamide). RNAseq performed on GBM26 tumors grown in vivo revealed that enzalutamide treatment caused minimal transcriptional changes when given as monotherapy but, when given in combination with radiation, blocked the ability of AR-positive GBMs to engage adaptive transcriptional programs related to multiple DNA repair pathways. We confirmed these mechanistic findings in vitro, as antiandrogens selectively impaired the repair of radiation-induced double strand DNA breaks in AR positive GBM cell lines. These results suggest that AR signaling may mediate therapy resistance in AR positive GBMs, and patients with these tumors could derive clinical benefit from combination therapies involving radiation and blood-brain-barrier permeable antiandrogens. Citation Format: Christian K. Werner, Uchechi Nna, Hanshi Sun, Kari Wilder-Romans, Joseph Dresser, Ayesha Kothari, Weihua Zhou, Yangyang Yao, Arvind Rao, Stefanie Stallard, Carl Koschmann, Tarik Bor, Waldemar Debinski, Alexander Hegedus, Meredith Morgan, Sriram Venneti, Edwina Baskin-Bey, Daniel Spratt, Howard Colman, Jann Sarkaria, Arul Chinnayain, Joel Eisner, Corey Speers, Theodore S. Lawrence, Roy Strowd, Daniel R. Wahl. Repurposing antiandrogens to overcome therapy resistance in androgen receptor-positive glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6267.

Abstract B15: The oral Chk1 inhibitor, SRA737, synergizes with immune checkpoint blockade in small-cell lung cancer (SCLC)

Abstract Background: Small-cell lung cancer (SCLC) is the most aggressive form of lung cancer. Despite the recent success of immunotherapy in other indications, only a minority of SCLC patients respond to immune checkpoint blockade (ICB) targeting programmed cell death protein 1 (PD-1) or programmed death ligand 1 (PD-L1) either as a monotherapy or combination. Therefore, there is a strong need to develop strategies to enhance the efficacy of immunotherapy in SCLC. Our group previously discovered that SCLC exhibits high expression of checkpoint kinase 1 (Chk1) and showed that preclinical in vivo models of SCLC respond to Chk1 inhibition. Based on data from others and our group, we hypothesized that targeting Chk1 can enhance antitumor immunity and synergize with ICB. Results: SRA737 treatment decreased cell viability with a range of potencies in a panel of SCLC cell lines in vitro. This was accompanied by an induction of double-strand breaks in sensitive cell lines as demonstrated by increased γ-H2AX. Intriguingly, SRA737 also led to an increase in micronuclei formation and STING activation in cells in vitro. Cell surface and total PD-L1 protein were increased following SRA737 treatment in vitro, further supporting a potential benefit of combining the drug with immune checkpoint blockade therapy in vivo. As hypothesized, SRA737 showed strong synergy with anti-PD-L1 antibody in an immunocompetent xenograft SCLC model. Triple-knockout SCLC cells generated from a GEMM mouse model with conditional deletion of Trp53, Rb1 and p130 were implanted into the flank of B6129F1 mice. The mice were treated for three weeks with either IgG (control), SRA737 (100mg/kg, either 3/7 or 5/7 days), anti-PD-L1 (300ug, 1/7 days) or the combination. While anti-PD1 antibody treatment was largely ineffective, SRA737 significantly delayed tumor growth (at Day 21: T/C=0.30 for 3/7 days and T/C=0.28 for 5/7 days). Combination treatment with SRA737 and anti-PD-L1 demonstrated remarkable antitumor efficacy, resulting in stable disease following SRA737 schedule of 3/7 days (T/C=0.12) and tumor regressions following SRA737 schedule of 5/7 days (T/C=0.1). These effects were sustained after treatment cessation and the long-term survival benefit is being assessed. Discussion: The intrinsic antitumor activity of the Chk1 inhibitor, SRA737, was significantly enhanced by addition of an anti-PD-L1 antibody, leading to tumor regressions in an immunocompetent SCLC model. Preliminary evidence suggests SRA737 induces micronuclei formation, STING activation and PD-L1 expression in tumor cells. Further studies to elucidate the mechanism of Chk1 inhibition-induced antitumor immunity in SCLC are ongoing. SRA737 is currently being tested in clinical trials both as a monotherapy and in combination with other agents. Given that the anti-PD-L1 antibody opdivo is now approved for SCLC, our data suggest intriguing possibilities for therapeutic synergy between the oral Chk1 inhibitor, SRA737, and ICB therapy that warrant further clinical investigation. Citation Format: Triparna Sen, Snezana Milutinovic, Robert J. Cardnell, Lixia Diao, Youhong Fan, Ryan J. Hansen, Bryan Strouse, Michael P. Hedrick, Christian Hassig, Jing Wang, Lauren A. Byers. The oral Chk1 inhibitor, SRA737, synergizes with immune checkpoint blockade in small-cell lung cancer (SCLC) [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B15.

MP51-09 THE NOVEL ROLE OF SPOP IN REGULATING TOPOISOMERASE 2A IN PROSTATE CANCER CELLS AS A POTENTIAL THERAPEUTIC MARKER FOR DNA REPAIR TARGETED THERAPY

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology II (MP51)1 Apr 2020MP51-09 THE NOVEL ROLE OF SPOP IN REGULATING TOPOISOMERASE 2A IN PROSTATE CANCER CELLS AS A POTENTIAL THERAPEUTIC MARKER FOR DNA REPAIR TARGETED THERAPY Ryuta Watanabe*, Masashi Maekawa, Miki Hieda, Tomohiko Taguchi, Noriyoshi Miura, Tadahiko Kikugawa, Shigeki Higashiyama, and Takashi Saika Ryuta Watanabe*Ryuta Watanabe* More articles by this author , Masashi MaekawaMasashi Maekawa More articles by this author , Miki HiedaMiki Hieda More articles by this author , Tomohiko TaguchiTomohiko Taguchi More articles by this author , Noriyoshi MiuraNoriyoshi Miura More articles by this author , Tadahiko KikugawaTadahiko Kikugawa More articles by this author , Shigeki HigashiyamaShigeki Higashiyama More articles by this author , and Takashi SaikaTakashi Saika More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000913.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: SPOP, speckle-type POZ protein, is a substrate recognizing receptor of the Cullin-3/RING ubiquitin E3 complex. Heterozygous point mutations in the substrate-binding domain of SPOP have been found in 10-15% of human prostate cancer patients. Although SPOP is essential for a DNA repair in exogenous DNA damage stresses, it still remains unclear whether SPOP is involved in the process of a DNA repair in endogenous DNA damage stresses. In this study, we focus on relationships the functions of SPOP, especially those involved in topoisomerase 1 (TOP1) and topoisomerase 2 (TOP2), to the DNA-protein crosslink repair from endogenous DNA damage in prostate cancer cell lines. METHODS: We first examined the level of γH2AX, a marker of DNA breaks in various prostate cancer cell lines; C4-2, LNCaP, PC3, DU145 upon SPOP knockdown (KD). To elucidate the site of action of SPOP during DNA repair, we treated control or SPOP-KD C4-2 cells with topoisomerase (TOP) inhibitors, which inhibit DNA repair. To investigate functions of SPOP in the regulation of TOP, we analyzed protein expression and cellular localization of TOPs by western blotting and immunofluorescence staining. We also measured the amounts of TOPs which covalently bound on DNA by cesium chloride-density gradient centrifugation. We finally detected the level of enzymes (TDP1, TDP2 and MRE11) which liberate TOPs from the TOP-DNA cleavage complex. RESULTS: We found that SPOP KD in androgen receptor (AR)-positive prostate cancer cells increased the amount of γH2AX. Because the level of γH2AX in SPOP-knockdown cells was not additive by treatment of TOP inhibitors, it was suggested that SPOP could act upstream of TOP1 and TOP2-dependent pathways. Immunofluorescence staining indicated that TOP2A, but not TOP1, was accumulated in the nuclei by SPOP knockdown. By cesium chloride-density gradient centrifugation, we found that SPOP KD increased the amount of TOP2A on genomic DNA. The amount of TDP1 and TDP2, which liberate TOP2A from the TOP2A-DNA complex was decreased by SPOP knockdown. We also confirmed that the increase of γH2AX in SPOP-KD C4-2 cells was dependent on both AR and TOP2A. CONCLUSIONS: Our results suggest that SPOP is involved in the DNA-protein crosslink repair process through the elimination of TOP2A from the TOP2A cleavage complex in prostate cancer cell lines. These results imply that TOP inhibitor might be effective to AR-positive, SPOP-wild type prostate cancer. Also, PARP inhibitor might be effective to SPOP-mutated prostate cancer. Source of Funding: JSPS KAKENHI Grant Number 19K18613 © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e767-e767 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ryuta Watanabe* More articles by this author Masashi Maekawa More articles by this author Miki Hieda More articles by this author Tomohiko Taguchi More articles by this author Noriyoshi Miura More articles by this author Tadahiko Kikugawa More articles by this author Shigeki Higashiyama More articles by this author Takashi Saika More articles by this author Expand All Advertisement PDF downloadLoading ...

Neuroprotection against Amyloid-β-Induced DNA Double-Strand Breaks Is Mediated by Multiple Retinoic Acid-Dependent Pathways.

In this study, we have investigated the role of all-trans-retinoic acid (RA) as a neuroprotective agent against Aβ1-42-induced DNA double-strand breaks (DSBs) in neuronal SH-SY5Y and astrocytic DI TNC1 cell lines and in murine brain tissues, by single-cell gel electrophoresis. We showed that RA does not only repair Aβ1-42-induced DSBs, as already known, but also prevents their occurrence. This effect is independent of that of other antioxidants studied, such as vitamin C, and appears to be mediated, at least in part, by changes in expression, not of the RARα, but of the PPARβ/δ and of antiamyloidogenic proteins, such as ADAM10, implying a decreased production of endogenous Aβ. Whereas Aβ1-42 needs transcription and translation for DSB production, RA protects against Aβ1-42-induced DSBs at the posttranslational level through both the RARα/β/γ and PPARβ/δ receptors as demonstrated by using specific antagonists. Furthermore, it could be shown by a proximity ligation assay that the PPARβ/δ-RXR interactions, not the RARα/β/γ-RXR interactions, increased in the cells when a 10 min RA treatment was followed by a 20 min Aβ1-42 treatment. Thus, the PPARβ/δ receptor, known for its antiapoptotic function, might for these short-time treatments play a role in neuroprotection via PPARβ/δ-RXR heterodimerization and possibly expression of antiamyloidogenic genes. Overall, this study shows that RA can not only repair Aβ1-42-induced DSBs but also prevent them via the RARα/β/γ and PPARβ/δ receptors. It suggests that the RA-dependent pathways belong to an anti-DSB Adaptative Gene Expression (DSB-AGE) system that can be targeted by prevention strategies to preserve memory in Alzheimer's disease and aging.

Lipophilic compounds, but not fucoxanthin, mediate the genotoxic effect of photoautotrophic grown Phaeodactylum tricornutum in Caco-2 and HT-29 cells

Abstract Phaeodactylum tricornutum (P. triconutum) gained high interest for use in human nutrition. We aimed to investigate the effect of photoautotrophically cultivated Phaeodactylum tricornutum on genotoxicity, cytotoxicity, formation of reactive oxygen species and apoptosis in Caco-2 and HT-29 cells. Furthermore, the effect of processing (sonication, ball-milling) on these parameters was assessed. P. triconutum dose-dependently induced DNA strand breaks (2 µg dry matter/mL – 200 µg dry matter/mL) in both cell lines. These breaks can mostly be repaired after a recovery time of 2 h. Using processing methods did not affect the genotoxicity. We found that a lipophilic fraction, but not fucoxanthin, might be responsible for the DNA damage. Neither the P. triconutum preparations nor fucoxanthin affected cell proliferation or revealed an apoptotic or necrotic activity. In conclusion, P. triconutum showed a genotoxic potential in intestinal cells, mainly mediated by lipophilic constituents. Sonication and ball-milling might be considered as safe methods for processing of P. triconutum.

Model Colibactins Exhibit Human Cell Genotoxicity in the Absence of Host Bacteria.

Colibactins are genotoxic secondary metabolites produced in select Enterobacteriaceae, which induce downstream DNA double-strand breaks (DSBs) in human cell lines and are thought to promote the formation of colorectal tumors. Although key structural and functional features of colibactins have been elucidated, the full molecular mechanisms regulating these phenotypes remain unknown. Here, we demonstrate that free model colibactins induce DSBs in human cell cultures and do not require delivery by host bacteria. Through domain-targeted editing, we demonstrate that a subset of native colibactins generated from observed module skipping in the nonribosomal peptide synthetase-polyketide synthase (NRPS-PKS) biosynthetic assembly line share DNA alkylation phenotypes with the model colibactins in vitro. However, module skipping eliminates the strong DNA interstrand cross-links formed by the wild-type pathway in cell culture. This product diversification during the modular NRPS-PKS biosynthesis produces a family of metabolites with varying observed mechanisms of action(DNA alkylation versus cross-linking)in cell culture. The presence of membranes separating human cells from model colibactins attenuated genotoxicity, suggesting that membrane diffusion limits colibactin activity and could account for thereported bacterium-human cell-to-cell contact phenotype. Additionally, extracellular supplementation of the colibactin resistance protein ClbS was able to intercept colibactins in an Escherichia coli-human cell transient infection model. Our studies demonstrate that free model colibactins recapitulate cellular phenotypes associated with module-skipped products in the native colibactin pathway and define specific protein domains that are required for efficient DNA interstrand cross-linking in the native pathway.