Prolonged Arrest Research Articles

Changes in the Proteome Profile of A549 Cells Following Helichrysetin-Induced Apoptosis Suggest the Involvement of DNA Damage Response and Cell Cycle Arrest-Associated Proteins.

Our previous findings demonstrated that Helichrysetin possessed promising anti-cancer activity. It was able to induce apoptosis in the A549 cell line. However, its mechanism of action is unknown. The present study aimed to unravel possible underlying molecular mechanisms of helichrysetin-induced apoptosis in A549 (human lung carcinoma) cells using comparative quantitative proteomics (iTRAQ labeled), followed by an exhaustive bioinformatics analysis. Our results suggested that DNA damage response (DDR) and cell cycle arrest were responsible for lung cancer cell death with helichrysetin treatment. Among proteins that changed in abundance were Nrf2 and HMOX1. They are oxidative stress-related proteins and were increased in abundance. BRAT1 was also increased in abundance, suggesting an increase in DNA damage repair, indicating the occurrence of DNA damage due to oxidative stress. However, several essential DDR downstream proteins such as p-ATM, BRCA1, FANCD2, and Rb1 that would further increase DNA damage were found to be dramatically decreased in relative abundance. Cell cycle-related proteins, p53, p21, and cyclin D1, were increased while cyclin A, cyclin E, and cdk2 were decreased. This is predicted to facilitate S-phase arrest. Furthermore, excessive DNA damage and prolonged arrest would in turn result in the induction of mitochondrial-mediated apoptosis. Based on these observations, we postulate that the effects of helichrysetin were in part via the suppression of DNA damage response which led to DNA damage and prolonged cell cycle arrest. Subsequently, this event initiated mitochondrial-mediated apoptosis in A549 lung cancer cells.

Development of new non-viral systems for genetic modification of senescent cells

Senescence is a process characterized by a prolonged irreversible cell-cycle arrest. The accumulation of senescent cells in tissues is related to aging and to the development of age-related diseases. Recently, gene therapy has emerged as a powerful tool for treating age-associated diseases by the transference of specific genes into the target cell population. However, thehigh sensitivity of senescent cells significantly precludes their genetic modification via classical viral and non-viral systems. Niosomes are self-assembled non-viral nanocarriers that exhibit important advantages due to their elevated cytocompatibility, versatility, and cost-efficiency, arising as a new alternative for genetic modification of senescent cells. In this work, we explore for the first time the use of niosomes for genetic modification of senescent umbilical cord-derived mesenchymal stem cells. We report that niosome composition greatly affected transfection efficiency; those formulations prepared in medium with sucrose and containing cholesterol as helper lipid being the most suitable to transfect senescent cells. Moreover, resulting niosome formulations exhibited a superior transfection efficiency with a markedly less cytotoxicity than the commercial reagent Lipofectamine. These findings highlight the potentiality of niosomes as effective vectors for genetic modification of senescent cells, providing new tools for the prevention and/or treatment of age-related diseases.

Cerebral Protection Strategies and Stroke in Surgery for Acute Type A Aortic Dissection.

Perioperative stroke remains a devastating complication in the operative treatment of acute type A aortic dissection. To reduce the risk of perioperative stroke, different perfusion techniques can be applied. A consensus on the preferred cerebral protection strategy does not exist. To provide an overview about the different cerebral protection strategies, literature research on Medline/PubMed was performed. All available original articles reporting on cerebral protection in surgery for acute type A aortic dissection and neurologic outcomes since 2010 were included. Antegrade and retrograde cerebral perfusion may provide similar neurological outcomes while outperforming deep hypothermic circulatory arrest. The choice of arterial cannulation site and chosen level of hypothermia are influencing factors for perioperative stroke. Deep hypothermic circulatory arrest is not recommended as the sole cerebral protection technique. Antegrade and retrograde cerebral perfusion are today's standard to provide cerebral protection during aortic surgery. Bilateral antegrade cerebral perfusion potentially leads to superior outcomes during prolonged circulatory arrest times between 30 and 50 min. Arterial cannulation sites with antegrade perfusion (axillary, central or carotid artery) in combination with moderate hypothermia seem to be advantageous. Every concept should be complemented by adequate intraoperative neuromonitoring.

SWR1 chromatin remodeling complex prevents mitotic slippage during spindle position checkpoint arrest.

Faithful chromosome segregation in budding yeast requires correct positioning of the mitotic spindle along the mother to daughter cell polarity axis. When the anaphase spindle is not correctly positioned, a surveillance mechanism, named as the spindle position checkpoint (SPOC), prevents the progression out of mitosis until correct spindle positioning is achieved. How SPOC works on a molecular level is not well understood. Here we performed a genome-wide genetic screen to search for components required for SPOC. We identified the SWR1 chromatin-remodeling complex (SWR1-C) among several novel factors that are essential for SPOC integrity. Cells lacking SWR1-C were able to activate SPOC upon spindle misorientation but underwent mitotic slippage upon prolonged SPOC arrest. This mitotic slippage required the Cdc14-early anaphase release pathway and other factors including the SAGA (Spt-Ada-Gcn5 acetyltransferase) histone acetyltransferase complex, proteasome components and the mitotic cyclin-dependent kinase inhibitor Sic1. Together, our data establish a novel link between SWR1-C chromatin remodeling and robust checkpoint arrest in late anaphase.

Detection of De Novo Dividing Stem Cells In Situ through Double Nucleotide Analogue Labeling.

Tissue-specific somatic stem cells are characterized by their ability to reside in a state of prolonged reversible cell cycle arrest, referred to as quiescence. Maintenance of a balance between cell quiescence and division is critical for tissue homeostasis at the cellular level and is dynamically regulated by numerous extrinsic and intrinsic factors. Analysis of the activation of quiescent stem cells has been challenging because of a lack of methods for direct detection of de novo dividing cells. Here, we present and experimentally verify a novel method based on double labeling with thymidine analogues to detect de novo dividing stem cells in situ. In a proof of concept for the method, we show that memantine, a drug widely used for Alzheimer's disease therapy and a known strong inducer of adult hippocampal neurogenesis, increases the recruitment into the division cycle of quiescent radial glia-like stem cells-primary precursors of the adult-born neurons in the hippocampus. Our method could be applied to assess the effects of aging, pathology, or drug treatments on the quiescent stem cells in stem cell compartments in developing and adult tissues.

Selected Articles from This Issue

The oncogenic transcription factor C/EBPβ is an emerging therapeutic target for many cancers. Rotolo and colleagues present data describing ST101, a peptide antagonist that prevents C/EBPβ dimerization and enhances its proteasome-dependent degradation. ST101 attenuates C/EBPβ target gene expression, resulting in tumor-specific cytotoxicity in glioblastoma, breast, melanoma, prostate, and lung cancer cells, while normal human epithelial cells are not impacted. In vivo xenograft models indicate that ST101 induces potent tumor growth inhibition or regression, both as a single agent and in combination studies. These data identify ST101 as a promising therapeutic against C/EBPβ-dependent cancers.Sarcomas are malignant mesenchymal tumors for which radical resection is standard of care for localized disease. However, complete resection is often not achieved and locoregional recurrence rates are as high as 84%. Although classically chemoresistant, including paclitaxel (PTX), Blessing and colleagues demonstrate prolonged cell-cycle arrest, mitotic catastrophe, subsequent apoptosis, and irreversible cell death in CS-1 as well as patient-derived liposarcoma and leiomyosarcoma when exposed to supratherapeutic PTX levels achievable via unique biocompatible, drug-eluting polymer films implanted during tumor resection. Re-evaluation of chemotherapeutics traditionally thought to be ineffective is warranted in the setting of enhanced supratherapeutic drug delivery platforms in vivo.Despite the promise of immune checkpoint inhibitors (ICIs) in tumor therapy, clinical trial outcomes report toxicities, including immune-related adverse effects (irAEs). Studies suggest that irAEs from ICI may be dose-dependent. By using a gene-based approach, Wang and colleagues report the potential of a systemic low-dose single-chain antibody (scFv) therapy against PD-L1 in conferring antitumor immunity in mice models of breast and colorectal cancers. The therapy effects of a one-time application of gene-based scFv therapy was comparable to multiple infusions of an anti-PD-L1 monoclonal antibody. Further validation of this approach on T cell homeostasis may offer an alternative for ICI therapy.The combination of DNA-damaging agents, like inhibitors of topoisomerase I, with inhibitors of the DNA damage response (DDR) have utility for treating DDR-deficient cancers. However, their combined toxicity has severely limited their use. Thomas, Santi, and co-workers investigated the use of a long-acting topoisomerase inhibitor, PLX038, with PARP inhibitors. PLX038 monotherapy and in combination with a PARP inhibitor potently inhibited the growth of an ATM-deficient xenograft. A clinical trial of PLX038 and rucaparib was initiated and during the dose escalation phase, a patient with an ATM deficiency showed a durable, complete response after only the first cycle of treatment.

Revealing microcystin-LR ecotoxicity to earthworm (Eisenia fetida) at the intestinal cell level

Potential adverse effects of microcystin-LR (MC-LR) on soil invertebrates have not been studied. Here we investigated the mechanism of MC-LR toxicity to earthworm (Eisenia fetida) intestine at the individual level and at the cellular level. The results showed an inverse relationship between the bodyweight and survival rate of earthworms over exposure time- and MC-LR doses in soil. Dose-dependent intestinal lesions and disturbances of enzymatic activities (e.g., cellulase, Na+/K+-ATPase, and AChE) were observed, which resulted in intestinal dysfunction. Excessive reactive oxygen species generation led to DNA damage and lipid peroxidation of intestinal cells. The oxidative damage to DNA prolonged cell cycle arrest at the G2/M-phase transition in mitosis, thus stimulating and accelerating apoptosis in earthworm intestine. MC-LR target earthworm intestine tissue. MC-LR at low concentrations can damage earthworm intestine regardless of exposure routes (oral or contact). High toxicity of MC-LR to earthworms delineates its ecological risks to terrestrial ecosystems.

Senescent cells in the brain and where to find them.

Cellular senescence is a process in which cells change their characteristic phenotype in response to stress and enter a state of prolonged cell cycle arrest accompanied by a distinct secretory phenotype. Cellular senescence has both beneficial and detrimental outcomes. With age, senescent cells progressively accumulate in tissues and might be the bridge connecting ageing to many age-related pathologies. In recent years, evidence emerged supporting the accumulation of brain senescent cells during neurological disorders and ageing. Here, we will discuss the different brain cell populations that exhibit a senescent phenotype. Subsequently, we will explore several senolytic strategies which have been developed to eliminate senescent cells. Finally, we will examine their potential to directly eliminate these senescent brain cells.

Risk factors for development of cerebral edema following cardiac arrest

BackgroundCerebral edema following cardiac arrest is a well-known complication of resuscitation and portends a poor outcome. We identified predictors of post-cardiac arrest cerebral edema and tested the association of cerebral edema with discharge outcome. MethodsWe performed a retrospective chart review including patients admitted at a single center between January 2015–March 2020 following resuscitation from in-hospital and out-of-hospital cardiac arrest who had head computed tomography imaging. Our primary outcome was moderate-to-severe cerebral edema, which we defined as loss of grey-white differentiation with effacement of the basal and ambient cisterns and radiographic evidence of uncal herniation. We used logistic regression to test associations of demographic information, clinical predictors and comorbidities with moderate-severe cerebral edema. ResultsWe identified 727 patients who met the inclusion criteria, of whom 102 had moderate-to-severe cerebral edema. We identified six independent predictors of moderate-to-severe cerebral edema: younger age, prolonged arrest duration, pulseless electrical activity/asystole as initial rhythm, unwitnessed cardiac arrest, hyperglycemia on admission, and lower Glasgow coma score on presentation. Of patients with moderate-to-severe cerebral edema, 2% survived to discharge, 56% had withdrawal of life-sustaining therapies and 42% progressed to death by neurological criteria. ConclusionsOur study identified several risk factors associated with the development of cerebral edema following cardiac arrest. Further studies are needed to determine the benefits of early interventions in these high-risk patients.

Treatment of Mantle Cell Lymphoma in Transplant Non-Eligible Patients

MCL is a rare but usually aggressive non-Hodgkin lymphoma that most commonly affects the elder population. It is now recognized as a heterogeneous disease with variable biologic and clinical behavior. MCL is considered incurable with current therapies and has historically been associated with a poor prognosis. . Large gains were made in the first decade of the new century when clinical trials established the importance of high-dose therapy and autologous stem-cell rescue and high-dose cytarabine in younger patients and the benefits of maintenance rituximab and bendamustine in older patients. Patients with mantle cell lymphoma (MCL) usually respond to initial combination chemotherapy, but the disease inevitably relapses and often follows an aggressive course. Treatment paradigms have evolved along two lines. Younger, fit mantle cell lymphoma (MCL) patients are generally treated with intensive strategies and older less fit patients with non-intensive strategies. Management of patients with newly diagnosed mantle cell lymphoma (MCL) depends on the age and fitness of the patient. For younger patients, the commonly accepted standard of care is a high-dose cytarabine-based induction chemotherapy followed by autologous stem cell transplantation (ASCT). In newly diagnosed patients with MCL ineligible for intensive therapy and ASCT, the standard-of-care has generally been R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone), followed by rituximab, maintenance. In recent years, bendamustine-based therapy has been increasingly adopted for older MCL patients and more recently, vincristine has been replaced by bortezomib in the R-CHOP combination as VR-CAP for previously untreated patients. Traditionally, the treatment of MCL has been determined by patients being deemed “transplant-eligible” or “transplant-ineligible”. In particular, greater depth of understanding of the molecular pathophysiology of MCL has resulted in an explosion of specifically targeted new efficacious agents. In particular, agents recently approved by the Food and Drug Administration include the proteasome inhibitor bortezomib, immunomodulator lenalidomide, and Bruton's tyrosine kinase inhibitor ibrutinib. Newer data suggest more tolerable front-line therapy, including regimens incorporating novel agents, may produce similar outcomes to intensive historical induction regimens. This may in turn preclude fewer patients from autologous stem cell transplant and produce better long-term outcomes in transplant-ineligible patients. In the relapsed/refractory setting, novel agents and combination regimens are improving outcomes and changing the landscape of treatment. New therapies with distinct mechanisms of action, including novel immunotherapeutics, antibody-drug conjugates, and non-covalent BTK inhibitors, have demonstrated great potential for improving outcomes post–BTK inhibitor failure in relapsed/refractory mantle cell lymphoma. Although cBTK inhibitor has transformed the treatment landscape in B-cell malignancies, the majority of patients will eventually experience disease progression or treatment intolerance. There are 2 oral BTK inhibitors approved for use in relapsed MCL: ibrutinib and acalabrutinib. Acalabrutinib, originally referred to as ACP-196, is a novel, irreversible BTK inhibitor that was designed to be more kinase-selective than ibrutinib. Orelabrutinib is an orally administered, potent, irreversible and highly selective BTK-inhibitor being developed the treatment of B cell malignancies and autoimmune diseases. Tirabrutinib irreversibly and covalently binds to BTK in B cells and inhibits aberrant B cell receptor signalling in B cell-related cancers and autoimmune diseases. Zanubrutinib received accelerated approval in the USA on 14 November 2019 for the treatment of adult patients with mantle cell lymphoma (MCL) who have received at least one prior therapy, based on overall response rate (ORR) seen in phase II and I/II clinical trials. Palbociclib is a specific, potent, oral inhibitor of CDK4/6 capable of inducing a complete, prolonged G1 cell cycle arrest (pG1) in Rb+ MCL cells. Zilovertamab vedotin is an antibodydrug conjugate, which binds specifically to receptor tyrosine kinase-like orphan receptor-1 (ROR-1), an oncoprotein that is pathologically expressed in mantle cell lymphoma and other malignancies. The development of anti-CD19 CAR T-cell therapy represents a major advance in the treatment of patients with chemorefractory B-cell malignancies.

Distinct elongation stalls during translation are linked with distinct pathways for mRNA degradation.

Key protein adapters couple translation to mRNA decay on specific classes of problematic mRNAs in eukaryotes. Slow decoding on non-optimal codons leads to codon-optimality-mediated decay (COMD) and prolonged arrest at stall sites leads to no-go decay (NGD). The identities of the decay factors underlying these processes and the mechanisms by which they respond to translational distress remain open areas of investigation. We use carefully designed reporter mRNAs to perform genetic screens and functional assays in Saccharomyces cerevisiae. We characterize the roles of Hel2, Syh1, and Smy2 in coordinating translational repression and mRNA decay on NGD reporter mRNAs, finding that Syh1 and, to a lesser extent its paralog Smy2, act in a distinct pathway from Hel2. This Syh1/Smy2-mediated pathway acts as a redundant, compensatory pathway to elicit NGD when Hel2-dependent NGD is impaired. Importantly, we observe that these NGD factors are not involved in the degradation of mRNAs enriched in non-optimal codons. Further, we establish that a key factor previously implicated in COMD, Not5, contributes modestly to the degradation of an NGD-targeted mRNA. Finally, we use ribosome profiling to reveal distinct ribosomal states associated with each reporter mRNA that readily rationalize the contributions of NGD and COMD factors to degradation of these reporters. Taken together, these results provide new insight into the role of Syh1 and Smy2 in NGD and into the ribosomal states that correlate with the activation of distinct pathways targeting mRNAs for degradation in yeast.

Uncoupling of Mitosis and Cytokinesis Upon a Prolonged Arrest in Metaphase Is Influenced by Protein Phosphatases and Mitotic Transcription in Fission Yeast.

Depletion of the Anaphase-Promoting Complex/Cyclosome (APC/C) activator Cdc20 arrests cells in metaphase with high levels of the mitotic cyclin (Cyclin B) and the Separase inhibitor Securin. In mammalian cells this arrest has been exploited for the treatment of cancer with drugs that engage the spindle assembly checkpoint and, recently, with chemical inhibitors of the APC/C. While most cells arrested in mitosis for prolonged periods undergo apoptosis, others skip cytokinesis and enter G1 with unsegregated chromosomes. This process, known as mitotic slippage, generates aneuploidy and increases genomic instability in the cancer cell. Here, we analyze the behavior of fission yeast cells arrested in mitosis through the transcriptional silencing of the Cdc20 homolog slp1. While depletion of slp1 readily halts cells in metaphase, this arrest is only transient and a majority of cells eventually undergo cytokinesis and show steady mitotic dephosphorylation. Notably, this occurs in the absence of Cyclin B (Cdc13) degradation. We investigate the involvement of phosphatase activity in these events and demonstrate that PP2A-B55Pab1 is required to prevent septation and, during the arrest, its CDK-mediated inhibition facilitates the induction of cytokinesis. In contrast, deletion of PP2A-B56Par1 completely abrogates septation. We show that this effect is partly due to this mutant entering mitosis with reduced CDK activity. Interestingly, both PP2A-B55Pab1 and PP2A-B56Par1, as well as Clp1 (the homolog of the budding yeast mitotic phosphatase Cdc14) are required for the dephosphorylation of mitotic substrates during the escape. Finally, we show that the mitotic transcriptional wave controlled by the RFX transcription factor Sak1 facilitates the induction of cytokinesis and also requires the activity of PP2A-B56Par1 in a mechanism independent of CDK.

Abstract 5380: Anticancer compound ONC201 induces prolonged stress and arrest of proliferation in BT474 human breast cancer cells without induction of cell death

Abstract Background and goals: ONC201 an experimental anticancer drug, targeting mitochondrial caseinolytic peptidase, ClpP is currently under investigation. Current data on the effect of ONC201 along with observations that ONC201 causes cytotoxic effect, there are data demonstrating that ONC201 is rather cytostatic and does not induce cell death. Recently, we demonstrated ONC201-induced arrest of proliferation and a decrease in the number of mitochondrial nucleoids. Here we extended our study to describe the effect of short-and long-term exposure of BT474 cells to ONC201. Observations: Proliferation and cell cycle arrest. ONC201 in a dose-dependent manner (0-50 µM) inhibited the proliferation of BT474 cells without induction of apoptotic cell death. Cells exposed to 10 µM ONC201 accumulated in G0/G1 phase (49.4 ± 11.5% vs 78.7 ± 5.1%) and declined in the S-phase of the cell cycle (39.2 ± 6.6% vs 9.0 ± 2.1%). In parallel, ONC201 exposure induced a decline in Cyclin E and Cdk2 protein levels. Nucleoids and mtDNA. Exposure of BT474 cells, to 10 µM ONC 201, decreased the number of mitochondrial nucleoids from 249 ± 52 to 155 ± 38 per cell after 24 h. Longer 72 h exposure further reduced the number of nucleoids to 84 ± 36 per cell. ONC201 exposure also induced reduction of mitochondrial size from 2.8 ± 0.5 µm2 to 1.5 ± 0.2 µm2 and depletion of mtDNA. Short-term and long-term consequences of cell exposure to ONC201. Short-term treatment (24 h exposure) demonstrated the reversible effect of ONC201 on the expression of stress proteins (ATF4, CHOP, and GDF-15), cell cycle regulatory proteins (cyclin E, Cdk2), and partial restoration of mitochondrial nucleoids (to 214 ± 46 nucleoids per cell). On the contrary, long-term treatment (72 h) resulted in the irreversible and persistent arrest of proliferation, decline of cell cycle regulatory proteins, and activation of stress proteins. Respiration. ONC201 exposure suppressed State 3, State 4, and uncoupled respiration in digitonin-permeabilized BT474 cells. Complex I activity was decreased from 3.3 ± 0.4 ng-atoms O/min/106 cells to 0.5 ± 0.1 and 0.4 ± 0.3 ng-atoms O/min/106 cells, in cells exposed to ONC201 for 24h and 72h, respectively. Similarly, ONC201 decreased the activity of Complex II from 3.5 ± 0.5 ng-atoms O/min/106 cells to 1.5 ± 0.6 and 0.3 ± 0.1 ng-atoms O/min/106. Conclusions: The consequences of ONC201 treatment were dependent on the duration of exposure to the drug. Short-term (24 h) exposure of BT474 cells resulted in reversible induction of stress proteins (ATF4, CHOP, and GDF-15), which were reversed following 120 h incubation in drug-free media. On the contrary, long-term (72 h) exposure of cells to ONC201 induced irreversible and persistent arrest of proliferation and elevation of expression of stress proteins. Thus, ONC201 can induce prolonged and irreversible elevation of stress proteins and persistent G1/S cell cycle arrest. Citation Format: Artem A. Mishukov, Irina V. Odinokova, Serazhutdin A. Abdullaev, Vitaly K. Zhalimov, Emily M. Fennel, Lucas J. Aponte-Collazo, Paul R. Graves, Lee M. Graves, Ekhson L. Holmuhamedov. Anticancer compound ONC201 induces prolonged stress and arrest of proliferation in BT474 human breast cancer cells without induction of cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5380.

Abstract 6057: Mechanisms mediating the radioresistance of human glioma cells growing in the murine olfactory bulb

Abstract We have previously reported (Int J Radiat Oncol Biol Phys. 107(1):194-201, 2020) that human GBM-stem like cells (GSCs) implanted into the right striatum of nude mice migrate/invade throughout the right hemisphere (RH) as well as into the olfactory bulb (OB). The highly invasive nature of the GSCs suggested a model system for evaluating the effects of specific brain microenvironments on glioma radioresponse and the potential for topographic heterogeneity in radioresistance. Subsequent analyses revealed that GSCs in the OB were significantly more radioresistant than those in the RH. Because the OB is highly enriched in neurons and given recent data suggesting that neuron/glioma interactions drive GBM growth, in this study we first defined contact between GSCs and neurons in the OB and RH using immunohistochemistry (IHC) and confocal microscopy. In the OB 56% of tumor cells were in direct contact with neurons as compared to 24% of tumor cells in the RH. Moreover, among the tumor cells in contact with neurons 25% had 2 or more neuronal contacts in the OB versus 2% in the RH. These data suggest a significantly greater level of glioma-neurons interaction in the OB. To further investigate the OB as a radioresistant niche, RNAseq was used to define the transcriptome of NSC11 GSCs located in the OB versus the RH. Of the greater than 15,000 genes evaluated, 1887 genes were differentially expressed between the 2 sites: 872 upregulated and 1015 down regulated in the OB versus the RH. Functional analyses (IPA and GSEA) indicated that among the genes overexpressed in the OB GSCs were a group associated with the cell cycle, specifically mitosis and the spindle assembly checkpoint (SAC). Comparison of the SAC genes overexpressed in the OB with GBM clinical outcomes (TCGA) showed a significant correlation with decreased patient survival. These analyses suggested that cell cycle regulation may play a role in the radioresistance of OB GSCs. Accordingly, radiation-induced cell cycle redistribution of GSCs in the OB and the RH was determined by IHC analysis of cell cycle phase markers and flow cytometry (propidium iodide) performed on dissociated tumor cells. Results showed a significantly higher percentage of cells in G2 in the OB at 24h and 48h after 10Gy, suggestive of prolonged G2 arrest. In addition, analysis of radiation-induced DNA DSBs using γH2AX and 53BP1 nuclear foci showed faster and more complete foci dispersal after 10Gy in the OB GSCs versus those in the RH, indicative of increased DSBs repair in the OB. These data suggest that GSC radioresistance in the OB is the result of a more effective DNA damage response. Moreover, results suggest that the murine OB may provide a model system for investigating GBM radioresistance as well as the potential role of glioma/neuron interactions. Citation Format: Charlotte M. Degorre, Ian C. Sutton, Stacey L. Lehman, Uma T. Shankavaram, Kevin A. Camphausen, Philip J. Tofilon. Mechanisms mediating the radioresistance of human glioma cells growing in the murine olfactory bulb [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6057.

DIPG-12. Induced mitotic abnormalities associated with BMI-1 modulation sensitize diffuse intrinsic pontine glioma cells to ionizing radiation

Abstract Diffuse intrinsic pontine glioma (DIPG) remains an incurable childhood brain cancer with a median overall survival of less than 12 months, affecting 200-300 children annually in the United States. Hence, there is an unmet need for the development of novel and effective targeted therapies. BMI-1 is a subunit of the multimeric protein complex Polycomb repressor complex 1 (PRC1) implicated in self-renewal of normal and cancer cells, and in DNA damage signaling. We have previously identified BMI-1 as a potential therapeutic target in DIPG and have shown that BMI-1 is highly expressed in DIPG tumors regardless of H3K27 mutational status. Treatment of DIPG cells with PTC596, a small molecule initially identified as a BMI-1 modulator, and ionizing radiation (IR) impairs the kinetics of DNA damage response. in vivo, treatment with PTC596 alone delayed tumor growth kinetics and induced in-tumor apoptosis. However, we observed tumor regrowth once PTC596 treatment is completed or discontinued. In the present study, we evaluated the use of PTC596 in combination with IR. Our in vivo results indicate that PTC596 sensitizes DIPG cells to IR inducing a prolonged cell growth arrest 14 days post-treatment compared to IR or PTC596 alone. The effectiveness of this combination is currently evaluated in murine orthotopic DIPG models and the results will be presented. PTC596 is being tested in newly diagnosed children with DIPG and high-grade gliomas (NCT03605550). Data collected from this study will support the development of a novel therapy including PTC596 in combination with radiotherapy to treat children with DIPG.

Stone heart syndrome after prolonged cardioplegia induced cardiac arrest in open-heart surgery – a pilot study on pigs

ObjectivesTo investigate Stone Heart Syndrome (SHS) as consequence of prolonged ischemic arrest in an experimental study on pigs in regards to onset of SHS and pathological changes. Outcomes defined as aortic cross clamp (ACC) time until onset of SHS and cellular changes characterized by SHS. MethodsEight pigs were included to undergo normothermic cardioplegia induced cardiac arrest ranging from 80 to 240 minutes of ACC. Duration of ACC was defined as time from initiation of aortic cross clamping until cessation. Normothermic, cardioplegic solution administered directly into the arterial system, though in a reduced dose compared to clinical practice. Myocardial contracture evaluated by palpation of the myocardium. Biopsies were collected from the left ventricle just after the induction of cardiac arrest and after reperfusion. Biopsies were evaluated for pathological changes indicative of SHS by electron microscopy. ResultsSix pigs completed the full trial, while two were lost to bleeding. Pigs undergoing 80 to 120 minutes of ACC regained heart rhythm either spontaneously or after defibrillation. Pigs undergoing more than 180 minutes of ACC had contracted hearts with no electrocardiographic response indicating the development of SHS. Electron microscopy findings after ACC of 80 to 120 minutes showed no or low degrees of cellular changes, whereas pig hearts with more than 180 minutes of ACC showed severe mitochondrial changes, endothelial damage, and shortening of sarcomeres consistent with SHS. ConclusionDevelopment of SHS in pigs was ACC time dependent and solely avoided when ACC was limited to a maximum of 120 minutes.

Serious Cardiac Arrhythmias Detected by Subcutaneous Long-term Cardiac Monitors in Patients With Drug-Resistant Epilepsy.

Epilepsy is associated with an increased risk of cardiovascular disease and premature mortality, including sudden unexpected death in epilepsy (SUDEP). Serious cardiac arrythmias might go undetected in routine epilepsy and cardiac investigations. This prospective cohort study aimed to detect cardiac arrhythmias in patients with chronic drug-resistant epilepsy (≥5 years duration) using subcutaneous cardiac monitors for a minimum follow-up duration of 12 months. Participants with known cardiovascular disease or those with abnormal 12-lead ECGs were excluded. The device was programmed to automatically record episodes of tachycardia ≥140 beats per minute (bpm), bradycardia ≤40 bpm for ≥3 seconds, or asystole ≥3 seconds. Thirty-one patients underwent subcutaneous cardiac monitoring for a median recording duration of 2.2 years (range 0.5-4.2). During this time, 28 patients (90.3%) had episodes of sustained (≥30 seconds) sinus tachycardia, 8/31 (25.8%) had sinus bradycardia, and 3 (9.7%) had asystole. Three patients (9.7%) had serious cardiac arrhythmias requiring additional cardiac interventions. Among them, 2 patients had prolonged sinus arrest and ventricular asystole (>6 seconds), leading to pacemaker insertion in one, and another patient with epileptic encephalopathy had multiple episodes of recurrent nonsustained polymorphic ventricular tachycardia and bundle branch conduction abnormalities. The time to first detection of a clinically significant cardiac arrhythmia ranged between 1.2 and 26.9 months following cardiac monitor insertion. Implantable cardiac monitors detected a high incidence of clinically significant cardiac arrhythmias in patients with chronic drug-resistant epilepsy, which may contribute to the incidence of premature mortality, including SUDEP.

Mutations in the acetylation hotspots of Rbl2 are associated with increased risk of breast cancer.

Retinoblastoma like protein-2 (Rbl2) is functionally regulated by phosphorylation and acetylation. Previously, we demonstrated that lysine K1083 (K1079 in human Rbl2) is a potential target for acetylation but its functional role remains elusive. We investigated alterations in human Rbl2 gene specifically targeting exons 19-22 harbouring acetylatable residues i.e. K1072, K1083 and K1115 through single stranded conformation polymorphism (SSCP) in breast cancer patients. The K1083 was found altered into arginine (R) in 51% of the cases but K1072 and K1115 remained conserved. The 'K1083R' mutation impairs the acetylation potential of this motif that may result in functional inactivation of Rbl2. These patients also showed poor survival outcome that highlights prognostic relevance of this residue. NIH3T3 cells expressing glutamine (K1083Q) mutated Rbl2 could not be arrested in G1 by serum starvation, whereas cells expressing Rbl2 with K1083R showed prolonged G1 arrest in fluorescence activated cell sorting (FACS) analysis. This suggests that K1083 acetylation is important for G1/S transition. Further, we performed molecular dynamic simulations (MDS) to analyse kinetics of residue K1083 with Cyc-D1/CDK4. Mutations at K1083 impaired this binding exposing neighbouring residues S1080, P1081, S1082 and R1084, hence enhancing the possibility of accelerated phosphorylation. S1080 has previously been reported as a promising candidate of cell cycle dependent phosphorylation in Rbl2. This highlights significance of mutations in the pocket domain of Rbl2 gene in breast cancer, and also strengthen the supposition that K1083 acetylation is pre-requisite for its phosphorylation.

Mechanodefibrillator dissociation due to prolonged sinus arrest

Mechanodefibrillator dissociation due to prolonged sinus arrest

Trade-off between sex and growth in diatoms: Molecular mechanisms and demographic implications.

Diatoms are fast-growing and winning competitors in aquatic environments, possibly due to optimized growth performance. However, their life cycles are complex, heteromorphic, and not fully understood. Here, we report on the fine control of cell growth and physiology during the sexual phase of the marine diatom Pseudo-nitzschia multistriata. We found that mating, under nutrient replete conditions, induces a prolonged growth arrest in parental cells. Transcriptomic analyses revealed down-regulation of genes related to major metabolic functions from the early phases of mating. Single-cell photophysiology also pinpointed an inhibition of photosynthesis and storage lipids accumulated in the arrested population, especially in gametes and zygotes. Numerical simulations revealed that growth arrest affects the balance between parental cells and their siblings, possibly favoring the new generation. Thus, in addition to resources availability, life cycle traits contribute to shaping the species ecological niches and must be considered to describe and understand the structure of plankton communities.