S Phase Research Articles

Root extract of Plumbago zeylanica L. induces cytotoxicity, inhibits cell migration and induces S-phase cell cycle arrest through down regulation of EGFR in HeLa cervical cancer cells

Plumbago zeylanica has been used in the traditional system of medicine from thousands of years owing to its potential therapeutic properties, however the anticancer and anti-metastatic effects are largely unknown against cervical cancer cells. In this study we demonstrated the cytotoxicity activity, inhibition of cell migration, induction of S-phase cell cycle arrest and the down regulation of expression level of epidermal growth factor receptor (EGFR) by ethanol extract of the root of P. zeylanica (ERPZ). The cytotoxicity effects were analyzed by MTT assay the IC50 was determined to be 10.49 ​μg/ml after 72 ​h of incubation. Besides, strong inhibition of wound healing activity was observed at 20 ​μg/ml concentration, where the wound size was reduced to 33%. The ethanol root extract of P. zeylanica also showed significant S-phase cell cycle arrest of 68.9% at 20 ​μg/ml concentration in HeLa cells which was accompanied by the down regulation of EGFR. Ethanol extracts of the root of P. zeylanica (ERPZ) treatment inhibited the growth of cervical cancer cells. The ERPZ arrested cells at the S-phase of the cell cycle. Besides the HRLC-MS analysis of ERPZ identified 15 compounds and among which the five major compounds such as 7,8-dihydroxy-4-methylcoumarin, neodiosmin, diosmetin, hispidulin, and formononetin were reported to possess antioxidant and anticancer activities, could plausibly induce cell death in HeLa cells.

Apoptosis and Cell Cycle Analysis of the Human Cancer Cell Lines; Breast Michigan Cancer Foundation 7, LS-174T Colon and HePG2 Liver in Response to the Methanolic and Butanolic Extracts ofProsopis juliflora

The phreatophytic tree;Prosopis juliflora(P. juliflora), of the Fabaceae family is a widespread invasive plant. The present study aimed to examine the characteristics of the anti-cancer effects potentially induced byP. julifloraleaves extracts, and to compare the bioactivity of its chemical composition in methanolic and butanolic solvents. MCF-7/breast, LS-174T/colorectal, and HepG2/liver, cancer cell lines were cultivated and incubated with various concentrations of the methanolic and butanolic extracts of the plant leaves, and hence the impact on cell viability, proliferation, and cell cycle stages have been investigated. Both extracts ofP. julifloraleave induced concentration-dependent cytotoxicity against the previously mentioned cancer cell lines. The calculated IC50 averaged 16.7, 18.04 and, 8.10μg/ml for methanolic extract, and 12.5, 32.8 and 5.17μg/ml for butanolic extract ofP. julifloraleaves on MCF-7, LS-174T and, HePG2, respectively. Detailed cellular analyses revealed that the cytotoxic action ofP. julifloraextracts was mainly via apoptosis but not necrosis for MCF7 in both extracts, either. However, apoptosis and necrosis reciprocally detected for LS-174T and HePG2 in response to the methanolic and butanolic extracts, either. The DNA-flow cytometry analysis showed cells specific antiproliferative action and cell cycle stages arrest in the tested cancer cell lines. The antiproliferative action has detected mostly at the G0/G1 rather than the S-phase of the cell cycle in both extracts. For identifying the chemical composition of the plant and evaluating the anticancer potency, both the methanolic and butanolic extracts were analyzed by liquid chromatography-mass spectrometry (LC-MS). Betaine and nicotinamide were the major constituents detected for both extracts showing potential anti-cancer effects against breast, hepatic, and colorectal cancerous cells.

Monitoring Cellular Proliferation, Migration, and Apoptosis Associated with Atherosclerosis Plaques In Vitro.

Bromodeoxyuridine/5-bromo-2'-deoxyuridine (BrdU) is a nucleoside analog of thymidine and its incorporation into DNA during replication within S-phase of the cell cycle is used to quantify cell proliferation. Quantification of incorporated BrdU is considered the most direct measure of cell proliferation, and here we describe BrdU incorporation into cultured vascular smooth muscle cells (VSMCs) and endothelial cells in vitro. Incorporation of fluorescent-labeled ethynyldeoxyuridine/5-ethynyl-2'-deoxyuridine (EdU) is a novel alternative to BrdU assays and presents significant advantages. This method of detection of EdU based on a simple "click" chemical reaction, which covalently bonds EdU to a fluorescent dye is also outlined in this chapter with a protocol for quantitative analysis of EdU incorporation using a Fiji-based macro. We also describe how proliferation can be assessed by quantification of classical proliferative markers such as phopsho-Ser807/811 retinoblastoma (Rb), proliferating cell nuclear antigen (PCNA) and cyclin D1 by Western blotting. As these markers are involved in different aspects of the cell cycle regulation, examining their expression levels can not only reveal the relative population of proliferating cells but can also improve our understanding of the mechanism of action of a given treatment or intervention. The scratch wound assay is a simple and cost-effective technique to quantify cell migration. A protocol which involves creating a wound in a cell cultured monolayer and measuring the distance migrated by the cells after a predefined time period is also described. Gap creation can also be achieved via physical cell exclusion where cells are seeded in distinct reservoirs of a cell culture insert which reveal a gap upon removal. Cell migration may then be quantified by monitoring the rate of gap closure. The presence of cleaved caspase-3 is a marker of programmed cell death (apoptosis). To detect cleaved caspase-3 in vitro, immunocytochemistry and fluorescence can be performed as outlined in this chapter.

Benefit of Serum Thymidine Kinase 1 Concentration for Risk Assessment from Gastric Neoplasms Progression to Carcinomas: A Systematic Review and Meta-analysis

Background: Human Thymidine kinase 1 (hTK1), a key enzyme involved in the DNA synthesis during S-phase of the cell cycle and upregulation of cell proliferation, thus it is reliable tumor proliferating biomarker for assessment of tumor proliferation rate in serum and in tissue in oncology. This meta-analysis is investigation whether the serum TK1 concentration（STK1p）based on hTK1-IgY-polyclonal-antibody can provide a benefit for risk assessment from gastric neoplasm progression to gastric carcinoma (GC) as well as for evaluation of treatment effect in GC. 1.2. Methods: Relevant studies were identified from the following electronic databases: PubMed, Embase, Chinese National Knowledge Infrastructure, Wanfang, the Chongqing VIP Chinese Science and Technology Periodical Database and Chinese Biomedical Literature Database from May 1, 2008, until November 30, 2021. Study quality was evaluated by the modified Newcastle-Ottawa Scale. Pooled estimates of weighted mean difference (MD), sensitivity analyses and publication bias were evaluated using Review manager and STATA software. RESULTS: Twenty-seven studies included to 1,909 GCs and 1,229 neoplasms (superficial gastritis, chronic gastritis, atrophic and gastric ulcers) and 2,260 tumor-frees. The results showed that STK1p levels increased significantly in the following manner (P<.0.006): tumor-free < superficial gastritis < chronic gastritis < atrophic gastritis < gastric ulcer < GC. The STK1p value of the GCs one-month-post-surgery decline significantly by 66% compared to pre-surgery. 1.3. Conclusion: STK1p value has the potential to be an early risk warning indicator for early gastric neoplasms progression to GCs and to be a helpful index for monitoring the response to surgery. 1.4. Core Tip: In spite of improvements in diagnosis and treatment the long-term survival rate for a large number of GCs is still dismal. Hence, it is important to explore serum biomarkers for early prediction of risk gastric neoplasms into GCs. In this meta-analysis, the STK1p as a prognostic biomarker was investigated showed significantly higher in GCs compere to different neoplasm and tumor-free people and was significantly reduced one-month post-surgery. It demonstrates that STK1p as tumor proliferating serum-biomarker, can be not only used for early risk gastric neoplasm progression and evaluation of treatment effect in GC patients, but also for a variety of other tumor types in clinical setting.

Ethyl Acetate Fraction in Hedyotis Diffusa Willd Inhibits T Cell Proliferation to Improve the Pathogenesis of Systemic Lupus Erythematosus

Abnormal proliferation of T cells plays an essential role in the pathogenesis of Systemic lupus erythematosus (SLE). The pharmaceutical effect of Hedyotis Diffusa Willd (HDW) on SLE has been investigated previously. Nevertheless, the biomedical mechanism is still left unclear. This study has been arranged to evaluate the therapeutic effect of the ethyl acetate fraction of HDW (EAHDW) on lupus mice and explore the potential therapeutic mechanism. EAHDW was prepared with 80% ethanol reflex extraction followed by successive extraction, and analyzed with HPLC and UPLC-Q/TOP-MS. The potential targets and STAT3 affinity regulators were predicted with network pharmacology. The pharmaceutic effect of EAHDW was studied with MRL/lpr mice. Cytokines and autoantibodies were quantified with ELISA assays. The pathological damage of glomerulus and STAT3 expression in the kidney was detected with histochemical and immunohistochemical techniques. The cell cycle properties in cell proliferation were identified with the flow cytometry. The western blot and dual-Luciferase reporter assay were applied to evaluate translational and transcriptional activity of STAT3, respectively. In this study, the extraction ratio of EAHDW was 2.7±1%, in which 19 ingredients were identified. Network pharmacological analysis showed that the target genes of EAHDW were highly focused on influencing the abnormal T cell proliferation in SLE. EAHDW showed the beneficial effects on pathological changes and STAT3 expression in the glomerulus of lupus mice, and the levels of cytokines and autoantibodies in serum. In cytological study, EAHDW treatment attenuated the transcription and phosphorylation of STAT3, which inhibited T cell proliferation by prolonged S-phase of the cell cycle. A total of 5 compounds in EAHDW exhibited high docking affinity to the DNA-binding site of STAT3. EAHDW could reduce the inflammatory response and inhibit the proliferation of T cells by interfering with the STAT3 signaling pathway, thereby playing a therapeutic effect on SLE.

Role of plasminogen activator inhibitor-1 in oral tongue squamous cell carcinoma: An immunohistochemical and in vitro analysis

ObjectiveTo evaluate the influence of plasminogen activator inhibitor-1 (PAI-1) on the biological behavior and prognosis of oral tongue squamous cell carcinoma (OTSCC). MethodsImmunoexpression of PAI-1 was analyzed in 60 OTSCC specimens and classified as low-expression (≤50% of positive cells) or high-expression (>50%). In vitro effects of recombinant human PAI-1 (rhPAI-1) were assessed through functional assays on the OTSCC-derived cell line SCC-25. Three cell groups were evaluated: G0 (control), G10 (10 nM rhPAI-1), and G20 (20 nM rhPAI-1). ResultsHigh membrane expression of PAI-1 was associated with tumor budding (p = 0.046) and high-risk cases (p = 0.043). Cytoplasmic and membrane expression of PAI-1 was not associated with patient survival. Cell viability (p = 0.020) and progression to the S-phase of the cell cycle (p = 0.024) were higher in G10 and G20 at 24 h. The percentages of apoptotic/necrotic cells were not affected by rhPAI-1. The presence of rhPAI-1 increased cell migration (p = 0.039) and invasion (p = 0.039) after 24 and 72 h, respectively. ConclusionOur findings indicate the involvement of PAI-1 in the biological behavior of OTSCC, although its expression may not predict patient survival. The in vitro results suggest that PAI-1 stimulates cell proliferation, migration and invasion and may contribute to the aggressive phenotype of OTSCC.

Abstract PO-034: CPSF3 inhibition halts pancreatic cancer cell proliferation by limiting core histone supplies

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies with few effective treatment options. Therefore, the need for new therapeutic interventions is urgent. Recently, we have revealed that the gene regulatory process alternative polyadenylation (APA) is highly dysregulated in PDAC patients. APA is a co-transcriptional process that generates distinct transcript isoforms with different 3’ untranslated region (UTR) length, which regulates mRNA stability and gene and protein expression. We found that APA promotes dysregulation of PDAC-promoting genes, predicts poor prognosis in PDAC patients and identified a novel APA-regulated drug target (Venkat et al., Genome Research, 2020). These findings raise the question of whether targeting APA could be a viable therapeutic strategy for PDAC treatment. APA is controlled by a large protein complex assembled on the pre-mRNA, including the cleavage and polyadenylation specificity factor 3 (CPSF3). CPSF3 is an endonuclease that cleaves the pre-mRNA before addition of the polyA tail. Notably, CPSF3 is also a subunit of the histone pre-mRNA processing complex and is critical for the cleavage and processing of core histones. Recently, it was determined that JTE-607, a drug with a long history of safety in humans but no known mechanism of action, is a CPSF3 inhibitor. Subsequently, CPSF3 has been shown to be required for growth of acute myeloid leukemia and Ewing sarcoma cells. However, whether CPSF3 is a potential target in adenocarcinoma is yet to be explored. In PDAC patients, CPSF3 is highly expressed and is associated with unfavorable prognosis. Knockdown or inhibition of CPSF3 decreases PDAC cell proliferation and clonogenicity while having no effect on the growth of non-transformed pancreatic cell lines. Cell cycle analysis and BrdU incorporation assays determined that CPSF3 inhibition arrests cells in early S-phase of the cell cycle. As maintenance of high histone levels are important for the packaging of newly synthesized DNA and progression through the cell cycle, we hypothesized that CPSF3 inhibition decreases PDAC cell proliferation by reducing histone levels. Indeed, CPSF3 inhibition decreased core histone protein levels in a time- and dose-dependent manner. High throughput screening revealed a synergistic effect between JTE-607 and Abexinostat, an HDAC inhibitor, suggesting a novel therapeutic strategy. Therefore, we identify CPSF3 as a druggable target in PDAC and reveal a novel mechanism by which CPSF3 disruption attenuates cell proliferation. Citation Format: Abdulrahman A. Alahmari, Carla Schwarz, Emily Paterson, Swati Venkat, Arwen Tisdale, Michael E. Feigin. CPSF3 inhibition halts pancreatic cancer cell proliferation by limiting core histone supplies [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-034.

The bioavailability time of commonly used thymidine analogues after intraperitoneal delivery in mice: labeling kinetics in vivo and clearance from blood serum.

Detection of synthetic thymidine analogues after their incorporation into replicating DNA during the S-phase of the cell cycle is a widely exploited methodology for evaluating proliferative activity, tracing dividing and post-mitotic cells, and determining cell-cycle parameters both in vitro and in vivo. To produce valid quantitative readouts for in vivo experiments with single intraperitoneal delivery of a particular nucleotide, it is necessary to determine the time interval during which a synthetic thymidine analogue can be incorporated into newly synthesized DNA, and the time by which the nucleotide is cleared from the blood serum. To date, using a variety of methods, only the bioavailability time of tritiated thymidine and 5-bromo-2'-deoxyuridine (BrdU) have been evaluated. Recent advances in double- and triple-S-phase labeling using 5-iodo-2'-deoxyuridine (IdU), 5-chloro-2'-deoxyuridine (CldU), and 5-ethynyl-2'-deoxyuridine (EdU) have raised the question of the bioavailability time of these modified nucleotides. Here, we examined their labeling kinetics in vivo and evaluated label clearance from blood serum after single intraperitoneal delivery to mice at doses equimolar to the saturation dose of BrdU (150mg/kg). We found that under these conditions, all the examined thymidine analogues exhibit similar labeling kinetics and clearance rates from the blood serum. Our results indicate that all thymidine analogues delivered at the indicated doses have similar bioavailability times (approximately 1h). Our findings are significant for the practical use of multiple S-phase labeling with any combinations of BrdU, IdU, CldU, and EdU and for obtaining valid labeling readouts.

Deficiency of the E3 Ligase Hoip Impairs Adult Hematopoiesis and Myeloid Leukemia

Ubiquitination is a post-translational modification that plays important roles in the regulation of various cellular processes. The linear ubiquitin chain assembly complex (LUBAC) is the only E3 ubiquitin ligase, which can specifically generate linear ubiquitin chains. LUBAC is composed of three subunits: catalytic Hoip (Rnf31), Hoil-1l (RBCK1), and Sharpin. LUBAC regulates canonical NF-κB signaling pathway and apoptosis. Several studies have shown that LUBAC is required for fetal hematopoiesis and tumorigenesis in several solid cancers, but the role of Hoip, which is the catalytic component of the LUBAC, in adult hematopoiesis and myeloid leukemia is unclear.To address whether Hoip is required for adult hematopoiesis, we used tamoxifen induced Hoip deleted mice (Hoip fl/fl; Rosa26-CreERT). Deletion of Hoip reduced the numbers of almost all cell fractions in bone marrow and peripheral blood, except for long-term hematopoietic stem cell (LT-HSC) fraction (CD34 - Flk2 - Lineage - c-Kit + Sca-1 +: LSK). Deletion of Hoip treated with tamoxifen markedly impaired colony formation of LT-HSCs in vitro. Among competitive transplantation assay using bone marrow cells from Hoip fl/fl; Rosa26-CreERT mice, deletion of Hoip treated with tamoxifen following stable reconstitution 8 weeks after transplantation led to a significant reduction of Hoip-deficient chimerism compared to control in vivo (Figure A). Deletion of Hoip induced apoptosis in bone marrow cells and increased the frequency of CD34 - LSK cells in S/G2/M phase of the cell cycle. Collectively, these data indicated that Hoip is required for adult hematopoiesis.To evaluate the consequences of Hoip deletion in myeloid leukemia propagation and maintenance, we utilized aggressive murine myeloid leukemia models driven by retroviral transduction of oncogenes: MLL-AF9 and NRAS G12V-driven acute myeloid leukemia (AML) and BCR-ABL and NUP98-HOXA9-driven blast crisis of chronic myelogenous leukemia (CML-BC) models. Deletion of Hoip in established leukemia cells treated with tamoxifen markedly impaired colony formation of leukemia cells in vitro and led to a significantly longer survival with reduced disease burden in bone marrow and spleen in vivo in both of leukemic models (Figure B,C). Deletion of Hoip also induced apoptosis in leukemia stem cells (LSCs: c-Kit + Lineage - cells) (Figure D) and increased the frequency of LSCs in S-phase of the cell cycle. Furthermore, we evaluated the contribution of HOIP to human myeloid leukemia. Knockdown of HOIP by small hairpin RNA using lentivirus in several myeloid leukemia cell lines (THP-1, SKM-1, and K562) and primary patient-derived AML cells exhibited reduced numbers of colony forming cells in vitro, and increased apoptosis. LUBAC inhibitor gliotoxin impaired the growth of murine and human leukemia cells. These results indicate that Hoip is essential for propagation and maintenance in murine and human myeloid leukemia.Because LUBAC regulates canonical NF-κB signaling pathway, we evaluated protein levels of p65 phosphorylation on Ser536 (p-p65) and IκBα in leukemia cells by flow cytometric analysis. Reduction in p-p65 and inhibition of IκBα degradation were observed in Hoip-deficient leukemia cells (Figure E), indicating that Hoip plays an important role in regulation of NF-κB signaling in myeloid leukemia.Since Hoip is the central component of LUBAC and the only catalytically active subunit of E3 ubiquitin ligase, we examined the importance of each domain of Hoip for myeloid leukemia propagation using Hoip mutant isoforms. Loss of colony-forming ability of Hoip-deficient AML cells could not be rescued by retroviral transduction of the Hoip mutants with deletion of UBA domain (which is required for interaction with the other LUBAC subunits) and with deletion of RBR domain and with mutation in C879A (which lack ligase activity) (Figure F), indicating that LUBAC ligase activity and interaction with the other LUBAC subunits are critical for leukemia propagation.Thus, our data demonstrated that Hoip is essential for adult hematopoiesis and myeloid leukemia. Given that patients with myeloid leukemia have shown increased activity of NF-κB signaling, inhibition of LUBAC ligase activity could serve as a promising strategy for treating myeloid leukemia. [Display omitted] DisclosuresJimbo: Japan Society for the Promotion of Science: Research Funding. Ito: Japan Society for the Promotion of Science: Research Funding; Institute for Frontier Life and Medical Sciences, Kyoto University: Research Funding. Iwama: Nissan Chemical Corporation: Research Funding. Nannya: Otsuka Pharmaceutical Co., Ltd.: Consultancy, Speakers Bureau; Astellas: Speakers Bureau. Konuma: Japan Society for the Promotion of Science: Research Funding; The Japanese Society of Hematology: Research Funding; SGH Foundation: Research Funding; Institute for Frontier Life and Medical Sciences, Kyoto University: Research Funding.

NFATC2 regulates Targets of MYC Signaling in MLL-AF9 AML

Background: Acute myeloid leukemia (AML) arises due to an accumulation of genetic lesions within myeloid progenitors and oncogenic transformation is often characterised by disordered transcription. Recently the histone lysine demethylase KDM4A was shown to be essential for AML blast survival and self-renewal. shRNA knockdown (KD) of KDM4A led to downregulated expression of the transcription factor NFATC2 an MLL-AF9 AML model, suggesting that it is a key target of KDM4A oncogenic function. The Nuclear Factor of Activated T Cells (NFAT) family of transcription factors control cell cycle genes and self-renewal pathways in hematopoietic tissues and are well-defined as oncogenic regulators in various malignancies. NFATs have recently been attributed roles in the development of FLT3 ITD AML and resistance to tyrosine kinase inhibitors (TKIs) in myeloid leukemias but there is little evidence detailing the role(s) of NFATC2 specifically in AML. We hypothesized that NFATc2 activity is essential for the survival of AML cells and the oncogenic transcriptional networks within these.Aims: To determine if AML cells are dependent on NFATC2 for survival and to elucidate the transcriptional and binding targets of NFATc2 in AML.Methods: NFATC2 was depleted using shRNA KD in numerous cell line models of AML and putative transcriptional targets were elucidated using RNA-seq following KD. Binding targets of NFATc2 were determined using ChIP-seq. Transcriptomic targets of NFATc2 were validated using the Fluidigm Biomark multiplex PCR system and real time quantitative PCR.Results: KD of NFATC2 significantly impaired the colony forming capacity and expansion in liquid cultures of AML cell lines from diverse (cyto)genetic backgrounds. MLL-AF9/TP53 mut THP-1 cells showed reduced entry to the S-phase of the cell cycle and downregulation of cyclin D1 following NFATC2 depletion, suggesting that NFATC2 is critical for cell cycle progression in these cells. Overexpression of human NFATC2 in THP-1 led to an increased rate of cell growth.RNA-seq analysis of THP-1 cells with NFATC2 KD revealed >20 genes with deregulated expression (FDR<0.1), which have been validated using PCR methods. Overexpression of human NFATC2 resulted in significant deregulation of 9 of these genes (FDR<0.1), defining a subset of genes which may regulate the observed phenotype. Additionally, these top genes were not all differentially regulated in other MLL-AF9 AML cell lines MOLM-13 and NOMO-1 following NFATC2 KD. Finally, in THP-1, gene set enrichment analysis (GSEA) of sequencing results revealed that targets of MYC and calmodulin kinase STK33 were enriched within the genes perturbed by NFATC2 depletion. Targets of MYC signaling were validated by PCR in THP-1 but were not found to be deregulated in MOLM-13 following NFATC2 KD.ChIP-seq analysis of NFATc2 binding in THP-1 cells showed that >30% of NFATc2 targets were at promoter regions within 5kb of the transcription start site. Motif analysis of precipitated DNA fragments discovered two novel motifs which were enriched at NFATc2 binding sites (p<0.0001).Discussion: NFATC2 was found to be essential for expansion of AML cells in various cell line models. In the MLL-AF9 driven THP-1 model a number of putative transcriptional and genomic targets were defined, which include novel targets not previously described in AML pathogenesis and targets of MYC, an established oncogenic protein in AML. The differing expression profiles observed across AML cell lines of diverse (cyto)genetic backgrounds with NFATC2 KD suggest that the regulatory targets of NFATc2 vary depending on the cellular signaling landscape. Together with the finding that NFATC2 is indispensable for AML cell survival this study has elucidated novel roles(s) for NFATC2 in AML oncogenesis. DisclosuresMassett: Kymab Ltd: Current Employment. Huang: Janssen Pharmaceutical Companies of Johnson & Johnson (China): Current Employment.

Ribonucleotide reductase: In-vitro S-glutathionylation of R2 and p53R2 subunits of mammalian class I ribonucleotide reductase protein.

Ribonucleotide reductases (RNR) catalyze the rate-limiting step in DNA synthesis during the S-phase of the cell cycle. Its constant activity in order to maintain dNTP homeostasis is a fascinating area of research and an attractive candidate for cancer research and antiviral drugs. Redox modification such as S-glutathionylation of the R1 subunit of mammalian RNR protein has been presumed to regulate the activity of RNR during catalytic cycles. Herein, we report S-glutathionylation of the R2 subunit. We have also shown Grx1 system can efficiently deglutathionylate the S-glutathionylated R2 subunit. Additionally, our data also showed for the very first time S-glutathionylation of mammalian p53R2 subunit that regulates DNA synthesis outside S-phase during DNA damage and repair. Taken together, these data will open new avenues for future research relating to exact physiological significance, target thiols, and/or overall RNR activity due to S-glutathionylation of R2 and p53R2 subunits and provide valuable insights for effective treatment regimes.

Feline thymidine kinase 1: molecular characterization and evaluation of its serum form as a diagnostic biomarker

BackgroundThymidine kinase 1 (TK1) catalyzes the initial phosphorylation of thymidine in the salvage pathway synthesis of dTTP, an essential building block of DNA. TK1 is a cytosolic enzyme with its highest level during the S-phase of the cell cycle. In cancer cells TK1 is upregulated and excess TK1 is leaked into the blood. Therefore, serum TK1 has been used as biomarker for cancer diagnosis and prognosis in human medicine. Feline TK1 shows high sequence similarity to TK1 from other species. The aim of this study was to characterize feline TK1 and evaluate if serum TK1 can be used as a diagnostic biomarker.ResultsFeline TK1 was cloned, expressed and affinity purified. The purified feline TK1 phosphorylated not only pyrimidine deoxyribonucleosides but also pyrimidine ribonucleosides and to some extent purine deoxynucleosides. A number of anticancer and antiviral nucleoside analogs also served as substrates with fairly high efficiency. ATP and dATP were the preferred phosphate donor. Serum TK1 activity in felines with malignant diseases was significantly higher than that in healthy individuals. ROC analysis revealed an area under the curve (AUC) of 0.98 with a sensitivity of 0.83 and a specificity of 0.95 for felines with lymphoma. Serum TK1 activity in felines with IBD or inflammatory disease was within the same range as healthy ones. Furthermore, in felines with lymphoma serum TK1 activity returned to normal levels in response to treatment.ConclusionFeline TK1 has high specific activity and a broader substrate specificity in comparison with TK1 from other species. Serum TK1 activity in felines with malignant diseases is significantly higher than that in normal felines and in felines with inflammatory diseases. These results suggest that serum TK1 may be a promising biomarker for the diagnosis and monitoring of malignant diseases and for the differential diagnosis of certain inflammatory disease.

SUMOylated Senataxin functions in genome stability, RNA degradation, and stress granule disassembly, and is linked with inherited ataxia and motor neuron disease.

BackgroundSenataxin (SETX) is a DNA/RNA helicase critical for neuron survival. SETX mutations underlie two inherited neurodegenerative diseases: Ataxia with Oculomotor Apraxia type 2 (AOA2) and Amyotrophic Lateral Sclerosis type 4 (ALS4).MethodsThis review examines SETX key cellular processes and we hypothesize that SETX requires SUMO posttranslational modification to function properly.ResultsSETX is localized to distinct foci during S‐phase of the cell cycle, and these foci represent sites of DNA polymerase/RNA polymerase II (RNAP) collision, as they co‐localize with DNA damage markers 53BP1 and H2AX. At such sites, SETX directs incomplete RNA transcripts to the nuclear exosome for degradation via interaction with exosome component 9 (Exosc9), a key component of the nuclear exosome. These processes require SETX SUMOylation. SETX was also recently localized within stress granules (SGs), and found to regulate SG disassembly, a process that similarly requires SUMOylation.ConclusionSETX undergoes SUMO modification to function at S‐phase foci in cycling cells to facilitate RNA degradation. SETX may regulate similar processes in non‐dividing neurons at sites of RNAP II bidirectional self‐collision. Finally, SUMOylation of SETX appears to be required for SG disassembly. This SETX function may be crucial for neuron survival, as altered SG dynamics are linked to ALS disease pathogenesis. In addition, AOA2 point mutations have been shown to block SETX SUMOylation. Such mutations induce an ataxia phenotype indistinguishable from those with SETX null mutation, underscoring the importance of this modification.

Intracellular oxidative stress induced by calcium influx initiates the activation of phagocytosis in keratinocytes accumulating at S-phase of the cell cycle after UVB irradiation

BackgroundPhagocytosis is an essential process that maintains cellular homeostasis. In the epidermis, the phagocytosis of melanosomes into keratinocytes is important to protect their DNA against damage from ultraviolet B (UVB) radiation. Furthermore, it is considered that UVB activates the phagocytosis by keratinocytes but the detailed mechanism involved is not fully understood. ObjectiveTo clarify the mechanism of UVB-enhanced phagocytosis in keratinocytes, we investigated the relationship between the phagocytic ability of keratinocytes and the cell cycle stage of keratinocytes. MethodsThe phagocytic ability of keratinocytes was evaluated using the incorporation of fluorescent beads after exposure to UVB or oxidative stress. S-phase was evaluated by BrdU incorporation and immunostaining of cyclin D1. Intracellular calcium levels of keratinocytes were measured using the probe Fluo-4AM. ResultsThe phagocytosis of fluorescent beads into keratinocytes was enhanced by UVB and also by oxidative stress. We found that keratinocytes exposed to UVB or oxidative stress were at S-phase of the cell cycle. Furthermore, keratinocytes synchronized to S-phase showed a higher phagocytic ability according to the increased intracellular ROS level. The UVB-enhanced phagocytosis and entrance into S-phase of keratinocytes was abolished by ascorbic acid, a typical antioxidant. Keratinocytes synchronized to S-phase and exposed to UVB or oxidative stress had increased levels of intracellular calcium and their enhanced phagocytic abilities were diminished by the calcium ion chelator BAPTA-AM. ConclusionTaken together, intracellular oxidative stress induced by intracellular calcium influx mediates the UVB-enhanced phagocytic ability of keratinocytes accumulating at S-phase of the cell cycle.

Cadmium (II)-Induced Oxidative Stress Results in Replication Stress and Epigenetic Modifications in Root Meristem Cell Nuclei of Vicia faba.

Among heavy metals, cadmium is considered one of the most toxic and dangerous environmental factors, contributing to stress by disturbing the delicate balance between production and scavenging of reactive oxygen species (ROS). To explore possible relationships and linkages between Cd(II)-induced oxidative stress and the consequent damage at the genomic level (followed by DNA replication stress), root apical meristem (RAM) cells in broad bean (V. faba) seedlings exposed to CdCl2 treatment and to post-cadmium recovery water incubations were tested with respect to H2O2 production, DNA double-strand breaks (γ-phosphorylation of H2AX histones), chromatin morphology, histone H3S10 phosphorylation on serine (a marker of chromatin condensation), mitotic activity, and EdU staining (to quantify cells typical of different stages of nuclear DNA replication). In order to evaluate Cd(II)-mediated epigenetic changes involved in transcription and in the assembly of nucleosomes during the S-phase of the cell cycle, the acetylation of histone H3 on lysine 5 (H3K56Ac) was investigated by immunofluorescence. Cellular responses to cadmium (II) toxicity seem to be composed of a series of interlinked biochemical reactions, which, via generation of ROS and DNA damage-induced replication stress, ultimately activate signal factors engaged in cell cycle control pathways, DNA repair systems, and epigenetic adaptations.

The Amazing Acrobat: Yeast's Histone H3K56 Juggles Several Important Roles While Maintaining Perfect Balance.

Acetylation on lysine 56 of histone H3 of the yeast Saccharomyces cerevisiae has been implicated in many cellular processes that affect genome stability. Despite being the object of much research, the complete scope of the roles played by K56 acetylation is not fully understood even today. The acetylation is put in place at the S-phase of the cell cycle, in order to flag newly synthesized histones that are incorporated during DNA replication. The signal is removed by two redundant deacetylases, Hst3 and Hst4, at the entry to G2/M phase. Its crucial location, at the entry and exit points of the DNA into and out of the nucleosome, makes this a central modification, and dictates that if acetylation and deacetylation are not well concerted and executed in a timely fashion, severe genomic instability arises. In this review, we explore the wealth of information available on the many roles played by H3K56 acetylation and the deacetylases Hst3 and Hst4 in DNA replication and repair.

Replication-Coupled Chromatin Remodeling: An Overview of Disassembly and Assembly of Chromatin during Replication.

The doubling of genomic DNA during the S-phase of the cell cycle involves the global remodeling of chromatin at replication forks. The present review focuses on the eviction of nucleosomes in front of the replication forks to facilitate the passage of replication machinery and the mechanism of replication-coupled chromatin assembly behind the replication forks. The recycling of parental histones as well as the nuclear import and the assembly of newly synthesized histones are also discussed with regard to the epigenetic inheritance.

Labeling and Purification of Temporally Expressed RNAs During the S-Phase of the Cell Cycle in Living Cells.

From high-throughput DNA and RNA sequencing technologies, it is evident that more than two-thirds of the mammalian genome is transcribed and nearly 98% of the transcriptional output in humans constitute noncoding RNA, comprising tens of thousands of small and long noncoding RNAs. These observations have put the study of RNA expression levels at the center of molecular biology research. The transcriptional output of cells changes temporally throughout different cell cycle phases, or in response to a large panel of stimuli. In such instances, the measure of induced RNA transcripts might be obscured by the presence of steady-state RNA levels in the total transcriptome. With this protocol, we provide a method for labeling and purification of the nascent RNAs transcribed over short periods of time in cultured cells. The supplementation of cell culture medium with a chemically modified analog of uridine, ethynyl-uridine, allows for the subsequent biotinylation of ethynyl-uridine residues with a click-chemistry reaction. The labeled RNA is then purified on streptavidin beads and eluted. The purified RNA is suitable for use in RT-qPCR assays as well as in deep sequencing applications.

Molecular mechanism of the MORC4 ATPase activation

Human Microrchidia 4 (MORC4) is associated with acute and chronic pancreatitis, inflammatory disorders and cancer but it remains largely uncharacterized. Here, we describe the structure–function relationship of MORC4 and define the molecular mechanism for MORC4 activation. Enzymatic and binding assays reveal that MORC4 has ATPase activity, which is dependent on DNA-binding functions of both the ATPase domain and CW domain of MORC4. The crystal structure of the ATPaseCW cassette of MORC4 and mutagenesis studies show that the DNA-binding site and the histone/ATPase binding site of CW are located on the opposite sides of the domain. The ATPase and CW domains cooperate in binding of MORC4 to the nucleosome core particle (NCP), enhancing the DNA wrapping around the histone core and impeding binding of DNA-associated proteins, such as transcription factors, to the NCP. In cells, MORC4 mediates formation of nuclear bodies in the nucleus and has a role in the progression of S-phase of the cell cycle, and both these functions require CW and catalytic activity of MORC4. Our findings highlight the mechanism for MORC4 activation, which is distinctly different from the mechanisms of action observed in other MORC family members.

STAT3 deficiency in B cells exacerbates uveitis by promoting expansion of pathogenic lymphocytes and suppressing regulatory B cells (Bregs) and Tregs

STAT3 transcription factor induces differentiation of naïve T cells into Th17 cells and loss of STAT3 in T cell prevents development of CNS autoimmune diseases. However, function of STAT3 in the B lymphocyte subset is not well understood. In this study, we have generated mice lacking STAT3 in CD19+ B cells (CD19-STAT3KO) and investigated intrinsic and extrinsic functions of STAT3 in B cells and its potential role in resistance or pathogenesis of organ-specific autoimmune diseases. We show that STAT3 regulates metabolic mechanisms in B cells with implications for bioenergetic and metabolic pathways that control cellular homeostasis in B cells. Thus, loss of STAT3 in CD19-STAT3KO cells perturbed growth and apoptosis by inducing rapid entry of B cells into the S-phase of the cell cycle, decreasing expression of cyclin-dependent kinase inhibitors and upregulating pro-apoptotic proteins. We further show that the CD19-STAT3KO mice develop severe experimental autoimmune uveitis (EAU), an animal model of human uveitis. Exacerbated uveitis in CD19-STAT3KO mice derived in part from enhanced expression of costimulatory molecules on B cells, marked increase of Th17 responses and increased recruitment of granulocytes into the neuroretina. The enhanced autoimmunity upon deletion of STAT3 in B cells is also recapitulated in experimental autoimmune encephalitis, a mouse model of multiple sclerosis and thus support our conclusion that STAT3 deletion in B cells enhanced inflammation and the effects observed are not model specific. Our data further indicate that STAT3 pathway modulates interactions between B and T cells during EAU resulting in alteration of lymphocyte repertoire by increasing levels of autoreactive pathogenic T cells while suppressing development and/or expansion of immune-suppressive lymphocytes (Bregs and Tregs). Taken together, STAT3 exerts diametrically opposite effects in lymphocytes, with loss of STAT3 in B cells exacerbating uveitis whereas Stat3 deletion in T cells confers protection.