G1 Phase Of Cell Cycle Research Articles

TLC388 Induces DNA Damage and G2 Phase Cell Cycle Arrest in Human Non-Small Cell Lung Cancer Cells.

TLC388, a camptothecin-derivative targeting topoisomerase I, is a potential anticancer drug. In this study, its effect on A549 and H838 human non-small cell lung cancer (NSCLC) cells was investigated. Cell viability and proliferation were determined by thiazolyl blue tetrazolium bromide and clonogenic assays, respectively, and cell cycle analysis and detection of phosphorylated histone H3 (Ser10) were performed by flow cytometry. γ-H2AX protein; G2/M phase-associated molecules ataxia-telangiectasia mutated (ATM), CHK1, CHK2, CDC25C, CDC2, and cyclin B1; and apoptosis were assessed with immunofluorescence staining, immunoblotting, and an annexin V assay, respectively. The effect of co-treatment with CHIR124 (a checkpoint kinase 1 [CHK1] inhibitor) was also studied. TLC388 decreased the viability and proliferation of cells of both NSCLC lines in a dose-dependent manner. TLC388 inhibited the viability of NSCLC cell lines with an estimated concentration of 50% inhibition (IC50), which was 4.4 and 4.1 μM for A549 and H838 cells, respectively, after 24 hours. Moreover, it resulted in the accumulation of cells at the G2/M phase and increased γ-H2AX levels in A549 cells. Levels of the G2 phase–related molecules phosphorylated ATM, CHK1, CHK2, CDC25C, and cyclin B1 were increased in TLC388-treated cells. CHIR124 enhanced the cytotoxicity of TLC388 toward A549 and H838 cells and induced apoptosis of the former. TLC388 inhibits NSCLC cell growth by inflicting DNA damage and activating G2/M checkpoint proteins that trigger G2 phase cell cycle arrest to enable DNA repair. CHIR124 enhanced the cytotoxic effect of TLC388 and induced apoptosis.

Hallmarks of cellular senescence more abundant in damaged compared to intact cartilage in osteoarthritis

Purpose: Cellular senescence is a consequence of aging, which is associated with cell cycle arrest and has recently been identified as a major factor in the pathogenesis of osteoarthritis. Senescence could contribute to OA by several mechanisms including joint tissue increase in pro-inflammatory senescence-associated secretory proteins (SASPs). Our goal was to quantify the extent and characteristics of cellular senescence of human OA cartilage. To this end, we developed a short enzymatic digestion protocol for primary chondrocyte isolation and compared intact and damaged regions of cartilage within each patient to minimize potential confounding by cell stress that could induce senescence phenotypes during the cell isolation and manipulation procedures. We hypothesized that differences between damaged and intact regions of cartilage from each patient will more reliably identify disease associated senescence than evaluation of damaged regions alone. We characterized senescence based on β-galactosidase (β-gal) activity and p16 INK4a protein expression (two accepted markers for senescent cell identification), quantification of MMP3 protein expression (a SASP) and characterization of cell cycle phases. Methods: Human knee joints were obtained as surgical waste tissue under IRB approval from 4 OA patients during total knee arthroplasty. To isolate primary chondrocytes from macroscopically damaged vs intact regions, human articular cartilage from selected regions was finely diced and digested in 0.3% type II collagenase solution for three hours at 37 °C. Chondrocytes were isolated by centrifugation (300G) and directly stored as cell pellets in -80oC for β-gal activity (fluorimetric quantification) or fixed in 80% methanol and stored in -20oC for flow-cytometric analysis of p16 INK4a(ROCHE #9517 ), MMP3 (Thermo #701285) and cell cycle phase (by propidium iodide, Miltenyi Biotec). β-gal was assessed by SensoLyte ® MUG β - Galactosidase Assay Kit (Anaspec AS-72132) and normalized to DNA content (Biotium #31008). Results: Our protocol yielded a mean of 2.78 x 106 (range: 1.94-3.65x106) chondrocytes from 1 gram of human cartilage. Compared to intact cartilage, β-gal activity and p16INK4a protein expression were higher in damaged cartilage (Figure 1A, N=4, P=0.034; Figure 1B, N=4, P=0.031). The proportion of p16INK4a+/MMP3+ cells was 2.8-fold higher in damaged cartilage compared with intact cartilage (Figure 1C, N=4, P=0.082). Overall, β-gal activity was positively correlated with p16INK4aprotein expression. (Figure 1D, N=8, r=0.57, p=0.072). Compared with chondrocytes from intact cartilage, a higher proportion of chondrocytes from damaged cartilage were in the tetraploid G2 cell cycle phase (P=0.04) and a lower proportion were in the diploid G1 phase (P=0.02) (Figure 2A, N=4). Compared to diploid (G1 phase) chondrocytes,p16INK4a protein was much more highly expressed in tetraploid (G2 phase) chondrocytes (Figure 2B, N=8, P=0.001). Conclusions: We successfully isolated primary human chondrocytes using a short enzymatic digestion protocol to minimize phenotypic changes from cell manipulation, a stressor that might artifactually increase senescence cell markers in chondrocytes. β-gal activity was positively correlated with p16 INK4a expression and both markers were higher in damaged cartilage, which suggested an association of senescent chondrocytes with OA. Moreover, MMP3 expression was positively correlated with p16 INK4a expression. These results suggest at least one means, namely increased cartilage degradation by MMP3, by which senescent chondrocytes could contribute to the pathogenesis of OA. A previous study reported a higher percentage of tetraploid chondrocytes within damaged cartilage; we further identified increased p16INK4a expression in tetraploid chondrocytes. In conclusion, this study provides supportive evidence for the role of cellular senescence in the pathogenesis of human OA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Natural extract and its fractions isolated from the marine bacterium Pseudoalteromonas flavipulchra STILL-33 have antioxidant and antiaging activities in Schizosaccharomyces pombe.

Investigations into the potential for pharmacological inhibition of the aging process and the onset of age-related disease are increasingly garnering attention. Here, we analyzed the antiaging properties of natural compounds derived from several marine bacteria in vitro and in vivo using the fission yeast Schizosaccharomyces pombe. The Pseudoalteromonas flavipulchra STILL-33 extract exhibited high antioxidant and antiglycation activities in vitro. We then characterized two antioxidant active fractions isolated from this extract. In addition, we showed that the P. flavipulchra STILL-33 extract or either of its two active fractions (Fractions 1 and 2) could extend the longevity of fission yeast. Moreover, the particular extract and two active fractions were found to induce mitochondrial activity and to delay the G1 phase of the fission yeast cell cycle, perhaps by improving the aging process. The P. flavipulchra STILL-33 extract and Fraction 1 also increased the expression of the catalase-encoding ctt1+ gene and thereby decreased the reactive oxygen species level. Structural analysis showed that Fraction 1 was dominated by l-arginine and ipriflavone, and we showed indeed that the two corresponding commercial products increase the fission yeast lifespan. As for Fraction 2 was identified as the putative structure of butamben. Together, these results should facilitate the discovery of additional antiaging compounds from P. flavipulchra and ultimately the development of novel antiaging compounds for pharmaceutical use.

Targeted Fluorescence Imaging and Biological Effects of Peptide Conjugated Quantum Dots on Pancreatic Cancer Cells.

Arginine-glycine-aspartic acid (RGD) peptide sequences exist in a variety of biological extracellular matrices and can specifically bind the cell-surface integrin αvβ3, which is overexpressed in cancer cells and plays important roles in tumor growth and invasion. Quantum dots (QDs) have been applied in the field of cell biology and can be physically conjugated to the surface of cancer cells for imaging. In this research, we developed QDs-RGD nanoparticles and investigated its application in pancreatic cancer cell imaging and its influence on the biological behavior of pancreatic cancer cells. The results of flow cytometric analysis showed that the αvβ3 receptor was markedly overexpressed on pancreatic cancer cells. In cellular uptake studies, the fluorescence signal of QDs-RGD nanoparticles in pancreatic cancer cells was higher than that of QDs without RGD conjugation, as determined by an inverted fluorescence microscope. Furthermore, the biological behavior of pancreatic cancer cells was affected by QDs-RGD nanoparticles, which inhibited proliferation, migration and invasion and induced G2-phase cell cycle arrest. With integrin αvβ3 as a target, QDs-RGD nanoparticles can generate high-quality images of pancreatic cancer cells and have immense potential for use in the targeted diagnosis and therapy of pancreatic cancer.

Cell trapping microfluidic chip made of Cyclo olefin polymer enabling two concurrent cell biology experiments with long term durability.

Cyclo Olefin Polymer (COP) based microbioreactors on a microfluidic chip were produced in house by hot-embossing and thermo-compression bonding methods. The chip allows two different experiments to be performed on trapped cells at the same time. On one side of the chip, red fluorescent protein (RFP) tagged nucleolar Nop56 protein was used to track changes in cell cycle as well as protein synthesis within the yeast cells under the application of the anti-tumor agent hydroxyurea (HU). Simultaneously, on the other side of the chip, the response of yeast cells to the drug metformin, mTOR inhibitor, was investigated to reveal the role of TOR signaling in ribosome biogenesis and cell proliferation. The results of 20h long experiments are captured by taking brightfield and fluorescent microscopy images of the trapped cells every 9min. The expression of Nop56 protein of ribosome assembly and synthesis was densely observed during G1 phase of cell cycle, and later towards the end of cell cycle the ribosomal protein expression slowed down. Under HU treatment, the morphology of yeast cells changed, but after cessation of HU, the biomass synthesis rate was sustained as monitored by the cell perimeter. Under metformin treatment, the perimeters of single cells were observed to decrease, implying a decrease in biomass growth; however these cells continued their proliferation during and after the drug application. The relation between ribosome biogenesis and cell cycle was successfully investigated on single cell basis, capturing cell-to-cell variations, which cannot be tracked by regular macroscale bioreactors.

Regulating of cell cycle progression by the lncRNA CDKN2B-AS1/miR-324-5p/ROCK1 axis in laryngeal squamous cell cancer.

To evaluate the function of long non-coding RNA ANRIL (CDKN2B-AS1) in laryngeal squamous cell cancer (LSCC), and to explore the underlying mechanism. The expression levels of CDKN2B-AS1 in LSCC tissues and cell lines (Tu177, HN4, AMC-HN-8 and NP69) were determined by reverse transcription quantitative PCR (RT-qPCR). AMC-HN-8 cells were then transfected with siRNAs of CDKN2B-AS1. The effects of CDKN2B-AS1 on cell proliferation, cell cycle, and apoptotic protein were determined by CCK-8 assay, flow cytometry analysis, and western blot, respectively. Dual luciferase reporter assay and RNA immunoprecipitation (RIP) assay were employed to verify the targets of CDKN2B-AS1. The miR-324-5p mimics or miR-324-5p inhibitor and ROCK1 over-expression plasmids were also transfected into AMC-HN-8 cells for further analysis. CDKN2B-AS1 was upregulated in LSCC tissues, and the upregulation of CDKN2B-AS1 was correlated with overall survival, advanced clinical stage, and lymph node metastasis. In AMC-HN-8 cells, the knockdown of CDKN2B-AS1 by siRNA inhibited cell viability, blocked cell cycle in G1 phase, and increased the expression levels of cyclin-dependent kinase inhibitor 1A (p21), cleaved caspase3, and cleaved PPoly (ADP-Ribose) polymerase 1. Results of dual luciferase reporter assay showed that miR-324-5p could bind to CDKN2B-AS1 or Rho-associated coiled-coil containing protein kinase 1 (ROCK1). Finally, over-expression of ROCK1 in AMC-HN-8 cells revised the inhibitory effect of CDKN2B-AS1 siRNA on cell growth. The upregulation of CDKN2B-AS1 was correlated with overall survival, advanced clinical stage, and lymph node metastasis and promoted LSCC cell growth via miR-324-5p/ROCK1 axis.

Cyclovirobuxine D Exerts Anticancer Effects by Suppressing the EGFR-FAK-AKT/ERK1/2-Slug Signaling Pathway in Human Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC), the sixth most common malignancy worldwide, is characterized by a dismal prognosis due to high recurrence and metastasis rates. Thus, the need for the development of novel chemotherapeutic drugs is urgent. Cyclovirobuxine D (CVB-D), a steroidal alkaloid extracted from Buxus microphylla that has been extensively used to relieve the symptoms of cardiovascular diseases, has shown promising antineoplastic effects in recent studies. However, the therapeutic effects and underlying mechanisms of CVB-D on HCC remain largely unelucidated. This study experimentally indicated that CVB-D can repress HCC cell proliferation by arresting the cell cycle in G2 phase and can facilitate apoptosis. In addition, the migratory and invasive capabilities of HCC cells were noticeably attenuated by a nonlethal dose of CVB-D, and this attenuation was correlated with the inhibition of epithelial-mesenchymal transition (EMT). Moreover, in vivo, CVB-D displayed excellent anticancer effects in HCC tumor-bearing nude mice. Regarding the molecular mechanisms of CVB-D activity, decreased Slug expression was determined to be associated with the aforementioned anti-HCC functions of this extract, which might be regulated by epidermal growth factor receptor (EGFR) through the focal adhesion kinase (FAK)-associated PI3K/AKT and MEK/ERK1/2 signaling pathways. Collectively, our results revealed the suppressive effects of CVB-D on progressive behaviors of HCC, including proliferation, migration, invasion, and EMT, in addition to its outstanding proapoptotic effects, which were correlated with the inhibition of the EGFR-FAK-AKT/ERK1/2-Slug signaling pathway. These discoveries provide an experimental and theoretical foundation for the use of CVB-D as a promising candidate for HCC therapy.

Efficient Generation of Large-Fragment Knock-In Mouse Models Using 2-Cell (2C)-Homologous Recombination (HR)-CRISPR.

Generating large-fragment knock-ins, such as reporters, conditional alleles, or humanized alleles, directly in mouse embryos is still a challenging feat. We have developed 2C-HR-CRISPR, a technology that allows highly efficient (10-50%) and rapid (generating founders in 2 months) targeting of large DNA fragments. Key to this strategy is the delivery of CRISPR reagents into 2-cell-stage mouse embryos, taking advantage of the high homologous recombination activity during the long G2 cell cycle phase at this stage. Furthermore, by exploiting a Cas9-monomeric streptavidin (Cas-mSA) and biotinylated PCR template (BioPCR) system to localize the repair template to specific double strand breaks, the efficiency can be further improved to up to 95%. Here we provide a procedure to generate large-fragment knock-in mouse models using 2C-HR-CRISPR. We first describe the principles for designing single guide RNAs and repair templates but refer to published manuscripts and protocols for molecular cloning methods or commercial sources for these reagents. We then describe two unique aspects of 2C-HR-CRISPR that are critical for success: (1) production of the CRISPR reagents for 2C-HR-CRISPR, particularly for applying the Cas9-mSA/BioPCR method, and (2) microinjection of mouse embryos at the 2-cell stage. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Single guide RNA and repair template design Basic Protocol 2: Preparing reagents for 2C-HR-CRISPR Basic Protocol 3: Microinjecting 2-cell-stage mouse embryos.

Comparison of Morphological and Functional Corneal Endothelial Changes after Cataract Surgery under DBCS Program at a Tertiary Care Centre

Introduction: Cataract extraction constitutes the largest workload through DBCS program in ophthalmic units in India. Both SICS & Phacoemulsification surgery are performed in a limited, confined space; however, securing adequate surgical space during cataract surgery can decrease the risk of corneal endothelial cell loss.1 Thus, anatomical and surgical factors, such as adequate Anterior Chamber Depth (ACD), are important for preserving these cells from the mechanical and thermal damage that can occur during the procedure. Under normal circumstances, corneal endothelial cells do not proliferate because they are stuck in G1 phase of cell cycle. The central corneal endothelial cell density gradually decreases at an average of about 0.6% per year, reducing approximately from 3400 cells/mm2 at age 15 to 2300 cells at age 80 years. Two important factors in maintaining corneal transparency are the number and the integrity of the corneal endothelial cells.3 Corneas with cell densities as low as 500 cells/mm2 and average cell area of approximately 2000-3000 μm2 can remain clear. Protection of corneal endothelium during cataract surgery is critical for achieving good visual outcomes. Objectives: To study the endothelial cell loss in SICS and Phacoemulsification and compare the cell loss between two surgeries and to compare morphological and functional changes in corneal endothelium between the two surgeries. Materials and methods: A comparative prospective study is performed in 200 eyes of 200 patients attending DBCS Camp at Babuji Eye Hospital and Chigateri General Hospital attached to JJM Medical College Hospital, Davanagere from July 2018 to October 2019 for cataract surgery. Patients were randomly allocated to two groups. One group underwent small incision cataract surgery and the other group underwent phacoemulsification. Corneal endothelial assessment was done for patients undergoing both the surgeries using non-contact specular microscopy Tomey EM 3000 preoperatively and postoperatively at 1 week and 6 weeks. Results: In SICS group, corneal endothelial count was 2303.0 ± 329.1 preoperatively, postoperatively it was reduced to 2068.9 ± 381.1 at 1 week and 1980.3 ± 401.5 at 6 weeks. In phacoemulsification group, it was found to be 2213.9 ± 442.3 preoperatively and which reduced to 1878.7 ± 458.3 at 1 week and 1796.4 ± 467.3 at 6 weeks postoperatively. Cases in SICS group showed 10.2% loss at 1 week and 14% cell loss at 6 weeks while phacoemulsification group showed 15.1% cell loss at 1 week and 18.9% cell loss at 6 weeks. Polymegathism was increased in both the groups while hexagonality was decreased in both groups. No significant difference in functional parameters like central corneal thickness (CCT) and best corrected visual acuity (BCVA) was observed in both SICS and Phacoemulsification group at the end of 6 weeks. Conclusion: The study shows that phacoemulsification is safe procedure in experienced hands. Compared to phacoemulsification, small incision cataract surgery is associated with lower post-operative endothelial damage. It is suggested that SICS should be used in patients at risk for endothelial damage in cataract surgery.

Radioimmunotherapy of B-cell lymphoma: In vitro investigations of 177Lu-rituximab on raji cells

Background: Rituximab is a chimeric monoclonal antibody, approved by the US Food and Drug Administration for the immunotherapy of non-Hodgkin's lymphoma (NHL).177Lu-labeled rituximab has been identified as a potential agent for the radioimmunotherapy of NHL and is presently under clinical investigations. The objective of the present study is to estimate the magnitude of apoptotic cell death and cell-cycle phase arrest. Materials and Methods: Characterization of177Lu-rituximab was performed by using instant thin-layer chromatography as well as by high-performance liquid chromatography. About 37 MBq (1 mCi) of177Lu-rituximab was incubated with Raji cells up to 48 h at 37°C in a humidified atmosphere of 5% CO2. Simultaneously, an equivalent amount of rituximab present in 37 MBq (1 mCi) of177Lu-rituximab complex was used as a vehicle control. All cell samples (treated, vehicle control, and control cells) were harvested post 24 and 48 h of incubation to perform different assays such as lactate dehydrogenase, XTT, cell viability by flowcytometer, apoptosis, and cell cycle analysis. Results: The studies revealed that177Lu-rituximab induced higher cell death and apoptosis compared to unlabeled rituximab. Similarly, an increase in cell population in G1-phase of cell cycle was observed, upon treatment of Raji cells with177Lu-rituximab complex for 24 h, while an increase in G2/M phase population was observed at 48 h of incubation. Conclusions: The present studies demonstrate that177Lu-rituximab is more effective in inducing apoptotic cell death and cell cycle-phase arrest compared to its unlabeled counterpart, indicating177Lu-rituximab may have better potential in the therapy of B-cell lymphoma.

RNA Polymerase II is Required for Spatial Chromatin Reorganization Following Exit from Mitosis

Mammalian chromosomes are three-dimensional entities shaped by converging and opposing forces. Mitotic cell division induces drastic chromosome condensation, but following reentry into the G1 cell cycle phase, condensed chromosomes unwind to reestablish interphase organization. Here, we use a cell line allowing auxin-mediated degradation of RNA polymerase II to test its role in this transition. In situ Hi-C showed that RNAPII is required for compartment and loop formation following mitosis. RNAPs often counteract loop extrusion and, in their absence, longer and more prominent loops arise. Evidence from chromatin fractionation, super-resolution imaging and in silico modeling attribute these effects to RNAPII-mediated cohesin loading at active promoters upon reentry into G1. Our findings reconcile the role of RNAPII in gene expression with that in chromatin architecture.

ASIC1a Inhibitor mambalgin-2 Suppresses the Growth of Leukemia Cells by Cell Cycle Arrest.

Although tyrosine kinase inhibitors have brought significant success in the treatment of chronic myelogenous leukemia, the search for novel molecular targets for the treatment of this disease remains relevant. Earlier, expression of acid-sensing ion channels, ASIC1a, was demonstrated in the chronic myelogenous leukemia K562 cells. Three-finger toxins from the black mamba (Dendroaspis polylepis) venom, mambalgins, have been shown to efficiently inhibit homo- and heteromeric channels containing the ASIC1a subunit; however, their use as possible antitumor agents had not been examined. In this work, using the patch-clamp technique, we detected, for the first time, an activation of ASIC1a channels in the leukemia K562 cells in response to an extracellular pH decrease. Recombinant mambalgin-2 was shown to inhibit ASIC1a activity and suppress the proliferation of the K562 cells with a half-maximal effective concentration (EC50) ~ 0.2 μM. Maximum mambalgin-2 inhibitory effect is achieved after 72 h of incubation with cells and when the pH of the cell medium reaches ~ 6.6. In the K562 cells, mambalgin-2 caused arrest of the cell cycle in the G1 phase and reduced the phosphorylation of G1 cell cycle phase regulators: cyclin D1 and cyclin-dependent kinase CDK4, without affecting the activity of CDK6 kinase. Thus, recombinant mambalgin-2 can be considered a prototype of a new type of drugs for the treatment of chronic myelogenous leukemia.

Crebanine N-oxide, a natural aporphine alkaloid isolated from Stephania hainanensis, induces apoptosis and autophagy in human gastric cancer SGC-7901 cells

Objective: To investigate the cytotoxic effects and the potential mechanisms of crebanine N-oxide in SGC-7901 gastric adenocarcinoma cells. Methods: The cytotoxicity of crebanine N-oxide was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay and cellular morphology was observed under a microscope. Cell apoptosis was determined by flow cytometry using propidium iodide staining. The expression levels of apoptotic-related proteins, cleaved caspase-3, cytochrome C, p53 and Bax, and autophagy- related proteins p62, beclin1 and LC3 were detected by Western blotting assays. Results: Crebanine N-oxide treatment significantly inhibited the proliferation of SGC-7901 cells in a dose-dependent and time- dependent manner via induction of G2-phase cell cycle arrest, apoptosis, and autophagy in SGC-7901 cells. Conclusions: Crebanine N-oxide could inhibit the growth of gastric cancer cells by promoting apoptosis and autophagy and could be used as a potential agent for treating gastric cancer.

Green synthesis and characterization of gold nanoparticles using endophytic fungi Fusarium solani and its in-vitro anticancer and biomedical applications.

The present study aimed to explore the anticancer potentials of the gold nanoparticles (NPs) obtained by green synthesis method using an endophytic strain Fusarium solani ATLOY – 8 has been isolated from the plant Chonemorpha fragrans. The formation of the NPs was analyzed by UV, FTIR, SEM and XRD. The synthesized NPs showed pink-ruby red colors and high peak plasmon band was observed between 510 and 560 nm. It is observed that intensity of absorption steadily increases the wavelength and band stabilizes at 551 nm. The XRD pattern revealed the angles at 19, 38.32, 46.16, 57.50, and 76.81° respectively. Interestingly, the FTIR band absorption noted at 1413 cm−1, 1041 cm−1 and 690 cm−1 ascribed the presence of amine II bands of protein, C-N and C-H stretching vibrations of the nanoparticles. SEM analysis indicated that the average diameter of the synthesized nanoparticles was between 40 and 45 nm. These NPs showed cytotoxicity on cervical cancer cells (He La) and against human breast cancer cells (MCF-7) and the NPs exhibited dose dependent cytotoxic effect. IC50 value was 0.8 ± 0.5 μg/mL on MCF-7 cell line and was found to be 1.3 ± 0.5 μg/mL on MCF-7 cell lines. The synthesized NPs induced apoptosis on these cancer cell lines. The accumulation of apoptotic cells decreased in sub G0 and G1 phase of cell cycle in the MCF-7 cancer cells were found to be 55.13%, 52.11% and 51.10% after 12 h exposure to different concentrations. The results altogether provide an apparent and versatile biomedical application for safer chemotherapeutic agent with little systemic toxicity.

MLL1 promotes myogenesis by epigenetically regulating Myf5.

ObjectivesMixed lineage leukaemia protein‐1 (MLL1) mediates histone 3 lysine 4 (H3K4) trimethylation (me3) and plays vital roles during early embryonic development and hematopoiesis. In our previous study, we found its expression was positively correlated with embryonic myogenic ability in pigs, indicating its potential roles in mammalian muscle development. The present work aimed to explore the roles and regulation mechanisms of MLL1 in myogenesis.Materials and methodsThe expression of MLL1 in C2C12 cells was experimentally manipulated using small interfering RNAs (siRNA). 5‐ethynyl‐2′‐deoxyuridine (EdU) assay, cell cycle assay, immunofluorescence, qRT‐PCR and Western blot were performed to assess myoblast proliferation and differentiation. Chromatin immunoprecipitation assay was conducted to detect H3K4me3 enrichment on myogenic factor 5 (Myf5) promoter. A cardiotoxin (CTX)‐mediated muscle regeneration model was used to investigate the effects of MLL1 on myogenesis in vivo.ResultsMLL1 was highly expressed in proliferating C2C12 cells, and expression decreased after differentiation. Knocking down MLL1 suppressed myoblast proliferation and impaired myoblast differentiation. Furthermore, knockdown of MLL1 resulted in the arrest of cell cycle in G1 phase, with decreased expressions of Myf5 and Cyclin D1. Mechanically, MLL1 transcriptionally regulated Myf5 by mediating H3K4me3 on its promoter. In vivo data implied that MLL1 was required for Pax7‐positive satellite cell proliferation and muscle repair.ConclusionMLL1 facilitates proliferation of myoblasts and Pax7‐positive satellite cells by epigenetically regulating Myf5 via mediating H3K4me3 on its promoter.

6-Gingerol induces cell-cycle G1-phase arrest through AKT\u2013GSK 3\u03b2\u2013cyclin D1 pathway in renal-cell carcinoma

Purpose6-Gingerol, a major biochemical and pharmacological active ingredient of ginger, has shown anti-inflammatory and antitumor activities against various cancers. Searching for natural products with fewer side effects for developing adjunctive therapeutic options is necessary.MethodsThe effects of 6-gingerol on proliferation, colony formation, and cell cycle in RCC cells were detected by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony formation assay, and propidium iodide (PI) staining, respectively. Western blotting, an immunofluorescence assay, and immunohistochemical staining were performed to assess the expression of relevant proteins. A subcutaneous tumor model was set up to investigate the 6-gingerol effects on tumor growth in vivo, and the pharmacokinetics of 6-gingerol in mice were detected by LC/MS assays.Results6-Gingerol treatment exerted time- and dose-dependent inhibition of the growth and colony formation of ACHN, 786-O, and 769-P cells, leading to a concomitant induction of cell-cycle G1-phase arrest and decrease in Ki-67 expression in the cell nucleus. Western-blotting results showed that 6-gingerol reduces phosphorylation of protein kinase B (AKT) Ser 473, cyclin-dependent kinases (CDK4), and cyclin D1 and, meanwhile, increases glycogen synthase kinase (GSK 3β) protein amount. Furthermore, the efficacy of 6-gingerol was demonstrated in an in vivo murine model of 786-O.ConclusionThe above results indicate that 6-gingerol can induce cell-cycle arrest and cell-growth inhibition through the AKT–GSK 3β–cyclin D1 signaling pathway in vitro and in vivo, suggesting that 6-gingerol should be useful for renal-cell carcinoma treatment.

α-Hederin induces the apoptosis of gastric cancer cells accompanied by glutathione decrement and reactive oxygen species generation via activating mitochondrial dependent pathway.

α-Hederin, a monodesmosidic triterpenoid saponin, exhibited promising antitumor potential against a variety of human cancer cell lines. However, few related studies about effects of α-hederin on gastric cancer are available. Herein, our results showed that α-hederin significantly inhibited the proliferation of gastric cancer cells and arrested the cell cycle in G1 phase in vitro (p < .05). Further research of the potential mechanism reflected that α-hederin could induce intracellular glutathione decrement, adenosine triphosphate level, and mitochondrial membrane potential variation via inducing reactive oxygen species accumulation during the apoptosis of gastric cancer cells. Moreover, the detection of mitochondrial and cytosol proteins with apoptosis-inducing factor, apoptosis protease activating factor-1, and cytochrome C showed an increase in the cytosol, followed by a decrease of Bcl-2 levels and increases of caspase-3, caspase-8, caspase-9, and Bax, which revealed that α-hederin induced apoptosis via triggering activation of the mitochondrial pathway. Furthermore, the above changes were amplified when pretreated with buthionine sulfoximine, whereas attenuated in the group pretreated with NAC than α-hederin alone (p < .05). In addition, α-hederin significantly inhibited the growth of xenografted gastric tumors with favorable safety. In conclusion, α-hederin could inhibit the proliferation and induce apoptosis of gastric cancer accompanied by glutathione decrement and reactive oxygen species generation via activating mitochondrial dependent pathway.

Design, Synthesis, and Anticancer Activity of Novel Trimethoxyphenyl-Derived Chalcone-Benzimidazolium Salts.

A series of novel trimethoxyphenyl-derived chalcone-benzimidazolium salts were synthesized. The biological properties of the compounds were screened in vitro against five different human tumor cell lines. The results suggest that the 5,6-dimethyl-benzimidazole or 2-methyl-benzimidazole ring as well as the 2-naphthylmethyl, 4-methylbenzyl, or 2-naphthylacyl substituent at position-3 of the benzimidazole ring was important to the cytotoxic activity. Notably, (E)-5,6-dimethyl-3-(naphthalen-2-ylmethyl)-1-(3-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenoxy)propyl)-1H-benzo[d]imidazol-3-ium bromide (7f) was more selective to HL-60, MCF-7, and SW-480 cell lines with IC50 values 8.0-, 11.1-, and 5.8-fold lower than DDP. Studies of the antitumor mechanism of action showed that compound 7f could induce cell-cycle G1 phase arrest and apoptosis in SMMC-7721 cells.

Hematopoietic Stem Cells Increase Quiescence during Aging

Introduction: Definitive hematopoietic stem cells (HSCs) sustain blood production from fetal development throughout life. In mice, most of steady state, young adult HSCs are in the G0 phase of cell cycle (quiescence), and are estimated to divide roughly once a month. Daily hematopoietic production is thus mainly sustained by highly proliferative downstream hematopoietic progenitor cells (HPCs). Aged haematopoiesis was demonstrated to be distinct from young haematopoiesis in various aspects such as i) a shift from lymphopoiesis to myelopoiesis, ii) functional decline of HSCs (self-renewal, homing), and iii) HSCs pool expansion. While several studies attempted to address whether changes in HSCs turnover during aging can account for the distinct aging associated phenotype and function, it remained to be determined whether aged HSCs overall cycle more or less frequently than young HSCs. Methods: To construct data-based, quantitative models, we measured turnover rates and compartment sizes of populations of HSCs, HSPCs and granulopoiesis/granulocytes, i.e. a post-mitotic mature hematopoietic linage with a short half-life. We examined four age groups: 3 week, 2 month, 1 year and 2 year old mice. Mice in each group were i.p. injected every 4 hours with 1 mcg EdU up to a maximum time of 48 hours. HSC, HSPC and granulopoiesis/granoulocyte compartment sizes and snapshot cell-cycle analysis was performed by FACS at multiple sampling points in BM and peripheral blood (PB), respectively. Based on this data, we built a mathematical model of HSC turn-over and HSPC differentiation during ageing. Moreover, we evaluated HSC cycling by CFSE dilution in steady-state transplantation experiments (as described before; Takizawa et al., J Exp Med 2011). Results: In line with previous reports, the HSCs compartment size gradually increased with age from 3wk old mice to 2 year old mice. In sharp contrast, cycling activity of HSCs as determined by EdU incorporation decreased gradually and significantly with increasing age. This was driven by decreased activation from the quiescent state, while the time that actively cycling HSCs require to progress through cell-division remains constant with age. Multipotent Progenitor (MPP) cycling showed a non-significant trend towards slower turn-over. These results were confirmed by complementary CFSE-dilution experiments. Mathematical modeling of HSC proliferation and differentiation revealed a higher probability of self-renewing divisions in 3 week old mice as compared to 2 month, 1 and 2 year old mice, with the latter both having nearly equal chances of self-renewing versus differentiating divisions. Conclusions: Our data clarifies the long-standing question, how the HSC pool increases with age. Instead of an increase in active cycling, an increase in HSC quiescence is responsible for the increased size of the HSCs pool in aging. Disclosures No relevant conflicts of interest to declare.

A feedback loop of conditionally stable circuits drives the cell cycle from checkpoint to checkpoint

We perform logic-based network analysis on a model of the mammalian cell cycle. This model is composed of a Restriction Switch driving cell cycle commitment and a Phase Switch driving mitotic entry and exit. By generalizing the concept of stable motif, i.e., a self-sustaining positive feedback loop that maintains an associated state, we introduce the concept of a conditionally stable motif, the stability of which is contingent on external conditions. We show that the stable motifs of the Phase Switch are contingent on the state of three nodes through which it receives input from the rest of the network. Biologically, these conditions correspond to cell cycle checkpoints. Holding these nodes locked (akin to a checkpoint-free cell) transforms the Phase Switch into an autonomous oscillator that robustly toggles through the cell cycle phases G1, G2 and mitosis. The conditionally stable motifs of the Phase Switch Oscillator are organized into an ordered sequence, such that they serially stabilize each other but also cause their own destabilization. Along the way they channel the dynamics of the module onto a narrow path in state space, lending robustness to the oscillation. Self-destabilizing conditionally stable motifs suggest a general negative feedback mechanism leading to sustained oscillations.