scholarly journals Mouse spleen lymphoblasts generated in vitro. Recovery in high yield and purity after floatation in dense bovine plasma albumin solutions.

1978 ◽  
Vol 147 (2) ◽  
pp. 279-296 ◽  
Author(s):  
R M Steinman ◽  
B G Machtinger ◽  
J Fried ◽  
Z A Cohn
Keyword(s):  
Cell Cycle ◽  
Cultured Cells ◽  
Mouse Spleen ◽  
High Yield ◽  
Cell Cycle Distribution ◽  
Low Density ◽  
Proliferating Cells ◽  
M Phase ◽  
Yield And Purity

Mouse spleen lymphoblasts, stimulated to divide in vitro, acquired a low cell density and could be separated by isopycnic techniques. Cultured cells were suspended in BPA columns, rho = 1.080, and spun to equilibrium. The method was simple, fast, accomodated large numbers of cells, and was reproducible. It provided lymphoblasts in high yield and purity (at least 80% of the low density cells were blasts). It allowed for the recovery of proliferating cells in their first cell cycle, and did not alter the subsequent ability of cells to proliferate when recultured in vitro. Certain properties of mouse spleen lymphoblasts were analyzed in detail. Lymphoblasts induced by LPS, FCS, con A (tetravalent and succinylated), and MLC were very similar except in the absolute numbers that were induced. The blasts exhibited the classic cytologic features of enlarged nucleoli and abundant cytoplasmic polyribosomes (basophilia). As a population, they were enlarged in size relative to nondividing cells, but this seemed to apply primarily to cells in the S and G2+ M phase of the cell cycle rather than G1. The cell cycle distribution of lymphoblasts was analyzed by flow microfluorometry. By analyzing low density cells obtained at varying intervals after mitogen stimulation, FMF indicated that lymphoblasts enter the S phase of their first cell cycle beginning at 20-24 h after stimulation.

scholarly journals 3,3′-Diindolylmethane: A Promising Sensitizer ofγ-Irradiation

2015 ◽  
Vol 2015 ◽  
pp. 1-6 ◽  
Author(s):  
Wenjing Wang ◽  
Maomin Lv ◽  
Chaoji Huangfu ◽  
Fang Wang ◽  
Jingang Zhang
Keyword(s):  
Cell Cycle ◽  
Phase Cell ◽  
Cell Cycle Distribution ◽  
Effective Treatment Modality ◽  
Intracellular Ros ◽  
M Phase ◽  
Induced Apoptosis ◽  
Mtt Assays ◽  
Mcf 7

Purpose. Radiotherapy is an effective treatment modality in the clinical treatment of breast cancer. The present work investigated the effect of 3,3′-diindolylmethane (DIM) onγ-irradiation sensitizing human breast carcinoma.Methods. Cell survival, intracellular ROS levels, cell cycle distribution, cell apoptosis, and expression of proteins related to apoptosis were measured with MTT assays, flow cytometry, and Western blot analysis, respectively.Results.In vitroDIM plusγ-irradiation arrested the activity of G2/M phase cell cycle, increased intracellular ROS level, significantly suppressed PARP (poly ADP-ribose polymerase), and enhancedγ-irradiation-induced apoptosis, thereby inhibiting the proliferation of MCF-7 cells.Conclusion. These data provide a rationale for the use of DIM as a promising sensitizer ofγ-irradiation.

scholarly journals Ionic Dissolution Products of NovaBone® Promote Osteoblastic Proliferation via Influences on the Cell Cycle

2009 ◽  
Vol 37 (3) ◽  
pp. 737-745 ◽  
Author(s):  
Z Qiu ◽  
H Yang ◽  
J Wu ◽  
L Wei ◽  
J Li
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Regulation ◽  
Cultured Cells ◽  
Bone Morphogenic Protein ◽  
Mg63 Cells ◽  
Silicon Ions ◽  
Cycle Regulation ◽  
Bone Morphogenic Protein 2 ◽  
Cells Cultured

This study investigated the effects of the ionic dissolution products of NovaBone® on osteoblastic proliferation and cell cycle regulation. MG63 osteoblast-like cells were cultured in NovaBone®-conditioned Dulbecco's Modified Eagle's Medium (DMEM) or control DMEM for 10 days. The concentration of silicon ions was significantly higher in NovaBone®-conditioned DMEM than control DMEM. MG63 cells cultured in NovaBone®-conditioned DMEM exhibited greater proliferation on days 1 and 4 than control cells. There were increased proportions of Novabone®-conditioned DMEM-cultured cells in the S and G2/M phases, and decreased proportions in the G0/G1 phase on days 1 and 4 versus control cells, while no differences were observed on days 7 and 10 between the two groups. Bone morphogenic protein 2 production increased in both groups, but was significantly higher for the NovaBone®-conditioned DMEM-cultured cells on day 10 compared with the controls. In conclusion, the NovaBone® ionic dissolution products, particularly the silicon ions, promoted proliferation of MG63 osteoblast-like cells in vitro via influences on the cell cycle.

The Xenopus protein kinase pEg2 associates with the centrosome in a cell cycle-dependent manner, binds to the spindle microtubules and is involved in bipolar mitotic spindle assembly

1998 ◽  
Vol 111 (5) ◽  
pp. 557-572 ◽  
Author(s):  
C. Roghi ◽  
R. Giet ◽  
R. Uzbekov ◽  
N. Morin ◽  
I. Chartrain ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Mitotic Spindle ◽  
Cultured Cells ◽  
Dependent Manner ◽  
Egg Extracts ◽  
Pericentriolar Material ◽  
Spindle Microtubules ◽  
Cycle Dependent

By differential screening of a Xenopus laevis egg cDNA library, we have isolated a 2,111 bp cDNA which corresponds to a maternal mRNA specifically deadenylated after fertilisation. This cDNA, called Eg2, encodes a 407 amino acid protein kinase. The pEg2 sequence shows significant identity with members of a new protein kinase sub-family which includes Aurora from Drosophila and Ipl1 (increase in ploidy-1) from budding yeast, enzymes involved in centrosome migration and chromosome segregation, respectively. A single 46 kDa polypeptide, which corresponds to the deduced molecular mass of pEg2, is immunodetected in Xenopus oocyte and egg extracts, as well as in lysates of Xenopus XL2 cultured cells. In XL2 cells, pEg2 is immunodetected only in S, G2 and M phases of the cell cycle, where it always localises to the centrosomal region of the cell. In addition, pEg2 ‘invades’ the microtubules at the poles of the mitotic spindle in metaphase and anaphase. Immunoelectron microscopy experiments show that pEg2 is located precisely around the pericentriolar material in prophase and on the spindle microtubules in anaphase. We also demonstrate that pEg2 binds directly to taxol stabilised microtubules in vitro. In addition, we show that the presence of microtubules during mitosis is not necessary for an association between pEg2 and the centrosome. Finally we show that a catalytically inactive pEg2 kinase stops the assembly of bipolar mitotic spindles in Xenopus egg extracts.

A novel PLK1 inhibitor onvansertib effectively sensitizes MYC-driven medulloblastoma to radiotherapy

2021 ◽  
Author(s):  
Dong Wang ◽  
Bethany Veo ◽  
Angela Pierce ◽  
Susan Fosmire ◽  
Krishna Madhavan ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Tumor Growth ◽  
Radiation Treatment ◽  
Tumor Regression ◽  
Tumor Growth Inhibition ◽  
Cell Cycle Distribution ◽  
Cell Renewal ◽  
Group 3

Abstract Background Group 3 medulloblastoma (MB) is often accompanied by MYC amplification. PLK1 is an oncogenic kinase that controls cell cycle and proliferation and has been preclinically validated as a cancer therapeutic target. Onvansertib (PCM-075) is a novel, orally available PLK1 inhibitor, which shows tumor growth inhibition in various types of cancer. We aim to explore the effect of onvansertib on MYC-driven medulloblastoma as a monotherapy or in combination with radiation. Methods Crisper-Cas9 screen was used to discover essential genes for MB tumor growth. Microarray and immunohistochemistry on pediatric patient samples were performed to examine the expression of PLK1. The effect of onvansertib in vitro was measure by cell viability, colony-forming assays, extreme limiting dilution assay and RNA-Seq. ALDH activity, cell-cycle distribution and apoptosis were analyzed by flow cytometry. DNA damage was assessed by immunofluorescence staining. Medulloblastoma xenografts were generated to explore the monotherapy or radio-sensitizing effect. Results PLK1 is overexpressed in Group 3 MB. The IC50 concentrations of onvansertib in Group 3 MB cell lines were in a low nanomolar range. Onvansertib reduced colony formation, cell proliferation, stem cell renewal and induced G2/M arrest in vitro. Moreover, onvansertib in combination with radiation increased DNA damage and apoptosis compare with radiation treatment alone. The combination radiotherapy resulted in marked tumor regression in xenografts. Conclusions These findings demonstrate the efficacy of a novel PLK1 inhibitor onvansertib in vitro and in xenografts of Group 3 MB, which suggests onvansertib is an effective strategy as monotherapy or in combination with radiotherapy in MB.

Apoptosis Susceptibility and Cell-Cycle Distribution in Cells from Myelodysplastic Syndrome Patients: Modulatory In-Vitro Effects of G-CSF and Interferon-α

2004 ◽  
Vol 45 (7) ◽  
pp. 1437-1443 ◽  
Author(s):  
Maria R Ricciardi ◽  
Maria T Petrucci ◽  
Chiara Gregorj ◽  
Vincenza Martini ◽  
Anna Levi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Myelodysplastic Syndrome ◽  
Interferon Α ◽  
Cell Cycle Distribution ◽  
In Vitro Effects ◽  
In Cells

Paralogs of Atlantic salmon myoblast determination factor genes are distinctly regulated in proliferating and differentiating myogenic cells

2010 ◽  
Vol 298 (6) ◽  
pp. R1615-R1626 ◽  
Author(s):  
Neil I. Bower ◽  
Ian A. Johnston
Keyword(s):  
Cell Cycle ◽  
Amino Acids ◽  
Amino Acid ◽  
Atlantic Salmon ◽  
Antigen Expression ◽  
Nuclear Antigen ◽  
Quiescent State ◽  
Mrna Levels ◽  
Proliferating Cells

The mRNA expression of myogenic regulatory factors, including myoD1 (myoblast determination factor) gene paralogs, and their regulation by amino acids and insulin-like growth factors were investigated in primary cell cultures isolated from fast myotomal muscle of Atlantic salmon ( Salmo salar). The cell cycle and S phase were determined as 28.1 and 13.3 h, respectively, at 18°C. Expression of myoD1b and myoD1c peaked at 8 days of culture in the initial proliferation phase and then declined more than sixfold as cells differentiated and was correlated with PCNA (proliferating cell nuclear antigen) expression ( R = 0.88, P < 0.0001; R = 0.70, P < 0.0001). In contrast, myoD1a transcripts increased from 2 to 8 days and remained at elevated levels as myotubes were formed. mRNA levels of myoD1c were, on average, 3.1- and 5.7-fold higher than myoD1a and myoD1b, respectively. Depriving cells of amino acids and serum led to a rapid increase in pax7 and a decrease in myoD1c and PCNA expression, indicating a transition to a quiescent state. In contrast, amino acid replacement in starved cells produced significant increases in myoD1c (at 6 h), PCNA (at 12 h), and myoD1b (at 24 h) and decreases in pax7 expression as cells entered the cell cycle. Our results are consistent with temporally distinct patterns of myoD1c and myoD1b expression at the G1 and S/G2 phases of the cell cycle. Treatment of starved cells with insulin-like growth factor I or II did not alter expression of the myoD paralogs. It was concluded that, in vitro, amino acids alone are sufficient to stimulate expression of genes regulating myogenesis in myoblasts involving autocrine/paracrine pathways. The differential responses of myoD paralogs during myotube maturation and amino acid treatments suggest that myoD1b and myoD1c are primarily expressed in proliferating cells and myoD1a in differentiating cells, providing evidence for their subfunctionalization following whole genome and local duplications in the Atlantic salmon lineage.

The Polo-like kinase Plx1 is a component of the MPF amplification loop at the G2/M-phase transition of the cell cycle in Xenopus eggs

1998 ◽  
Vol 111 (12) ◽  
pp. 1751-1757 ◽  
Author(s):  
A. Abrieu ◽  
T. Brassac ◽  
S. Galas ◽  
D. Fisher ◽  
J.C. Labbe ◽  
...  
Keyword(s):  
Phase Transition ◽  
Cell Cycle ◽  
Cyclin A ◽  
Cyclin B ◽  
Cdc2 Kinase ◽  
C Terminus ◽  
M Phase ◽  
Egg Extracts ◽  
Amplification Loop

We have investigated whether Plx1, a kinase recently shown to phosphorylate cdc25c in vitro, is required for activation of cdc25c at the G2/M-phase transition of the cell cycle in Xenopus. Using immunodepletion or the mere addition of an antibody against the C terminus of Plx1, which suppressed its activation (not its activity) at G2/M, we show that Plx1 activity is required for activation of cyclin B-cdc2 kinase in both interphase egg extracts receiving recombinant cyclin B, and cycling extracts that spontaneously oscillate between interphase and mitosis. Furthermore, a positive feedback loop allows cyclin B-cdc2 kinase to activate Plx1 at the G2/M-phase transition. In contrast, activation of cyclin A-cdc2 kinase does not require Plx1 activity, and cyclin A-cdc2 kinase fails to activate Plx1 and its consequence, cdc25c activation in cycling extracts.

Novel Thiadiazoline Spiro Quinoline Analogues Induced Cell death in MCF-7 cells via G2/M Phase Cell Cycle Arrest

2021 ◽  
Author(s):  
Selvaraj Shyamsivappan ◽  
Raju Vivek ◽  
Thangaraj Suresh ◽  
Adhigaman Kaviyarasu ◽  
Sundarasamy Amsaveni ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Cell Cycle Arrest ◽  
Spectroscopic Studies ◽  
Phase Cell ◽  
Quinoline Derivatives ◽  
Cycle Arrest ◽  
M Phase ◽  
Mcf 7

Abstract A progression of novel thiadiazoline spiro quinoline derivatives were synthesized from potent thiadiazoline spiro quinoline derivatives . The synthesized compounds portrayed by different spectroscopic studies and single X-ray crystallographic studies. The compounds were assessed for in vitro anticancer properties towards MCF-7 and HeLa cells. The compounds showed superior inhibition action MCF-7 malignant growth cells. Amongst, the compound 4a showed significant inhibition activity, the cell death mechanism was evaluated by fluorescent staining, and flow cytometry, RT-PCR, and western blot analyses. The in vitro anticancer results revealed that the compound 4a induced apoptosis by inhibition of estrogen receptor alpha (ERα) and G2/M phase cell cycle arrest. The binding affinity of the compounds with ERα and pharmacokinetic properties were confirmed by molecular docking studies.

scholarly journals Synthesis and In Vitro Antitumor Activity of Naringenin Oxime and Oxime Ether Derivatives

2019 ◽  
Vol 20 (9) ◽  
pp. 2184 ◽  
Author(s):  
Ahmed Dhahir Latif ◽  
Tímea Gonda ◽  
Máté Vágvölgyi ◽  
Norbert Kúsz ◽  
Ágnes Kulmány ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Antioxidant Activities ◽  
Biological Activities ◽  
Gynecological Cancer ◽  
Human Leukemia ◽  
Oxime Ether ◽  
M Phase ◽  
Induced Apoptosis ◽  
Mcf 7

Naringenin is one of the most abundant dietary flavonoids exerting several beneficial biological activities. Synthetic modification of naringenin is of continuous interest. During this study our aim was to synthesize a compound library of oxime and oxime ether derivatives of naringenin, and to investigate their biological activities. Two oximes and five oxime ether derivatives were prepared; their structure has been elucidated by NMR and high-resolution mass spectroscopy. The antiproliferative activity of the prepared compounds was evaluated by MTT assay against human leukemia (HL-60) and gynecological cancer cell lines isolated from cervical (HeLa, Siha) and breast (MCF-7, MDA-MB-231) cancers. Tert-butyl oxime ether derivative exerted the most potent cell growth inhibitory activity. Moreover, cell cycle analysis suggested that this derivative caused a significant increase in the hypodiploid (subG1) phase and induced apoptosis in Hela and Siha cells, and induced cell cycle arrest at G2/M phase in MCF-7 cells. The proapoptotic potential of the selected compound was confirmed by the activation of caspase-3. Antioxidant activities of the prepared molecules were also evaluated with xanthine oxidase, DPPH and ORAC assays, and the methyl substituted oxime ether exerted the most promising activity.

scholarly journals Mapping choline metabolites in normal and transformed cells

Metabolomics ◽  
2020 ◽  
Vol 16 (12) ◽  
Author(s):  
Irena Roci ◽  
Jeramie D. Watrous ◽  
Kim A. Lagerborg ◽  
Mohit Jain ◽  
Roland Nilsson
Keyword(s):  
Cell Lines ◽  
Cell Cycle Progression ◽  
High Resolution Mass Spectrometry ◽  
Proliferating Cells ◽  
Rnai Silencing ◽  
Choline Dehydrogenase ◽  
Choline Metabolism ◽  
Metabolic Products ◽  
M Phase

Abstract Introduction Choline is an essential human nutrient that is particular important for proliferating cells, and altered choline metabolism has been associated with cancer transformation. Yet, the various metabolic fates of choline in proliferating cells have not been investigated systematically. Objectives This study aims to map the metabolic products of choline in normal and cancerous proliferating cells. Methods We performed 13C-choline tracing followed by liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis of metabolic products in normal and in vitro-transformed (tumor-forming) epithelial cells, and also in tumor-derived cancer cell lines. Selected metabolites were quantified by internal standards. Results Untargeted analysis revealed 121 LCMS peaks that were 13C-labeled from choline, including various phospholipid species, but also previously unknown products such as monomethyl- and dimethyl-ethanolamines. Interestingly, we observed formation of betaine from choline specifically in tumor-derived cells. Expression of choline dehydrogenase (CHDH), which catalyzes the first step of betaine synthesis, correlated with betaine synthesis across the cell lines studied. RNAi silencing of CHDH did not affect cell proliferation, although we observed an increased fraction of G2M phase cells with some RNAi sequences, suggesting that CHDH and its product betaine may play a role in cell cycle progression. Betaine cell concentration was around 10 µM, arguing against an osmotic function, and was not used as a methyl donor. The function of betaine in these tumor-derived cells is presently unknown. Conclusion This study identifies novel metabolites of choline in cancer and normal cell lines, and reveals altered choline metabolism in cancer cells.

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