scholarly journals Combinatorial Histone H3 Modifications Are Dynamically Altered in Distinct Cell Cycle Phases

2021 ◽  
Author(s):  
Congcong Lu ◽  
Mariel Coradin ◽  
Kevin A. Janssen ◽  
Simone Sidoli ◽  
Benjamin A. Garcia
Keyword(s):  
Cell Cycle ◽  
Histone H3 ◽  
Distinct Cell ◽  
Cell Cycle Phases

Flow Cytometric Quantification of All Cell Cycle Phases and Apoptosis in a Two-Color Fluorescence Plot

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4720-4720
Author(s):  
Olivier Herault ◽  
Christine Vignon
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Histone H3 ◽  
Leukemic Cells ◽  
Becton Dickinson ◽  
Alexa Fluor ◽  
Flow Cytometric ◽  
M Phase ◽  
Cell Cycle Phases ◽  
The Impact

Abstract Abstract 4720 An optimal technology for cell cycle analysis would allow measuring concomitantly apoptosis, G0, G1, S, G2 and M phases in combination with cell surface phenotyping. We propose an easy method in flow cytometry allowing this discrimination in an only two-color fluorescent plot. It is based on the concomitant use of 7-amino-actinomycin D and antibodies anti-Ki67 and anti-phospho(Ser10)-histone H3 conjugated to Alexa Fluor.. 488 to discriminate G0 et M phases, respectively. The proposed method is particularly valuable in a clinical setting as verified by analyzing human leukemic cells from marrow samples or exposed to cell cycle modifiers. The method was established using the human KG1a cell line and was applied to charcterize the cell cycle and apoptosis of fresh human marrow leukemic cells. The cells were permeabilized with 1 mL of ice cold ethanol (1 h, 4°C). Following two washes with PBS, 1% FBS and 0.25% Triton X-100 (PFT), the cells were stained in 200 μL of PFT for 30 min at room temperature in the dark with 1 μg 7-AAD (Sigma-Aldrich), 5 μL Alexa Fluor.. 488-conjugated anti-human Ki67 mAb (B56, Becton-Dickinson) and 3 μL Alexa Fluor.. 488-conjugated anti-phospho(ser10)-histone H3 polyclonal antibody (Cell Signaling Technology). A control tube was prepared with 1 μg 7-AAD and 5 μL of mouse IgG1 Alexa Fluor.. 488 (Becton-Dickinson). After 2 washes with PFT, the cells were stained with 10 μL of APC-Cy7-conjugated anti-CD45 (A20, Becton-Dickinson) followed by incubation for 20 min at 4°C. Cells were then washed twice with PBS, centrifuged for 5 min at 500 g and resuspended in 300 μL of PBS. This flow cytometric method allows for a precise analysis of the impact on the cell cycle of various functional modulators. As an example, it was applied to analyze the pro-quiescent effects of contact with bone marrow MSCs as well as and the apoptosis induction and mitosis inhibition of the human KG1a leukemic cell line by camptothecin. The cell cycle characteristics of untreated KG1a cells were clearly quantified as follows: 0.4% sub-G1, 0.8% G0, 67.9% G1, 14.9% S, 14.2% G2 and 1.8% M phase. The contact with marrow MSCs during 72 h induced an increase in G0 phase and a decrease in M phase (5.3% and 0.4%, respectively). We verified the anti-proliferative and pro-apoptotic effects of 24 h exposure to camptothecin, which induced a decrease in S, G2 and M phases (6.1%, 6.2% and 0.4%, respectively) and an increase in sub-G1 phase (1.7%). Moreover, it is interesting to note that the staining protocol preserved the integrity of the plasma membrane and allows for the analysis of heterogeneous cell populations. We document here the successful utilization of this method to discriminate concomitantly apoptosis and the cell cycle phases in a model of leukemic cells exposed to inducers of cell cycle perturbations. The value of this method to analyze heterogeneous populations was shown by discriminating the marrow cells from acute myeloid leukemia. Disclosures: No relevant conflicts of interest to declare.

Hydrogen peroxide and quercetin induced changes on cell viability, apoptosis and oxidative stress in HepG2 cells

2020 ◽  
Vol 01 ◽  
Author(s):  
Ayşe Mine Yılmaz ◽  
Gökhan Biçim ◽  
Kübra Toprak ◽  
Betül Karademir Yılmaz ◽  
Irina Milisav ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Cycle ◽  
Hydrogen Peroxide ◽  
Cell Viability ◽  
Hepg2 Cells ◽  
Proteasome Activity ◽  
Cell Cycle Phases ◽  
Induced Changes ◽  
And Oxidative Stress ◽  
Quercetin Treatment

Background: Different cellular responses influence the progress of cancer. In this study, we have investigated the effect of hydrogen peroxide and quercetin induced changes on cell viability, apoptosis and oxidative stress in human hepatocellular carcinoma (HepG2) cells. Methods: The effects of hydrogen peroxide and quercetin on cell viability, cell cycle phases and oxidative stress related cellular changes were investigated. Cell viability was assessed by WST-1 assay. Apoptosis rate, cell cycle phase changes and oxidative stress were measured by flow cytometry. Protein expressions of p21, p27, p53, NF-Kβ-p50 and proteasome activity were determined by Western blot and fluorometry, respectively. Results: Hydrogen peroxide and quercetin treatment resulted in decreased cell viability and increased apoptosis in HepG2 cells. Proteasome activity was increased by hydrogen peroxide but decreased by quercetin treatment. Conclusion: Both agents resulted in decreased p53 protein expression and increased cell death by different mechanisms regarding proteostasis and cell cycle phases.

miRNA-mediated Drug Repurposing Unveiled Potential Candidate Drugs for Prolactinoma Treatment

2021 ◽  
Author(s):  
Büşra Aydin ◽  
Sema Arslan ◽  
Fatih Bayraklı ◽  
Betül Karademir ◽  
Kazim Yalcin Arga
Keyword(s):  
Cell Cycle ◽  
Data Integration ◽  
Cell Viability ◽  
Drug Repurposing ◽  
Serum Prolactin ◽  
Drug Candidates ◽  
Mirna Level ◽  
Level Data ◽  
Cell Cycle Phases

Introduction: Prolactinomas, also called lactotroph adenomas, are the most encountered type of hormone-secreting pituitary neuroendocrine tumors (PitNET) in the clinic. The preferred first-line therapy is a medical treatment with dopamine agonists (DA), mainly cabergoline, to reduce serum prolactin levels, tumor volume, and mass effect. However, in some cases, patients have displayed DA-resistance with aggressive tumor behavior or are faced with recurrence after drug withdrawal. Also, currently used therapeutics have notorious side effects and impair the life quality of the patients. Methods: Since the amalgamation of clinical and laboratory data besides tumor histopathogenesis and transcriptional regulatory features of the tumor emerge to exhibit essential roles in the behavior and progression of prolactinomas, in this work, we integrated mRNA and microRNA (miRNA) level transcriptome data that exploit disease-specific signatures in addition to biological and pharmacological data to elucidate a rational prioritization of pathways and drugs in prolactinoma. Results: We identified eight drug candidates through drug repurposing based on mRNA-miRNA level data integration and evaluated their potential through in vitro assays in the MMQ cell line. Seven re-purposed drugs including 5-flourocytosine, nortriptyline, neratinib, puromycin, taxifolin, vorinostat, and zileuton were proposed as potential drug candidates for the treatment of prolactinoma. We further hypothesized possible mechanisms of drug action on MMQ cell viability through analyzing PI3K/Akt signaling pathway and cell cycle arrest via flow cytometry and western blotting. Discussion: We presented the transcriptomic landscape of prolactinoma through miRNA and mRNA level data integration and proposed repurposed drug candidates based on this integration. We validated our findings through testing cell viability, cell cycle phases, and PI3K/Akt protein expressions. Effects of the drugs on cell cycle phases and inhibition of PI3K/Akt pathway by all drugs gave us promising output for further studies using these drugs in the treatment of prolactinoma. This is the first study that reports miRNA-mediated repurposed drugs for prolactinoma treatment via in vitro experiments.

scholarly journals Modification of Histones by Sugar β-N-Acetylglucosamine (GlcNAc) Occurs on Multiple Residues, Including Histone H3 Serine 10, and Is Cell Cycle-regulated

2011 ◽  
Vol 286 (43) ◽  
pp. 37483-37495 ◽  
Author(s):  
Suisheng Zhang ◽  
Kevin Roche ◽  
Heinz-Peter Nasheuer ◽  
Noel Francis Lowndes
Keyword(s):  
Cell Cycle ◽  
Histone H3

Q-FIHC: Quantification of fluorescence immunohistochemistry to analysep63 isoforms and cell cycle phases in human limbal stem cells

2006 ◽  
Vol 69 (12) ◽  
pp. 983-991 ◽  
Author(s):  
Enzo Di Iorio ◽  
Vanessa Barbaro ◽  
Stefano Ferrari ◽  
Claudio Ortolani ◽  
Michele De Luca ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Limbal Stem Cells ◽  
Cell Cycle Phases ◽  
Fluorescence Immunohistochemistry

Histone H3 transcription in Saccharomyces cerevisiae is controlled by multiple cell cycle activation sites and a constitutive negative regulatory element

1992 ◽  
Vol 12 (12) ◽  
pp. 5455-5463 ◽  
Author(s):  
K B Freeman ◽  
L R Karns ◽  
K A Lutz ◽  
M M Smith
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cell Division ◽  
Transcriptional Activation ◽  
Regulatory Element ◽  
Histone H3 ◽  
Regulatory Elements ◽  
Cell Division Cycle ◽  
Cell Cycle Activation ◽  
Multiple Cell

The promoters of the Saccharomyces cerevisiae histone H3 and H4 genes were examined for cis-acting DNA sequence elements regulating transcription and cell division cycle control. Deletion and linker disruption mutations identified two classes of regulatory elements: multiple cell cycle activation (CCA) sites and a negative regulatory site (NRS). Duplicate 19-bp CCA sites are present in both the copy I and copy II histone H3-H4 promoters arranged as inverted repeats separated by 45 and 68 bp. The CCA sites are both necessary and sufficient to activate transcription under cell division cycle control. A single CCA site provides cell cycle control but is a weak transcriptional activator, while an inverted repeat comprising two CCA sites provides both strong transcriptional activation and cell division cycle control. The NRS was identified in the copy I histone H3-H4 promoter. Deletion or disruption of the NRS increased the level of the histone H3 promoter activity but did not alter the cell division cycle periodicity of transcription. When the CCA sites were deleted from the histone promoter, the NRS element was unable to confer cell division cycle control on the remaining basal level of transcription. When the NRS element was inserted into the promoter of a foreign reporter gene, transcription was constitutively repressed and did not acquire cell cycle regulation.

scholarly journals Adenovirus type 2 activates cell cycle-dependent genes that are a subset of those activated by serum.

1985 ◽  
Vol 5 (11) ◽  
pp. 2936-2942 ◽  
Author(s):  
H T Liu ◽  
R Baserga ◽  
W E Mercer
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Thymidine Kinase ◽  
Histone H3 ◽  
Adenovirus Type ◽  
3T3 Cells ◽  
Cycle Dependent ◽  
Cellular Dna ◽  
Adenovirus Type 2

We have studied a panel of 10 genes and cDNA sequences that are expressed in a cell cycle-dependent manner in different types of cells from different species and that are inducible by different mitogens. These include five sequences (c-myc, 4F1, 2F1, 2A9, and KC-1) that are preferentially expressed in the early part of the G1 phase, three genes (ornithine decarboxylase, p53, and c-rasHa) preferentially expressed in middle or late G1, and two genes (thymidine kinase and histone H3) preferentially expressed in the S phase of the cell cycle. We have studied the expression of these genes in nonpermissive (tsAF8) and semipermissive (Swiss 3T3) cells infected with adenovirus type 2. Under the conditions of these experiments, adenovirus type 2 infection stimulates cellular DNA synthesis in both tsAF8 and 3T3 cells. However, four of the five early G1 genes (c-myc, 4F1, KC-1, and 2A9) and one of the late G1 genes (c-ras) are not induced by adenovirus infection, although they are strongly induced by serum. The other sequences (2F1, ornithine decarboxylase, p53, thymidine kinase, and histone H3) are activated by both adenovirus and serum. We conclude that the cell cycle-dependent genes activated by adenovirus 2 are a subset of the cell cycle-dependent genes activated by serum. The data suggest that the mechanisms by which serum and adenovirus induce cellular DNA synthesis are not identical.

scholarly journals Continued initiation of DNA synthesis in arginine-deprived Chinese hamster ovary cells.

1977 ◽  
Vol 73 (1) ◽  
pp. 200-205 ◽  
Author(s):  
A S Weissfeld ◽  
H Rouse
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cell Multiplication ◽  
Ovary Cells ◽  
Cell Cycle Phases

When exponentially growing CHO cells were deprived of arginine (Arg), cell multiplication ceased after 12 h, but initiation of DNA synthesis continued: after 48 h of starvation with continuous [3H]thymidine exposure, 85% of the population had incorporated label, as detected autoradiographically. Consideration of the distribution of exponential cells in the various cell cycle phases leads to a calculation that most cells in G1 at the time that Arg was removed, as well as those in S, engaged in some DNA synthesis during starvation. In contrast, isoleucine (Ile)-starved cells did not initiate DNA synthesis, as has been reported by others. Experiments with cells synchronized by mitotic selection confirmed this difference in Arg- and Ile- deprived behavior, but also showed that cells which underwent the mitosis leads to G1 transition during Arg starvation remained arrested in G1 (G0?). The results suggest that Arg-deprived cells continue to maintain some proliferative function(s) while Ile-deprived cells do not.

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