Effect of 1-(4-amino-2-methyl-5-pyrimidil)methyl-3-(2-chloroethyl)3-nitrosourea hydrochloride (ACNU) on glioma cells: ultrastructural observation using cell-cycle-specific cell sorting by flow cytometry

Flow cytometry and electronic cell sorting are being investigated to screen gynecologic specimens for cervical neoplasia. Cellular DNA content is quantitated by Chromomycin A3 fluorescence and cell size is quantitated by 90 degrees light scatter; the logarithms of the measured intensities are used to produce a two parameter histogram. To determine the cell types responsible for signals in various histogram regions, systematic electronic cell sorting is performed. The sorted fractions are sedimented into microscope slides and stained by the Papanicolaou technique. The cells in each fraction are identified by conventional cytomorphologic criteria. Morphologic analysis of sorted cells reveals histogram regions corresponding to specific cell types. One very important region contains the highest concentration of signals from abnormal cells and is therefore the best region to analyze for specimen abnormality. However, because a significant number of signals in this region are from normal cells, specimens cannot be diagnosed by their analysis. Another important histogram region is composed primarily of signals from endocervical columnar and metaplastic cells. The presence of such cells is a good criterion for specimen adequacy, therefore analysis of signals in this region is essential to assess specimen adequacy for automatic screening.

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Dual excitation multi-fluorescence flow cytometry for detailed analyses of viability and apoptotic cell transition

European Journal of Histochemistry ◽

10.4081/838 ◽

2009 ◽

Vol 47 (4) ◽

pp. 289 ◽

Cited By ~ 17

Author(s):

G Mazzini ◽

C Ferrari ◽

E Erba

Keyword(s):

Cell Cycle ◽

Flow Cytometry ◽

Cell Membrane ◽

Apoptotic Cell ◽

Intact Cell ◽

Specific Cell ◽

Apoptotic Cells ◽

Apoptotic Pathway ◽

Culture Models ◽

Cell Cycle Phases

The discrimination of live/dead cells as well as the detection of apoptosis is a frequent need in many areas of experimental biology. Cell proliferation is linked to apoptosis and controlled by several genes. During the cell life, specific events can stimulate proliferation while others may trigger the apoptotic pathway. Very few methods (i.e. TUNEL) are now available for studies aimed at correlation between apoptosis and proliferation. Therefore, there is interest in developing new methodological approaches that are able to correlate apoptosis to the cell cycle phases. Recently new approaches have been proposed to detect and enumerate apoptotic cells by flow cytometry. Among these, the most established and applied are those based on the cell membrane modifications induced in the early phases of the apoptotic process. The dye pair Hoechst 33342 (HO) and Propidium Iodide (PI), thanks to their peculiar characteristics to be respectively permeable and impermeable to the intact cell membrane, seems to be very useful. Unfortunately the spectral interaction of these dyes generates a consistent “energy transfer” from HO to PI. The co-presence of the dyes in a nucleus results in a modification in the intensity of both the emitted fluorescences. In order to designate the damaged cells (red fluorescence) to the specific cell cycle phases (blue fluorescence), we have tested different staining protocols aimed to minimize the interference of these dyes as much as possible. In cell culture models, we are able to detect serum-starved apoptotic cells as well as to designate their exact location in the cell cycle phases using a very low PI concentration. Using a Partec PAS flow cytometer equipped with HBO lamp and argon ion laser, a double UV/blue excitation has been performed. This analytical approach is able to discriminate live blue cells from the damaged (blue-red) ones even at 0.05 ?g/mL PI. The same instrumental setting allows performing other multi-colour analyses including AnnexinV-FITC as well as the possibility to make a correlated analysis to phenotype markers.

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Data Driven Cell Cycle Model to Quantify the Efficacy of Cancer Therapeutics Targeting Specific Cell-Cycle Phases From Flow Cytometry Results

Frontiers in Bioinformatics ◽

10.3389/fbinf.2021.662210 ◽

2021 ◽

Vol 1 ◽

Author(s):

David W. James ◽

Andrew Filby ◽

M. Rowan Brown ◽

Huw D. Summers ◽

Lewis W. Francis ◽

...

Keyword(s):

Cell Cycle ◽

Flow Cytometry ◽

Phase Transitions ◽

Mechanism Of Action ◽

Cell Cycle Phase ◽

Data Driven ◽

Cycle Phase ◽

Cell Populations ◽

Specific Cell ◽

Cell Cycle Phases

Many chemotherapeutic drugs target cell processes in specific cell cycle phases. Determining the specific phases targeted is key to understanding drug mechanism of action and efficacy against specific cancer types. Flow cytometry experiments, combined with cell cycle phase and division round specific staining, can be used to quantify the current cell cycle phase and number of mitotic events of each cell within a population. However, quantification of cell interphase times and the efficacy of cytotoxic drugs targeting specific cell cycle phases cannot be determined directly. We present a data driven computational cell population model for interpreting experimental results, where in-silico populations are initialized to match observable results from experimental populations. A two-stage approach is used to determine the efficacy of cytotoxic drugs in blocking cell-cycle phase transitions. In the first stage, our model is fitted to experimental multi-parameter flow cytometry results from untreated cell populations to identify parameters defining probability density functions for phase transitions. In the second stage, we introduce a blocking routine to the model which blocks a percentage of attempted transitions between cell-cycle phases due to therapeutic treatment. The resulting model closely matches the percentage of cells from experiment in each cell-cycle phase and division round. From untreated cell populations, interphase and intermitotic times can be inferred. We then identify the specific cell-cycle phases that cytotoxic compounds target and quantify the percentages of cell transitions that are blocked compared with the untreated population, which will lead to improved understanding of drug efficacy and mechanism of action.

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Cell Cycle Progression Kinetics of Regenerating Mouse Epidermal Cells: An In Vivo Study Combining DNA Flow Cytometry, Cell Sorting, and [3H]dThd Autoradiography

Journal of Investigative Dermatology ◽

10.1111/1523-1747.ep12285672 ◽

1986 ◽

Vol 86 (4) ◽

pp. 402-405 ◽

Cited By ~ 17

Author(s):

Ole Petter Fraas Clausen ◽

Bente Kirkhus ◽

Aasa Rambæk Schjølberg

Keyword(s):

Cell Cycle ◽

Flow Cytometry ◽

Cell Sorting ◽

Cell Cycle Progression ◽

Epidermal Cells ◽

In Vivo Study ◽

Dna Flow Cytometry ◽

Cycle Progression ◽

Kinetics Of

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MicroRNA-490-5P Targets CCND1 to Suppress Cellular Proliferation in Glioma Cells and Tissue Through Cell Cycle Arrest

Current Neurovascular Research ◽

10.2174/1567202615666180813130143 ◽

2018 ◽

Vol 15 (3) ◽

pp. 246-255 ◽

Cited By ~ 4

Author(s):

Long Zhao ◽

Xiaoping Tang ◽

Renguo Luo ◽

Jie Duan ◽

Yuanchuan Wang ◽

...

Keyword(s):

Cell Cycle ◽

Cell Cycle Arrest ◽

Cellular Proliferation ◽

Glioma Cells ◽

Cycle Arrest

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Pisosterol Induces G2/M Cell Cycle Arrest and Apoptosis via the ATM/ATR Signaling Pathway in Human Glioma Cells

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520620666200203160117 ◽

2020 ◽

Vol 20 (6) ◽

pp. 734-750

Author(s):

Wallax A.S. Ferreira ◽

Rommel R. Burbano ◽

Claudia do Ó. Pessoa ◽

Maria L. Harada ◽

Bárbara do Nascimento Borges ◽

...

Keyword(s):

Cell Cycle ◽

Cell Cycle Arrest ◽

Signaling Pathway ◽

Glioma Cell ◽

Molecular Mechanisms ◽

Glioma Cells ◽

Dependent Manner ◽

M Cell ◽

Trypan Blue Exclusion ◽

Cycle Arrest

Background: Pisosterol, a triterpene derived from Pisolithus tinctorius, exhibits potential antitumor activity in various malignancies. However, the molecular mechanisms that mediate the pisosterol-specific effects on glioma cells remain unknown. Objective: This study aimed to evaluate the antitumoral effects of pisosterol on glioma cell lines. Methods: The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and trypan blue exclusion assays were used to evaluate the effect of pisosterol on cell proliferation and viability in glioma cells. The effect of pisosterol on the distribution of the cells in the cell cycle was performed by flow cytometry. The expression and methylation pattern of the promoter region of MYC, ATM, BCL2, BMI1, CASP3, CDK1, CDKN1A, CDKN2A, CDKN2B, CHEK1, MDM2, p14ARF and TP53 was analyzed by RT-qPCR, western blotting and bisulfite sequencing PCR (BSP-PCR). Results: Here, it has been reported that pisosterol markedly induced G2/M arrest and apoptosis and decreased the cell viability and proliferation potential of glioma cells in a dose-dependent manner by increasing the expression of ATM, CASP3, CDK1, CDKN1A, CDKN2A, CDKN2B, CHEK1, p14ARF and TP53 and decreasing the expression of MYC, BCL2, BMI1 and MDM2. Pisosterol also triggered both caspase-independent and caspase-dependent apoptotic pathways by regulating the expression of Bcl-2 and activating caspase-3 and p53. Conclusions: It has been, for the first time, confirmed that the ATM/ATR signaling pathway is a critical mechanism for G2/M arrest in pisosterol-induced glioma cell cycle arrest and suggests that this compound might be a promising anticancer candidate for further investigation.

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A Novel Camptothecin Derivative 3j Inhibits Nsclc Proliferation Via Induction of Cell Cycle Arrest By Topo I-Mediated DNA Damage

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520619666181207102037 ◽

2019 ◽

Vol 19 (3) ◽

pp. 365-374 ◽

Cited By ~ 1

Author(s):

Yang Liu ◽

Jingyin Zhang ◽

Shuyun Feng ◽

Tingli Zhao ◽

Zhengzheng Li ◽

...

Keyword(s):

Cell Cycle ◽

Flow Cytometry ◽

Dna Damage ◽

Cyclin D ◽

Dna Relaxation ◽

Cycle Arrest ◽

H460 Cell ◽

H1975 Cell

Objective: The aim of this study is to investigate the inhibitory effect of camptothecin derivative 3j on Non-Small Cell Lung Cancer (NSCLCs) cells and the potential anti-tumor mechanisms. Background: Camptothecin compounds are considered as the third largest natural drugs which are widely investigated in the world and they suffered restriction because of serious toxicity, such as hemorrhagic cystitis and bone marrow suppression. Methods: Using cell proliferation assay and S180 tumor mice model, a series of 20(S)-O-substituted benzoyl 7- ethylcamptothecin compounds were screened and evaluated the antitumor activities in vitro and in vivo. Camptothecin derivative 3j was selected for further study using flow cytometry in NSCLCs cells. Cell cycle related protein cyclin A2, CDK2, cyclin D and cyclin E were detected by Western Blot. Then, computer molecular docking was used to confirm the interaction between 3j and Topo I. Also, DNA relaxation assay and alkaline comet assay were used to investigate the mechanism of 3j on DNA damage. Results: Our results demonstrated that camptothecin derivative 3j showed a greater antitumor effect in eleven 20(S)-O-substituted benzoyl 7-ethylcamptothecin compounds in vitro and in vivo. The IC50 of 3j was 1.54± 0.41 µM lower than irinotecan with an IC50 of 13.86±0.80 µM in NCI-H460 cell, which was reduced by 8 fold. In NCI-H1975 cell, the IC50 of 3j was 1.87±0.23 µM lower than irinotecan (IC50±SD, 5.35±0.38 µM), dropped by 1.8 fold. Flow cytometry analysis revealed that 3j induced significant accumulation in a dose-dependent manner. After 24h of 3j (10 µM) treatment, the percentage of NCI-H460 cell in S-phase significantly increased (to 93.54 ± 4.4%) compared with control cells (31.67 ± 3.4%). Similarly, the percentage of NCI-H1975 cell in Sphase significantly increased (to 83.99 ± 2.4%) compared with control cells (34.45 ± 3.9%) after treatment with 10µM of 3j. Moreover, increased levels of cyclin A2, CDK2, and decreased levels of cyclin D, cyclin E further confirmed that cell cycle arrest was induced by 3j. Furthermore, molecular docking studies suggested that 3j interacted with Topo I-DNA and DNA-relaxation assay simultaneously confirmed that 3j suppressed the activity of Topo I. Research on the mechanism showed that 3j exhibited anti-tumour activity via activating the DNA damage response pathway and suppressing the repair pathway in NSCLC cells. Conclusion: Novel camptothecin derivative 3j has been demonstrated as a promising antitumor agent and remains to be assessed in further studies.

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