scholarly journals Cell cycle analysis of MDA-MB-157 and APC knockdown cells

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Jamie Brown ◽  
Emily Astarita ◽  
Camden Hoover ◽  
Jenifer R. Prosperi
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Chemotherapy Resistance ◽  
Cell Cycle Analysis ◽  
Alternative Methods ◽  
Serum Starvation ◽  
G1 Arrest ◽  
Breast Cancers ◽  
Cell Synchronization ◽  
M Phase

Background and Hypothesis: A majority of sporadic breast cancers include deficits in the expression of Adenomatous Polyposis Coli (APC), a tumor suppressor. Deficits in APC are more common in patients with TNBC (triple negative breast cancer), which is also a cancer prone to chemotherapy resistance. The Prosperi lab previously found that APC knockdown cells (APCKD) were resistant to Paclitaxel (PTX). We hypothesize that APCKD cells are resistant to PTX treatment through avoidance of G2/M arrest. This summer, my goal was to investigate the mechanism by which PTX works to arrest the cell cycle at G2/M.  Experimental Design or Project Methods: Cell Synchronization: To synchronize MDA-MB-157 and APCKD cells, we first tried serum starvation for 24-72 hours. Second, we tried synchronizing the cells using a double thymidine block as described (Bostock, 1971). In either protocol, cells were then stained with Propidium Iodide (PI) and flow cytometry was performed. Paclitaxel (PTX) treatment and cell cycle analysis: MDA-MB-157 and APCKD cells were grown to confluency and then treated with 0.078µM PTX for 12, 24, and 48 hours. Cells were stained with PI and flow cytometry was performed. Results: Cell synchronization: APCKD cells cells have an increased cell population in the G2/M phase than the parental cells after serum starvation. Importantly, APC knockdown cells are not impacted by serum starvation up to 72 hours. Additionally, a double thymidine block is insufficient to synchronize MDA-MB-157 and APCKD cells. A double thymidine block did shorten the S phase and move MDA-MB-157 and APCKD cells closer to G0-G1 arrest, but did not synchronize. Paclitaxel (PTX) treatment and cell cycle analysis: MDA-MB-157 and APCKD cells treated for longer intervals experienced more cell death and were further arrested in G2/M. Conclusion and Potential Impact: We learned that MDA-MB-157 and APCKD cannot be easily synchronized using serum starving or a double thymidine block. Future investigations will require alternative methods of synchronization or will proceed without synchronization. Furthermore, APCKD cells do not avoid G2/M arrest when treated with Paclitaxel, indicating a different mechanism of PTX resistance.

scholarly journals Adenomatous Polyposis Coli Loss Controls Cell Cycle Regulators and Response to Paclitaxel

2020 ◽  
Author(s):  
Emily M. Astarita ◽  
Camden A. Hoover ◽  
Sara M. Maloney ◽  
T. Murlidharan Nair ◽  
Jenifer R. Prosperi
Keyword(s):  
Breast Cancer ◽  
Cell Cycle ◽  
Adenomatous Polyposis Coli ◽  
Cyclin B1 ◽  
Cell Cycle Analysis ◽  
Specific Cell ◽  
Breast Cancers ◽  
Cell Cycle Regulators ◽  
Adenomatous Polyposis ◽  
Polyposis Coli

AbstractAdenomatous Polyposis Coli (APC) is lost in approximately 70% of sporadic breast cancers, with an inclination towards triple negative breast cancer (TNBC). TNBC is treated with traditional chemotherapy, such as paclitaxel (PTX); however, tumors often develop drug resistance. We previously created APC knockdown cells (APC shRNA1) using the human TNBC cells, MDA-MB-157, and showed that APC loss induces PTX resistance. To understand the mechanisms behind APC-mediated PTX response, we performed cell cycle analysis and analyzed cell cycle related proteins. Cell cycle analysis indicated increased G2/M population in PTX-treated APC shRNA1 cells compared to PTX-treated controls, suggesting that APC expression does not alter PTX-induced G2/M arrest. We further studied the subcellular localization of the G2/M transition proteins, cyclin B1 and CDK1. The APC shRNA1 cells had increased CDK1, which was preferentially localized to the cytoplasm, and increased CDK6. RNA-sequencing was performed to gain a global understanding of changes downstream of APC loss and identified a broad mis-regulation of cell cycle-related genes in APC shRNA1 cells. Our studies are the first to show an interaction between APC and taxane response in breast cancer. The implications include designing combination therapy to re-sensitize APC-mutant breast cancers to taxanes using the specific cell cycle alterations.

Cell Cycle Analysis by Flow Cytometry

2001 ◽  
Author(s):  
Zbigniew Darzynkiewicz
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Cycle Analysis

scholarly journals MicroRNA-101 inhibits growth and metastasis of human ovarian cancer cells by targeting PI3K/AKT

2021 ◽  
Vol 17 (1) ◽  
pp. 127-134
Author(s):  
Min Wei ◽  
Hongjuan Jin ◽  
ShuLi Yang ◽  
Zhuo Li ◽  
Xinlei Wang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Ovarian Cancer ◽  
Cancer Cells ◽  
Quantitative Polymerase Chain Reaction ◽  
Pten Expression ◽  
Cell Cycle Analysis ◽  
Ovarian Cancer Cells ◽  
G1 Arrest ◽  
Down Regulation ◽  
Over Expression

IntroductionOvarian cancer is the most frequent cause of gynecological cancer related mortality in woman. This study was designed to investigate the role and therapeutic potential of miRNA-101 in ovarian cancer.Material and methodsExpression analysis was carried out by real-time quantitative polymerase chain reaction. Transfections were performed with the help of Lipofectamine 2000 reagent. AO/EB and annexin V/PI staining was used to detect apoptosis and flow cytometry was used for cell cycle analysis. Western blotting was employed for cell cycle analysis.ResultsIt was found that miRNA-101 was significantly down-regulated in ovarian cancer cells. The over-expression of miRNA-101 causes a significant decrease in the viability of ovarian cancer cells via the initiation of apoptosis and sub-G1 arrest of OVACAR-3 cells. It was indicated that PTEN was the potential target of miRNA-101 in OVACAR-3 cells. There was 4.5-fold up-regulation of PTEN expression in ovarian cancer cell lines and the over-expression of miRNA-101 in OVACAR-3 cells resulted in the down-regulation of PTEN expression. The inhibition of PTEN in the OVACAR-3 cells arrested the proliferation of these cells. The over-expression of miRNA-101 causes significant down-regulation in PI3K and AKT expression of OVACAR-3 cells.ConclusionsIt can be concluded that miRNA-101 acts as a tumor suppressor which may be beneficial in the treatment of ovarian cancer.

scholarly journals Cell-Cycle Analysis of Fission Yeast Cells by Flow Cytometry

PLoS ONE ◽  
2011 ◽  
Vol 6 (2) ◽  
pp. e17175 ◽  
Author(s):  
Jon Halvor Jonsrud Knutsen ◽  
Idun Dale Rein ◽  
Christiane Rothe ◽  
Trond Stokke ◽  
Beáta Grallert ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Fission Yeast ◽  
Yeast Cells ◽  
Cell Cycle Analysis

Chapter 21 Cell-Cycle Analysis Using Continuous Bromodeoxyuridine Labeling and Hoechst 33358—Ethidium Bromide Bivariate Flow Cytometry

1994 ◽  
pp. 327-340 ◽  
Author(s):  
Martin Poot ◽  
Holger Hoehn ◽  
Manfred Kubbies ◽  
Angelika Grossmann ◽  
Yuchyau Chen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Ethidium Bromide ◽  
Cell Cycle Analysis

BrdU—Hoechst flow cytometry: A unique tool for quantitative cell cycle analysis

1988 ◽  
Vol 174 (2) ◽  
pp. 309-318 ◽  
Author(s):  
P.S. Rabinovitch ◽  
M. Kubbies ◽  
Y.C. Chen ◽  
D. Schindler ◽  
H. Hoehn
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Cycle Analysis

Apoptosis Was Induced by Casticin in Acute Myelocytic Leukemia Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3991-3991
Author(s):  
Jie Jin ◽  
Jia-Kun Shen ◽  
Hua-ping Du ◽  
Min Yang ◽  
Yun-Gui Wang
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Death ◽  
Leukemic Cell ◽  
K562 Cells ◽  
Acute Myelocytic Leukemia ◽  
Myelocytic Leukemia ◽  
Cycle Arrest ◽  
M Phase ◽  
Ic50 Values

Abstract Casticin, a component from Vitex rotundifolia wich was widely used as an anti-inflammatory agent in Chinese traditional medicine, was reported to have anti-tumor activities in lung cancer and breast cancer. There are yet no reports on roles against acute myelocytic leukemia (AML). This study aims to elucidate the anti-leukemic activity of casticin on AML cells. We investigated the efficient efficacy and the mechanisms by which casticin triggers cell death in AML cells by analyzing cell cycle perturbations, apoptosis-related marker expression. Cell viability was measured by MTT method; apoptosis and cell cycle arrest were determined by flow cytometry and AV-PI assay. Western blot was performed to measure the apoptosis-related marker. Concentration-dependant cell deaths were observed in AML cell lines including K562, U937 and THP-1, with IC50 values of 24h (hours) being 47.4μM, 67.8μM and 61.7μM, respectively. Time-dependant cell deaths were also observed. At the concentration of 20μM casticin, 45.7%, 76.1% and 80.9% of K562 cells were inhibited at 24h, 48h and 72h, respectively; 24.7%, 30% and 61% of U937 cells were inhibited at 24h, 48h and 72h, respectively; while for THP-1, 29%, 41.8% and 53.9% were inhibited at 24h, 48h and 72h, respectively. Apoptosis was found using AV-PI staining by flow cytometry analysis. We observed an obvious G2/M phase increase prolongation in casticin treated K562 cells. BThe distribitions of G2/M phase were 2.9%, 33.6%, 75.3%, 54.9%, 29.7% and 27.0% in K562 cells after treated by 20μM casticin for 0h, 6h, 12h, 24h, 36h and 48h, respectively. Furthermore, apoptosis-related proteins, PARP and caspase 3, were cleaved in casticin treated K562 cells. Taken together, these results demonstrated that casticin can induce leukemic cell death through apoptosis, suggesting that casticin could be a promising therapeutic agent against acute myeloid leukemia.

Utility of Multiparameter Flow Cytometry Immunophenotypic Studies In Patients with Systemic Light Chain (AL) Amyloidosis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4051-4051
Author(s):  
Bruno Paiva ◽  
María-Belén Vidriales ◽  
Jose J. Perez ◽  
Maria-Consuelo López-Berges ◽  
Ramón García-Sanz ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Differential Diagnosis ◽  
Plasma Cell ◽  
Light Chain ◽  
Cell Cycle Analysis ◽  
Disease Assessment ◽  
Multiparameter Flow Cytometry ◽  
Al Amyloidosis

Abstract Abstract 4051 Multiparameter flow cytometry (MFC) immunophenotyping has shown to be of value for differential diagnosis and minimal residual disease assessment in multiple myeloma. However, the clinical value of MFC immunophenotyping in other plasma cell disorders (PCD) remains largely unexplored. Systemic light chain (AL) amyloidosis is a rare PCD characterized by the accumulation of monoclonal light chain fragments leading to end-organ damage and short survival. Bone marrow (BM) plasma cell (PC) infiltration in AL is usually low and thus the identification of clonal PC can be often difficult by immunohistochemistry and/or immunofluorescence. In the present study we focused on 34 BM samples sent to our institution with a suspected diagnosis of AL. MFC immunophenotypic studies were performed using the following 4-color combinations of MoAbs (FITC/PE/PerCP-Cy5.5/APC): CD38/CD56/CD19/CD45 (n=34); in addition cy-Kappa/cy-Lambda/CD19/CD38 staining was add to confirm the clonal or polyclonal nature of BMPC in equivocal cases. Ploidy and cell cycle analysis were additionally performed in a subset of cases (n=12/34). From the total 34 cases included in the present study, 28 had a confirmed diagnosis of AL. The remaining 6 cases were finally diagnosed with localized - amyloidoma - (n=2) and familial (n=1) forms of amyloidosis, multiple myeloma-associated amyloid (n=2) and congestive pericarditis (n=1). Interestingly, the presence of clonal PC was detected by MFC in 27 of the 28 (96%) patients with AL; in turn, clonal PC were undetectable in the BM of all cases with localized and familial forms of amyloidosis. The median overall level of PC (M-PC plus N-PC) seen in MFC immunophenotypic analyses of BM samples of the 28 patients with AL was 1.9% (range: 0.1% - 15%), with a significant positive correlation between PC enumerated by MFC and conventional morphology (r=0.5; p=.01). Within the BMPC compartment, the median proportion of clonal PC was of 94% (mean 81% ± 29%); in 6 cases all BMPC were clonal while in the remaining 22 patients residual normal PC persisted (median of normal PC/BMPC 13% ± 31%). The most common aberrant phenotypes were down-regulation of CD19 (92%) and CD45 (83%), followed by overexpression of CD56 (56%) and infra-expression of CD38 (42%). Aneuploidy was only found in 18% of cases, all of them hyperdiploid. Cell cycle analysis showed a median % of S-phase and G2-Mitosis PC of 0.7% and 3.5%, respectively. Concerning patients' outcome, cases with undetectable normal PC (6/28, 21%) had a significantly decreased overall survival (OS) compared to patients with persistent BM normal PC at diagnosis (22/28, 79%) with 3-year OS rates of 0% vs. 59%, respectively (p=.001). In summary, these preliminary data suggests that MFC immunophenotyping investigations may be clinically relevant in patients with suspected amyloidosis for i) differential diagnosis between AL and other forms of amyloidosis and, ii) prognostication of patients with AL according to the presence or absence of baseline persistent normal PC. Disclosures: No relevant conflicts of interest to declare.

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