A Large-Scale Automated Method for Hepatocyte Isolation: Effects on Proliferation in Culture

2005 ◽  
Vol 14 (5) ◽  
pp. 291-299 ◽  
Author(s):  
M. J. Nieuwoudt ◽  
E. Kreft ◽  
B. Olivier ◽  
S. Malfeld ◽  
J. Vosloo ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Viability ◽  
Large Scale ◽  
Centrifuge Method ◽  
Automated Method ◽  
Bioartificial Liver Support ◽  
Albumin Production ◽  
Hepatocyte Isolation ◽  
Pyruvate Ratio

Large-scale sterile methods for isolating hepatocytes are desirable for the development of bioartificial liver support systems. In this study the traditional centrifuge method was compared with the use of a Baylor Rapid Autologous Transfusion (BRAT) machine for isolating large quantities of porcine hepatocytes. After isolating hepatocytes, the methods were evaluated in terms of cell viability and yield per liver, proliferation over 7 days, and the effects on the cell cycle using the trypan blue exclusion test, conventional phase-contrast light microscopy, the lactate to pyruvate ratio, the leakage of lactate dehydrogenase (LD) and aspartate aminotransferase (AST), lidocaine clearance, albumin production, and flow cytometry. With the centrifuge method the mean cell viability was 92.5%, while with the BRAT method the viability was 95.9%. The minimal cell yields with the BRAT procedure were 7.3 × 109 for 250-ml centrifuge bowls and 2.8 × 109 for 165-ml bowls, which compares well with that found by other authors. Because the same initial procedures were employed in both methods the total hepatocyte yield per liver was comparable. Flow cytometry confirmed that the proliferation of hepatocytes was facilitated by oxygenation during the isolation procedure. The recovery of hepatocytes in culture following isolation was similar after either method. Daily microscopic investigation indicated that cytoplasmic vacuolization and granularities were present after either procedure and these disappeared following 3–4 days of culturing. Flow cytometry indicated that the hepatocyte cell cycle was similar after either method; at 7 days the profile indicated that the cells were still proliferating. Trends in the lactate to pyruvate ratio and the leakage of LD and AST indicated that the functional polarity of hepatocytes was regained after approximately 3 days. Lidocaine clearance at 4 days indicated that the cytochrome P450 system was active, while significant albumin production was apparent at day 5. The benefit of using BRAT technology in hepatocyte isolation lies in guaranteed sterility, convenience, speed, and the ability to oxygenate media and cell suspensions during the procedure.

scholarly journals Effects of curcumin in combination with cyclophosphamide on canine mammary tumour cell lines 

2014 ◽  
Vol 59 (No. 11) ◽  
pp. 553-572 ◽  
Author(s):  
F. Ustun Alkan ◽  
C. Anlas ◽  
S. Cinar ◽  
F. Yildirim ◽  
O. Ustuner ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Protein Expression ◽  
Cell Viability ◽  
Dna Fragmentation ◽  
Mammary Cancer ◽  
Combined Treatment ◽  
Cytotoxic Agents ◽  
Immunocytochemical Staining ◽  
Bax Protein

In recent years, significant emphasis has been placed on combination chemotherapy in cancer using cytotoxic agents and plant derived-bioactive substances that are capable of selectively arresting cell growth and inducing apoptosis in tumour cells. The present study was undertaken to evaluate the possibility that the combination of curcumin and cyclophosphamide could show synergistic anti-proliferative effects towards CMT-U27 and CMT-U309 canine mammary cancer cells and, if so, to clarify the mechanism involved. The anti-proliferative activities of curcumin, cyclophosphamide and a combined treatment on CMT cells were determined using the MTT and LDH assays. The concentration of drug required for 50% inhibition of cell viability (IC<sub>50</sub>) and combination index (CI) values were calculated from log dose-response curves of fixed-combinations of curcumin and cyclophosphamide generated from MTT assays. Apoptosis was detected using a DNA fragmentation assay and Annexin-V/propidium iodide staining followed by flow cytometry. Cell cycle analyses were also performed using flow cytometry. The expression of the apoptosis-related proteins Bax and Bcl-2 was determined by immunocytochemical staining. MTT and LDH assays showed that curcumin and cyclophosphamide induced a dose- and a time-dependent decrease in cell viability. Isobole analysis revealed that the substances exhibited a synergistic interaction when IC<sub>50</sub> and 1/2 IC<sub>50 </sub>concentrations of curcumin and cyclophosphamide were added concurrently to the cultures. This synergy was characterised by a significant increase in the percentage of early and late apoptotic CMT-U27 and CMT-U309 cells. However, internucleosomal fragmentation of DNA was not observed in the DNA fragmentation assay. Cells treated with curcumin and cyclophosphamide arrested at the G<sub>2</sub>/M and S phases of the cell cycle, respectively. In combined treatments cells were arrested in both phases of the cell cycle. Furthermore, immunocytochemical stainings demonstrated that the curcumin induced apoptosis in CMT cells by the modulation of Bcl-2/Bax protein expression, as the expression of Bcl-2 was decreased and that of Bax increased. This effect was more pronounced in combination treatments. In conclusion, our study shows that a combination of curcumin and cyclophosphamide shows synergistic growth inhibitory activity on CMT cells via induction of apoptosis and cell cycle arrest accompanied by modulation of Bcl-2/Bax protein expression. This finding provides a molecular basis for the development of natural compounds as novel anticancer agents and will allow lowering the dose of cytotoxic agents, which will in turn lead to more specific and less toxic therapies for mammary cancer in dogs.

MLN4924, an Investigational Small Molecule Inhibitor of NEDD8-Activating Enzyme (NAE), Inhibits NF-κB Activity and Induces G1 Cell Cycle Arrest and Apoptosis in Pre-Clinical Models of Hodgkin Lymphoma (HL)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3717-3717
Author(s):  
Matthew J. Barth ◽  
Cory Mavis ◽  
Francisco J. Hernandez-Ilizaliturri ◽  
Myron S. Czuczman
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Western Blot ◽  
Cell Viability ◽  
Tumor Cells ◽  
Cell Lines ◽  
Small Molecule ◽  
Wild Type ◽  
Cycle Arrest ◽  
Molecule Inhibitor

Abstract Abstract 3717 The incorporation of combined-modality therapy, risk-stratified chemotherapy selection, high-dose chemotherapy and autologous stem cell support (HDC-ASCS), and monitoring treatment response by functional imaging are factors that have contributed to the improvement in clinical outcomes in HL patients. Unfortunately, those patients not eligible for or that have failed HDC-ASCS remain a challenge for the treating oncologist, stressing the need for novel therapeutic strategies. Significant improvements in the understanding of the biology of HL have been achieved, including cellular pathways altered in HL (e.g. the ubiquitin-proteasome system) and the role of the tumor microenvironment. MLN4924 is an investigational small-molecule inhibitor of NEDD8-activating enzyme (NAE). NAE is an enzyme responsible for activating NEDD8, an ubiquitin-like molecule in the neddylation cascade that is responsible for cullin-ring ligase (CRL) mediated polyubiquitination of proteins targeting them for proteasomal degradation. In order to better understand the activity of MLN4924 in HL, we performed pre-clinical testing in IkB wild type (L-1236), IkB mutated (KM-H2 and L-428) HL cell lines, and in primary tumor cells derived from a HL patient. Malignant cells were exposed to escalating doses of MLN4924 and changes in cell viability were quantified at different time periods by alamar Blue reduction assay. Patient tumor cells were incubated with MLN4924 for 48 hrs and cell viability was determined using the CellTiterGlo assay. Induction of apoptosis in HL cell lines following exposure to MLN4924 was determined by flow cytometry for Annexin-V and propidium iodide (PI) staining and western blot for caspase-3 and PARP cleavage. Cell cycle analysis was performed by flow cytometry using PI staining. Inhibition of NAE by MLN4924 in HL cell lines was measured by western blot for NEDD8-cullin. Finally, changes in NF-kB activity following MLN4924 exposure were determined by p65 nuclear localization using Image stream technology. MLN4924 exhibited a dose- and time-dependent decrease in cell viability in all HL cell lines at nM concentrations. No differences in anti-tumor activity were observed between IkB-wild type (L-1236 IC50 = 250nM) and IkB–mutated HL cell lines (KM-H2 IC50 = 250nM and L-428 IC50 = 300nM). MLN4924 induced apoptosis in a dose-dependent manner in all cell lines tested. In addition, MLN4924 induced cell cycle arrest in G1 phase and inhibition of NAE was demonstrated by a decrease in NEDD8 conjugated CRL. L1236 cells exposed to MLN4924 also demonstrated a decrease in degradation of IκBα as evidenced by increased levels of p-IκBα following exposure to MLN4924 with a corresponding decrease in p65 nuclear translocation. Surprisingly KMH-2 cells, which carry a mutated IκBα protein that is truncated and non-functional, had a decrease in nuclear p65 following exposure to MLN4924, suggesting an alternative mechanism of NF-kB inhibitory activity by MLN4924. In summary, MLN4924 demonstrates activity against HL cells in vitro through inhibition of NF-kB, and is a promising novel agent for the treatment of HL. We continue to investigate the pre-clinical activity of MLN4924 both as a single-agent and in combination with traditional chemotherapy and other novel agents. Disclosures: No relevant conflicts of interest to declare.

The Acquirement Of Rituximab Resistance Is Associated With De-Regulation Of The PI3K/Akt/mTOR Signaling Pathway Leading To Chemotherapy Resistance In Burkitt Lymphoma Pre-Clinical Models

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5144-5144 ◽  
Author(s):  
Sarah Frys ◽  
Andrew Skomra ◽  
Natalie M Czuczman ◽  
Cory Mavis ◽  
Delphine C.M. Rolland ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Signal Transduction ◽  
Flow Cytometry ◽  
Cell Viability ◽  
Cell Lines ◽  
Burkitt Lymphoma ◽  
Chemotherapy Resistance ◽  
Signal Transduction Pathways ◽  
Mtor Signaling Pathway ◽  
Cycle Arrest

Abstract Treatment with multi-agent chemotherapy regimens has significantly improved survival in pediatric Burkitt Lymphoma (BL) leading to long-term survival in over 80% of cases. The incorporation of rituximab in the treatment of pediatric B-cell non-Hodgkin lymphoma (B-NHL) has been slower than in adults, but may improve clinical outcomes in high risk pediatric BL patients when combined with chemotherapy regimens. On the other hand, BL patients with primary refractory or relapsed disease have a dismal prognosis, stressing the need to identify the mechanism(s) resulting in chemotherapy/rituximab resistance and to develop novel therapeutic approaches. To this end, we exposed a BL cell line (Raji) to escalating doses of rituximab with or without human serum and generated/characterized several BL rituximab-resistant (Raji 7R and Raji 8RH) (RRCL) or rituximab-chemotherapy resistant (Raji 2R and Raji 4RH) (RCRCL) cell lines. Subsequently, we screened for aberrant activation of signal transduction pathways between RSCL (Raji), RRCL (Raji 7R and Raji 8RH), or RCRCL (Raji 2R and Raji 4RH) in an attempt to define what pathways were associated with resistance to both rituximab and chemotherapy agents. This was accomplished by the analysis of phosphorylation patterns on key-regulatory members of pre-defined signal transduction pathways using Western blotting, phospho-flow cytometry studies and phosphoproteomics. Effects on cell viability or cell cycle distribution of RRCL or RCRCL following pharmacological inhibition of key-regulatory pathways identified was then performed using the alamar blue reduction assay or flow cytometry respectively. While total Akt (Protein kinase B) expression was similar between all the types of BL cell lines tested, we found that RCRCL (Raji 2R and Raji 4RH) had an increase in basal phosphorylation levels of Akt at the Ser473 and Thr308 phosphorylation sites when compared to RSCL (Raji cells) or RRCL (Raji 7R and Raji 8RH). These findings were confirmed by phospho-flow cytometry studies. Phosphoproteomic analysis comparing Raji (RSCL) against Raji 4RH (RCRCL) cells identified an increase of at least 2 fold in the phosphorylation of 315 proteins in RCRCL including several direct targets of AKT such as GSK3B, WEE1, FOXO1 and PRAS40. Altered phosphorylation of multiple Akt/mTOR downstream proteins (BAD, 4EBP1, GSK3B and ERK) was detected by western blot in RCRCLs (Raji 2R and Raji 4RH) compared to RSCL and RRCLs. In vitro exposure of RCRCL (Raji 2R and Raji 4RH) to escalating doses of MK-2206, a selective Akt inhibitor, or idelalisib, a selective PI3 kinase delta inhibitor, resulted in a dose- and time-dependent decrease in cell viability of RCRCL and to a lesser degree RRCL and RSCL (RCRCL vs. RRCL/RSCL, p<0.05). The IC50 of MK-2206 was lower in RCRCL (Raji-2R = 2.6µM and Raji-4RH = 3.2µM) than in RSCL (Raji= 4.4µM) or RRCL (Raji 7R = 4.0µM and Raji 8RH = 5.2µM). Similarly, the IC50 for idelalisib was lower in RCRCL (Raji-2R = 61µM and Raji-4RH = 149µM) than in RSCL (Raji= 341µM) or RRCL (Raji 7R = 195µM and Raji 8RH = 318µM). In addition, PI3K/Akt/m-TOR inhibition with either MK-2206 or idelalisib induced cell cycle arrest in G1 phase in RSCL/RRCL, but G2/M cell cycle arrest was observed in RCRCL. In BL cells pre-treated with idelalisib (10µM and 50µM) for 24 hours prior to exposure to doxorubicin (1µM, 10µM or 20µM) for 48 hours, the RCRCL Raji 2R exhibited an increased sensitivity to doxorubicin compared to non-idelalisib exposed controls (untreated vs. idelalisib 10µM vs. idelalisib 50µM: doxorubicin 10µM=78% vs. 69% vs. 56%, p<0.05; doxorubicin 20µM=61% vs. 49% vs. 43%, p<0.05). Raji and Raji 7R cells pre-treated with idelalisib did not exhibit an increase in doxorubicin sensitivity. Together our data suggest that constitutive phosphorylation/activation of the PI3K/Akt signal transduction pathway is associated with the development of resistance and may play a role in shared resistant pathways that lead to the acquirement of chemotherapy resistance observed in some rituximab-resistant cell lines. Additionally, inhibition of the PI3K/Akt/mTOR pathway may partially re-sensitize chemotherapy resistant cells to the cytotoxic effects of chemotherapeutic agents. Targeting the PI3K/Akt/mTOR signaling pathway may be clinically relevant in some patients with relapsed/refractory BL. (Research supported by a grant from Hyundai Hope on Wheels and a St. Baldrick’s Foundation Scholar Award) Disclosures: Czuczman: Genetech, Onyx, Celgene, Astellas, Millennium, Mundipharma: Advisory Committees Other.

PI3K/Akt/mTOR Pathway Inhibition in Chemotherapy-Sensitive and -Resistant Models of Burkitt Lymphoma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1558-1558 ◽  
Author(s):  
Maria Bhatti ◽  
Thomas Ippolito ◽  
Cory Mavis ◽  
Matthew J. Barth
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Viability ◽  
Cell Line ◽  
Cell Lines ◽  
Propidium Iodide ◽  
Mtor Pathway ◽  
Propidium Iodide Staining ◽  
Induction Of Apoptosis ◽  
Pre Treatment

Abstract Introduction: Burkitt lymphoma (BL) is the most common form of B-cell non-Hodgkin lymphoma (B-NHL) in children. Despite significant improvements in survival with de novo disease, treatment of relapsed or refractory BL remains a significant hurdle with survival in only about 20% of patients. Novel therapeutic approaches are necessary to improve outcomes in this group of childhood B-NHL patients with the worst prognosis. Recent literature has identified a high rate of recurrent mutations that result in activation of the PI3K/Akt pathway in BL and have implicated activation of PI3K/Akt in coordination with Myc in BL lymphomagenesis. Our laboratory has developed rituximab and chemotherapy resistant cell line models and subsequently found that these cell lines exhibit increased activation of Akt. We hypothesized that increased activation of Akt may be contributing to chemoresistance and that targeting the PI3K/Akt/mTOR pathway may increase chemoresponsiveness. To that end, we have investigated the effect of inhibiting the PI3K/Akt/mTOR pathway with either the PI3K-delta inhibitor idelalisib or the pan-PI3K/mTOR inhibitor BEZ-235 in cell line models of BL. Methods: The in vitro effect of idelalisib or BEZ-235 was investigated in BL cell lines including Raji, Raji 2R and Raji 4RH (rituximab-chemotherapy resistant), Raji 7R and Raji 8RH (rituximab resistant), Ramos and Daudi. Cell viability following inhibitor exposure was assessed by Alamar blu and cell-titer glo assays. The effect of inhibitor exposure on cell cycle progression was determined by flow cytometry using propidium iodide staining. Inhibition of Akt activation following inhibitor exposure was determined using phospho-flow cytometry. The activity of cytotoxic chemotherapeutic agents following inhibition by idelalisib or BEZ-235 was assessed using Alamar blu and cell titer glo assays. Results: In vitro exposure of BL cell lines to idelalisib in concentrations from 0.1-100µM for 24, 48 or 72 hours resulted in a dose and time-dependent decrease in viable cells in all cell lines tested with IC50 concentrations of 60-300uM. Pre-treatment with the pan-caspase inhibitor QVD resulted in a small reversal in the decrease in cell viability suggesting only a minimal portion of the activity was caspase dependent. When induction of apoptosis was measured using annexin V-propidium iodide staining, little induction of apoptosis was observed with single agent idelalisib at concentrations up to 100uM. Determination of cell cycle progression following exposure to idelalisib at 1, 10, 50 or 100 uM for 24, 48 or 72 hours indicated a time and dose dependent cell cycle arrest in all cell lines. In chemotherapy-sensitive cell lines the arrest was primarily noted in G1, while the chemotherapy-resistant Raji 2R and Raji 4RH cell lines exhibited arrest primarily in G2/M. A significant reduction in cell viability following chemotherapy exposure for 48 hours was noted in chemotherapy resistant Raji 2R cells following pre-treatment for 48 hours with idelalisib 10uM compared to non-idelalisib exposed cells (doxorubicin 10uM 55% vs 77%, p<0.001; vincristine 0.05uM, 48% vs 61%, P<0.001). At higher idelalisib pre-treatment concentrations (50uM) additional synergistic activity was observed in Raji 2R cells (cisplatin 48% vs 61%, p<0.001; dexamethasone 67% vs 87%, p<0.01). To further assess the effect of dual inhibition of PI3K and mTOR, cell lines were exposed to the dual inhibitor BEZ-235. BEZ-235 exhibited a more potent decrease in cell viability compared to idelalisib with activity at nM concentrations. Unlike idelalisib, exposure to BEZ-235 resulted in significant induction of apoptosis by Annexin V-propidium iodide staining. BEZ-235 also exhibited synergistic activity in combination with chemotherapy in all cell lines. At equivalent dosing, BEZ-235 exposure resulted in a more significant decrease in Akt phosphorylation compared to idelalisib as determined by flow cytometry for p-Akt at Ser and Thr phosphorylation sites. Conclusions: Chemotherapy sensitive and resistant BL cell line models are susceptible to inhibition of the PI3K/Akt/mTOR pathway. Targeted inhibition of this pathway leads to a decrease in AKT activation, decrease in cell viability, cell cycle arrest and an increase in sensitivity to cytotoxic chemotherapeutic agents. Broader inhibition of both PI3K and mTOR is more effective than more targeted inhibition of PI3K-delta alone. Disclosures No relevant conflicts of interest to declare.

Clarithromycin Interacts with Lenalidomide in the Combination Regimen Bird and Overcomes Drug Resistance in Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2125-2125
Author(s):  
Fengyan Jin ◽  
Shuang Li ◽  
Lijuan Chen ◽  
Chuan Wu ◽  
Wei Li ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Drug Resistance ◽  
Cell Viability ◽  
Cell Cycle Arrest ◽  
Parp Cleavage ◽  
Newly Diagnosed ◽  
Cycle Arrest ◽  
Caspase Cascade

Abstract Introduction:Although the macrolide antibiotic clarithromycin (CAM, or Biaxin) has only minimal single agent activity in MM, the regimens with addition of CAM to IMiDs and steroids, including BLT-D (Biaxin/low-dose thalidomide/dexamethasone [Dex]), BiRD (Biaxin/Revlimid [lenalidomide, Lena]/Dex), t-BiRD (thalidomide/BiRD), Car-BiRD (carfilzomib/BiRD), and ClaPD (CAM/pomalidomide/Dex), appear highly effective in treatment of newly-diagnosed and probably relapsed/refractory multiple myeloma (MM). In this context, two phase 3 trials are currently ongoing to evaluate the efficacy of BiRD vs. RD in newly-diagnosed MM in the United States and Europe, respectively. Of note, recent findings also suggests that addition of CAM to RD might overcome resistance to RD. However, despite increasing clinical evidence for its promising activity, the exact mechanism for such a combination strategy still remain largely unclear. Here, we investigated the mechanisms of action underlying the interaction between CAM and Lena and their capability to overcome drug resistance in MM cells, focusing on the cereblon (CRBN)/IKZF1,3/IRF4/Myc signaling cascade, recently identified as the novel target of IMiDs, Materials and Methods: To test our hypothesis whether and how BiRD overcomes resistance to RD (Lena/Dex), human MM cells lines employed in this study included Dex-sensitive (MM.1S) vs -resistant (MM.1R) cells, drug-naïve RPMI8226 cells vs their Lena-resistant (R10R) or bortezomib (Btz)-resistant counterparts (DR), as well as primary CD138+MM cells isolated from bone marrow samples of newly-diagnosed and relapsed/refractory patients who had received prior IMiDs (including Lena) or Btz. Cells were exposed (72 hr) to CAM (50-100 mg/ml) ± Lena (1-10 mM), after which the CCK-8 assay and flow cytometry with annexin V/7AAD staining were performed to monitor cell viability and apoptosis, respectively. Mechanistic studies included Western blot analyses of the CRBN/IKZF1,3/IRF4/Myc signaling pathway, as well as the apoptotic caspase cascade. Cell cycle was also assessed by flow cytometry. Results: Whereas Lena (1-10 mM) had almost no direct effects on cell viability, CAM (≥ 100 mg/ml) displayed a dose-dependent toxicity in various MM cell lines. Notably, subtoxic concentrations of CAM (e.g., 50 mg/ml) significantly potentiated lethality of Lena in MM.1S (CI value = 0.40-0.86, indicating synergism). Significantly, this effect was even more robust in Dex-resistant MM.1R cells. These events were associated with marked activation of caspase 3, 8, and 9 and PARP cleavage, accompanied by down-regulation of the anti-apoptotic proteins Bcl-2 and Bcl-xL. While add-on of CAM significantly increased lethality of Lena in RPMI8226 cells, combined treatment was strikingly more effective against Lena-resistant R10R cells. In addition, Btz-resistant RPMI8226 cells were also more sensitive to both CAM alone and in combination with Lena, compared to parental RPMI8226 cells. Consistently, markedly enhanced cell killing by the combination was also observed in primary CD138+ cells, particularly those obtained from patients relapsed after prior IMiDs. Exposure to Lena with or without CAM sharply down-regulated CRBN in MM cells, accompanied by reduced expression of IKZF1, IKZF3, IRF4, and Myc. Interestingly, Lena failed to down-regulate CRBN/IKZF1/IRF4/Myc in Lena-resistant R10R cells, while addition of CAM dramatically resensitized these cells to the action of Lena. Moreover, Lena in the presence or absence of CAM induced cell cycle arrest at G0/G1, in association with marked up-regulation of p21Cip1 and p27Kip1. Last, Lena induced LC3A-II expression (a marker of autophagy), which was clearly increased in the presence of CAM, likely in association with the capability of CAM to impair the late stage process of autophagy e.g., autophagosome clearance by lysosome. Conclusion: Together, these findings indicate that CAM significantly increases the anti-MM activity of Lena in MM cells, especially those resistant to the first-line therapy (e.g., Dex and Btz), and notably overcomes Lena resistance. The mechanisms involves disruption of the CRBN/IKZF1/IRF4/Myc pathway, as well as activation of the apoptotic caspase cascade, induction of cell cycle arrest, and attenuation of autophagy. Collectively, these mechanistic findings support exploring the BiRD regimen in MM, particularly to overcome RD resistance. Disclosures No relevant conflicts of interest to declare.

scholarly journals Usnea Acid as Multidrug Resistance (MDR) Reversing Agent against Human Chronic Myelogenous Leukemia K562/ADR Cells via an ROS Dependent Apoptosis

2019 ◽  
Vol 2019 ◽  
pp. 1-7 ◽  
Author(s):  
Wenjing Wang ◽  
Shubin Niu ◽  
Luxin Qiao ◽  
Feili Wei ◽  
Jiming Yin ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Multidrug Resistance ◽  
Cell Viability ◽  
Leukemia Cell ◽  
Myelogenous Leukemia ◽  
Leukemia Cell Line ◽  
Phase Cell ◽  
Cell Cycle Distribution ◽  
Promising Therapeutic Approach

Purpose. Multidrug resistance (MDR) is a major obstacle in chemotherapy of leukemia treatments. In this paper, we investigated Usnea Acid (UA) as MDR reversal agent on hematologic K562/ADR cells via ROS dependent apoptosis. Methods. CCK8 assay was used to measure cell viability rate of K562/ADR. Intracellular reactive oxygen species (ROS) generation, cell cycle distribution, cell apoptosis were measured with flow cytometry, respectively. Proteins related to apoptosis were measured by Western blot. Intracellular Adriamycin accumulation was observed by confocal microscopy and measured by flow cytometry. Results. In vitro study showed intracellular Adriamycin accumulation was remarkably increased by UA. Cell viability treated with Adr (4 μM) was decreased from 89.8%  ± 4.7 to 32%  ± 8.9 by combined with UA (4 μM). Adr-induced apoptosis and G1/G0 phase cell cycle arrest were remarkably increased by UA, as well as, intracellular ROS level. However, MDR reversing activity of UA was inhibited by N-acetyl cysteine (NAC), a ROS scavenger. Conclusion. These data provide compelling evidence that UA is a promising agent against MDR in leukemia cell line and suggest a promising therapeutic approach for leukemia.

scholarly journals A combined characterization of coelomic fluid cell types in the spiny starfish Marthasterias glacialis – inputs from flow cytometry and imaging

2020 ◽  
Author(s):  
Bárbara Oliveira ◽  
Silvia Guatelli ◽  
Pedro Martinez ◽  
Beatriz Simões ◽  
Claúdia Bispo ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Viability ◽  
Cell Types ◽  
Coelomic Fluid ◽  
Transmission Electron ◽  
Marthasterias Glacialis ◽  
Combined Microscopy ◽  
Diploid Cells

ABSTRACTCoelomocytes is a generic name for a collection of cellular morphotypes, present in many coelomate animals, that has been reported as highly variable across echinoderm classes. The roles attributed to the major types of the free circulating cells present in the coelomic fluid of echinoderms include immune response, phagocytic digestion and clotting. The main aim of the present study is the thorough characterization of coelomocytes present in the coelomic fluid of Marthasterias glacialis (class Asteroidea) through the combined use of flow cytometry (FC) and fluorescence plus transmission electron microscopy. Two coelomocyte populations (here named P1 and P2) were identified by flow cytometry and subsequently studied in terms of abundance, morphology, ultrastructure, cell viability and cell cycle profiles. Ultrastructurally, P2 diploid cells showed two main morphotypes, similar to phagocytes and vertebrate thrombocytes, whereas the small P1 haploid cellular population was characterized by a low mitotic activity, relatively undifferentiated cytotype and a high nucleus/cytoplasm ratio. These last cells resemble stem-cell types present in other animals. P1 and P2 cells differ also in cell viability and cell cycle profiles. Additionally, two other morphotypes were only detected by fluorescence microscopy and a third one when using a combined microscopy/FC approach.

Effect of Threonine on the Proliferation and Viability of Porcine Granulosa Cells

2017 ◽  
Vol 17 (1) ◽  
pp. 1-6
Author(s):  
Zhou Ke-Rou ◽  
Zhang Qian ◽  
Jin Er-Hui ◽  
Hu Qian-Qian ◽  
Gu You-Fang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Proliferation ◽  
Cell Viability ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Cell Morphology ◽  
S Phase ◽  
Control Group ◽  
Porcine Granulosa Cells

The proliferation and viability of granulosa cells directly affects the development and function of follicles. The current study examined the effect of threonine on cell morphology and cell cycle of porcine granulosa cells. It would help to understand hormone secretion, reproductive health, and disease prevention and treatment in females. Granulosa cells were isolated from pig ovaries, and different passages of cells were stained and identified by FSHR antibody. Threonine was added to the culture medium, and cells were stained with HE to examine the changes in granulosa cell morphology. Cell viability was measured using the CCK-8 assay, and cell cycle phase was determined by flow cytometry. Immunofluorescence staining showed >98% FSHR-positive cells demonstrating that the isolated granulosa cells were >95% pure. HE-staining of different passages of granulosa cells showed that cells from the second to fourth passage have stable morphology, whereas those from the fifth and subsequent passages began to degenerate and degrade. CCK-8 results showed that =0.5 mM threonine can significantly enhance granulosa cell viability after 0.5, 1 and 2 h of treatment, and cells treated with 1 mM threonine for 4 h had the highest cell viability. In contrast, 5 and 10 mM threonine significantly reduced cell viability, indicating that only a certain amount of threonine can enhance granulosa cell viability. Flow cytometry results demonstrated that the percentage of G1 phase cells was higher in the 0.1, 0.5 and 1 mM groups than in the control group, but lower in the 10 mM group than in control group. While the 5 mM group had the highest percentage of S phase cells, the 10 mM group had no S phase cells. In contrast, the percentage of G2 phase cells was much higher in the 10 mM group than in the control group and other dose groups, indicating that an appropriate amount of threonine can promote granulosa cell proliferation, but excessive threonine can inhibit cell proliferation.

scholarly journals Effect of Huangkui on mouse podocytes under high glucose environment

2021 ◽  
Vol 20 (5) ◽  
pp. 515-523
Author(s):  
Chun-Li Yu ◽  
◽  
Xiu-Li Fei ◽  
Ding-Zhong Tang ◽  
Kun Liu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Diabetic Nephropathy ◽  
Protein Expression ◽  
Cell Viability ◽  
Mtt Assay ◽  
High Glucose ◽  
Group Iv ◽  
Normal Glucose ◽  
Mrna And Protein Expression

To explore a new underlying molecular mechanism of Huangkui Extract Powder (HKEP) in the alleviation of diabetic nephropathy (DN). Murine immortalized podocytes were divided into (i) normal glucose (NG, 5.6 mM), (ii) NG + HKEP (0.45 g/L), (iii) HG, and (iv) HG + HKEP (0.45 g/L) groups. MTT assay and flow cytometry were used to detect the podocyte proliferation, apoptosis and cell cycle. Cell viability was inhibited, and apoptosis increased in (iii) HG group compared with (i) NG group (p<0.05). mRNA and protein expression of nephrin and podocin significantly decreased in (iii) HG group compared with (i) NG group (p<0.05). When compared with (iii) HG group, (iv) HG + HKEP group had higher cell viability, lower apoptotic rate and higher mRNA and protein expression of nephrin and podocin (p<0.05). HKEP can attenuate HG-induced podocyte damage, which may be one of the mechanisms of HKEP for attenuating DN.

Silencing of TEL/AML1 In Definitive Leukemic Cells Does Not Impair Cell Survival

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3229-3229
Author(s):  
Marketa Zaliova ◽  
Jozef Madzo ◽  
Gunnar Cario ◽  
Jan Trka
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Viability ◽  
Fusion Gene ◽  
Global Gene Expression ◽  
Leukemic Cells ◽  
Cell Cycle Distribution ◽  
Wild Type ◽  
Egfp Reporter

Abstract Abstract 3229 The most frequent structural chromosomal aberration in childhood acute lymphoblastic leukemia t(12;21) generates TEL/AML1 fusion gene. Resulting TEL/AML1 protein probably acts as an aberrant transcription factor that deregulates AML1-dependent transcription but its target genes and thus also the exact role in leukemic cells remain unknown. In vivo studies showed that TEL/AML1 itself is not sufficient to cause leukemia but may induce a preleukemic state characterized by the increased numbers of multipotent or B-cell progenitors with an incomplete block of differentiation. Despite its role for leukemia establishment the relevance of TEL/AML1 fusion gene for leukemia persistance has not been studied enough.To address this question and to explore the possibility of TEL/AML1-targeted therapy, we studied the effects of RNAi-mediated TEL/AML1 silencing on leukemic cells. As the only siRNA used for TEL/AML1 silencing published so far (Diakos et al, Blood, 2007) targets also the wild type AML1 (46% transcript reduction, our data), our first goal was to identify efficient and TEL/AML1-specific siRNA. We designed eleven different siRNAs spanning the fusion point of TEL/AML1 lacking the total sequence homology to wild type TEL and AML1 alleles to avoid their silencing. These 11 siRNAs were tested in HeLa cells transgenic for TEL/AML1-ires2-EGFP reporter. After lipofection into HeLa cells the efficiency of individual siRNAs was measured as a decrease of EGFP reporter fluorescence by flow cytometry. The best five siRNAs, that induced 50–58% silencing of the EGFP reporter, were tested at the mRNA level in TEL/AML-positive leukemic cell line. 24h after electroporation of siRNAs, when the silencing reached its maximum, two most efficient siRNAs induced 58% and 57% TEL/AML1 transcript reduction, respectively. We achieved 61% TEL/AML1 transcript reduction with the pool of both siRNAs while there was only slight reduction (14%) of wild type AML1 transcript. We used this efficient and specific siRNA pool to silence TEL/AML1 in REH and UOC-B6 TEL-AML1 positive cell lines and studied its effect on cell viability, proliferation and global gene expression. Applying two rounds of siRNA electroporation within 48 hours interval we achieved 74% and 86% TEL/AML1 protein knockdown in REH and UOC-B6 cells, respectively. We used trypan blue staining followed by optical microscopy to monitor cell viability and staining with annexin V and propidium idode to assess apoptosis rate by flow cytometry. Analysis of DNA content using staining with propidium iodide was performed to assess cell-cycle distribution. Incorporation of nucleoside analog was measured by flow cytometry to analyse de novo DNA synthesis as an indicator of proliferation rate. Despite the common expectation derived from studies on other fusion oncogenes (BCR/ABL, AML1/ETO, E2A/PBX), TEL/AML1 silencing neither decreased cell viability, nor induced apoptosis. On the contrary, TEL/AML1 depletion was accompanied by the slight but significant increase in the fraction of S-phase cells and corresponding rise in proliferation rate. Opposite effects on cell cycle distribution and proliferation were induced when we silenced wild type AML1. These findings support our hypothesis that TEL/AML1 may block previously established AML1 function in G1/S progression through the cell cycle. In line with the lack of effect on cell viability and discreet effect on cell-cycle distribution and proliferation we found no significant changes in global gene expression pattern upon TEL/AML1 depletion. Our data indicate, that TEL/AML1 is dispensable for the survival of definitive leukemic cells. This work was supported by grants MSM0021620813 and MZOFNM2005. Disclosures: No relevant conflicts of interest to declare.

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