scholarly journals Monitoring and modeling of lymphocytic leukemia cell bioenergetics reveals decreased ATP synthesis during cell division

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Joon Ho Kang ◽  
Georgios Katsikis ◽  
Zhaoqi Li ◽  
Kiera M. Sapp ◽  
Max A. Stockslager ◽  
...  
Keyword(s):  
Cell Division ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Leukemia Cell ◽  
Atp Synthesis ◽  
Electrical Circuit ◽  
Lymphocytic Leukemia ◽  
Time Dynamics ◽  
Lymphocytic Leukemia Cell

Abstract The energetic demands of a cell are believed to increase during mitosis, but the rates of ATP synthesis and consumption during mitosis have not been quantified. Here, we monitor mitochondrial membrane potential of single lymphocytic leukemia cells and demonstrate that mitochondria hyperpolarize from the G2/M transition until the metaphase-anaphase transition. This hyperpolarization was dependent on cyclin-dependent kinase 1 (CDK1) activity. By using an electrical circuit model of mitochondria, we quantify mitochondrial ATP synthesis rates in mitosis from the single-cell time-dynamics of mitochondrial membrane potential. We find that mitochondrial ATP synthesis decreases by approximately 50% during early mitosis and increases back to G2 levels during cytokinesis. Consistently, ATP levels and ATP synthesis are lower in mitosis than in G2 in synchronized cell populations. Overall, our results provide insights into mitotic bioenergetics and suggest that cell division is not a highly energy demanding process.

scholarly journals Time-dynamics of mitochondrial membrane potential reveal an inhibition of ATP synthesis in mitosis

2019 ◽  
Author(s):  
Joon Ho Kang ◽  
Georgios Katsikis ◽  
Max A. Stockslager ◽  
Daniel Lim ◽  
Michael B. Yaffe ◽  
...  
Keyword(s):  
Cell Division ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Atp Synthesis ◽  
Electrical Circuit ◽  
Time Dynamics ◽  
Delay Cell ◽  
Transport Chain ◽  
A Cell

AbstractThe energetic demands of a cell are believed to increase during mitosis 1–7. As cells transit from G2 into mitosis, mitochondrial electron transport chain (ETC) activity increases 4,8,9, and cellular ATP levels progressively decrease until the metaphase-anaphase transition 3,7,10, consistent with elevated consumption. The rates of ATP synthesis during mitosis, however, have not been quantified. Here, we monitor mitochondrial membrane potential of single lymphocytes and demonstrate that cyclin-dependent kinase 1 (CDK1) activity causes mitochondrial hyperpolarization from G2/M until the metaphase-anaphase transition. By using an electrical circuit model of mitochondria, we quantify the time-dynamics of mitochondrial membrane potential under normal and perturbed conditions to extract mitochondrial ATP synthesis rates in mitosis. We found that mitochondrial ATP synthesis decreases by approximately 50 % during early mitosis, when CDK1 is active, and increases back to G2 levels during cytokinesis. Consistently, acute inhibition of mitochondrial ATP synthesis failed to delay cell division. Our results provide a quantitative understanding of mitochondrial bioenergetics in mitosis and challenge the traditional dogma that cell division is a highly energy demanding process.

Metabolic Profile Of Leukemia Cells Influences Treatment Efficacy Of L‑Asparaginase

2019 ◽  
Author(s):  
Katerina Hlozkova ◽  
Alena Pecinova ◽  
David Pajuelo Reguera ◽  
Marketa Simcikova ◽  
Lenka Hovorkova ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Cell Lines ◽  
Mitochondrial Membrane ◽  
Metabolic Profile ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Lower Sensitivity ◽  
Leukemia Cell Lines

Abstract Background Effectiveness of L-asparaginase administration in acute lymphoblastic leukemia treatment is mirrored in overall outcome of patients. Generally, leukemia patients differ in their sensitivity to L-asparaginase; however, the mechanism underlying their inter-individual differences is still not fully understood. We have previously shown that L-asparaginase rewires the biosynthetic and bioenergetic pathways of leukemia cells to activate both anti-leukemic and pro-survival processes. Herein, we investigated the relationship between the metabolic profile of leukemia cells and their sensitivity to currently used cytostatic drugs.Methods Altogether, 19 leukemia cell lines and primary leukemia cells from 11 patients were used. Glycolytic function and mitochondrial respiration were measured using Seahorse bioanalyzer. Sensitivity to cytostatics was measured using MTS assay and/or absolute count and flow cytometry. Mitochondrial membrane potential was determined as TMRE fluorescence.Results We characterized the basal metabolic state of the cells derived from different leukemia subtypes using cell lines and primary samples and assessed their sensitivity to cytostatic drugs. We found that leukemia cells cluster into distinct groups according to their metabolic profile, which is mainly driven by their hematopoietic lineage of origin from which they derived. However, majority of lymphoid leukemia cell lines and patients with lower sensitivity to L-asparaginase clustered regardless their hematopoietic phenotype together with myeloid leukemias. Furthermore, we observed a correlation of specific metabolic parameters with sensitivity to L-asparaginase. Greater ATP-linked respiration and lower basal mitochondrial membrane potential in cells significantly correlated with higher sensitivity to L-asparaginase. No such correlation was found in other tested cytostatic drugs.Conclusions These data support the prominent role of the cell metabolism in the treatment effect of L-asparaginase. Based on these findings metabolic profile could identify leukemia patients with lower sensitivity to L-asparaginase with no specific genetic characterization.

Nanoparticles – a Novel Approach to Chronic Lymphocytic Leukemia Treatment?

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4601-4601 ◽  
Author(s):  
Ida Franiak-Pietryga ◽  
Ewelina Ziolkowska ◽  
Barbara Ziemba ◽  
Dietmar Appelhans ◽  
Brigitte Voit ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Membrane Potential ◽  
Chronic Lymphocytic Leukemia ◽  
Mitochondrial Membrane Potential ◽  
Targeted Delivery ◽  
Mitochondrial Membrane ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Amino Groups ◽  
Medical University

Abstract Abstract 4601 Treatment of chronic lymphocytic leukemia (CLL), including conventional therapy based on alkylating agents, purine nucleoside analogues (cladribine, fludarabine (FA)) or monoclonal antibodies (rituximab (Rit), alemtuzumab), significantly improved overall and complete responses. Although there are many treatment possibilities, this disease still remains incurable. Therefore, searching for new therapeutical strategies in CLL is vital. Nanotechnology, a new and promising field of scientific research, may be of use in medicine and the pharmaceutical industry. Dendrimers, nanoparticles (polymers) of dendritic architecture, used as carriers of drugs, nucleic acid and photosensitizers for targeted delivery, contrast agents in magnetic resonance imaging (MRI) have already been reported. It seems likely that dendrimers themselves might be damaging for neoplastic cells. The aim of our study was to preliminarly assess the clinical value of treating CLL patients with poly(propylene imine) (PPI) dendrimers. The assessment was based on the in vitro induction of cytotoxicity and apoptosis by fourth generation PPI dendrimers, with the surface amino groups substituted with maltotriose [Mal-III] residues. PPI-G4-OS-Mal-III dendrimers have, in contrast to the parental cationic PPI-G4, a virtually neutral surface charge. Therefore, they are preferentially involved in H-bond driven interactions. The study was conducted in 15 untreated CLL patients (7 men, 8 women) who were diagnosed in accordance with IWCLL criteria and followed at the Hematology Department, Medical University, Lodz, Poland. The Ethics Committee of the Medical University of Lodz, Poland approved the study (RNN/75/10/KE). Informed consent was obtained from all patients involved in the study. The mean age of CLL patients was 63.80 yrs. Peripheral blood mononuclear cells (MNCs) were separated from EDTA fresh blood. MNCs were incubated with dendrimers at concentrations of 4, 6 and 8 mg/ml. The cultures without dendrimers served as controls. Dendrimers in which approximately 35% of peripheral amino groups were coated with Mal-III (Leibniz Institute of Polymer Research, Dresden, Germany) have been defined as PPI-G4-OS-Mal-III. The abbreviation OS describes the open shell structure of carbohydrate-modified dendrimers (Fig. 1). The molar mass of this PPI dendrimer was 31000 g/mol. Apoptosis was measured by the Annexin-V/Propidium Iodide test. A decrease in mitochondrial membrane potential (one of the earliest events in the apoptotic pathway) was evaluated by the CMXRos technique, using flow cytometry. The percentage of cells with lower mitochondrial membrane potential (MMP) (DYmlow/Gly-A−) was determined. The percentage of apoptotic MNCs induced by PPI-G4-OS-Mal-III after 24 h and 48 h incubations was significantly higher than the percentage of spontaneous apoptotic leukemic cells. PPI glycodendrimers induced MNC apoptosis to a greater degree after 48 h than 24 h. The largest differences were observed for Ann-V+JP+ cells at a concentration of 8 mg/ml (p = 0.007). The IC50 value after 48-h-incubation was calculated as 8.24 mg/ml. Moreover, the CMXRos technique revealed apoptosis induction by PPI-G4-OS-Mal-III at each examined concentration in comparison with control cultures (Table 1). Table 1. 24h Control PPI-G4-OS-Mal-III 4 mg/ml PPI-G4-OS-Mal-III 6 mg/ml PPI-G4-OS-Mal-III 8 mg/ml FA 1.6 μM Rit 10 μg/ml X 23.7 51.0 60.1 64.4 62.9 28.0 48h X 28.3 67.8 71.9 79.3 89.0 29.4 X – mean percentage of cells with lower mitochondrial potential (DYmlow/Gly-A− [%]) Finally, PPI-G4-OS-Mal-III was not observed to have any harmful effects on erythrocytes (RBCs) or platelets (PLTs). The studied dendrimer induced PLT aggregation at a concentration of 50 mg/ml. Hemolysis induced by the PPI-G4-OS-Mal-III dendrimer is not important from the biological point of view. To summarise, the PPI-G4-OS-Mal-III dendrimer demonstrated higher cytotoxicity towards CLL cells than healthy donor cells. Its potency to trigger apoptosis is similar to many PNAs and monoclonal antibodies widely used in CLL therapy. Thus, dendrimers are a potential tool for CLL treatment. The study was partially supported by Grant No. DEC-2011/01/B/NZ5/01371 from the National Science Centre, Poland. Fig. 1. Fig. 1. Disclosures: No relevant conflicts of interest to declare.

Functional Characterization of NAD Dependent De-Acetylases SIRT1 and SIRT2 in B-Cell Chronic Lymphocytic Leukemia (CLL)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4141-4141
Author(s):  
Savita Bhalla ◽  
Leo I. Gordon
Keyword(s):  
Data Mining ◽  
Membrane Potential ◽  
B Cell ◽  
Mitochondrial Membrane Potential ◽  
Cell Lines ◽  
Mitochondrial Membrane ◽  
Functional Characterization ◽  
Lymphocytic Leukemia ◽  
Microarray Gene Expression ◽  
Knock Down

Introduction: CLL is characterized by abnormal proliferation and accumulation of mature CD5 positive B-lymphocytes in blood, bone marrow, spleen and lymph nodes. Outcome and survival is determined in part by the presence of 11q deletions and17p deletion/TP53 mutation with complex karyotype. Sirtuins are NAD+ dependent ADP-ribosyl transferases with evolutionary conserved function in cellular metabolism and chromatin regulation. Seven sirtuins (SIRT1-SIRT7) have been identified in mammals at distinct subcellular locations and targeting different substrates. SIRT1, 2, 6, and 7 are primarily found in the nucleus, SIRT2 in the cytoplasm and SIRT3, 4, 5 in the mitochondria. Sirtuins are associated with cancer as they deacetylate cancer associated transcription factors, and SIRT1 is overexpressed in acute myeloid leukemia, colon and prostate cancers. Several studies reported SIRT2 as a tumor suppressor as it is down regulated in human gliomas. SIRT1 and SIRT6 are reported to be significantly increased in CLL. We hypothesized that sirtuins play an important role in the development and maintenance of CLL and might therefore be a target. Methods: We measured SIRT 1 and 2 expression in fresh primary CLL cells, in the B-cell pro-lymphocytic cell lines JVM-3 and MEC-2 and by data-mining of the Oncomine microarray gene expression datasets. Oncomine is a bioinformatics initiative that collects, standardizes, analyzes, and delivers cancer transcriptome data to the biomedical research community. We then inhibited SIRT activity in primary CLL cells and cell lines by pharmacologic inhibitors EX-529 and sirtinol, and by knock down using shRNA in cell lines and then measured cell viability, apoptosis, reactive oxygen species formation and mitochondrial membrane potential. To determine the metabolic contribution to SIRT activity, we studied the effect of SIRT inhibition under conditions of nutrient deprivation. Results: We observed an increase in SIRT1mRNA expression in CLL by data mining of the independent microarray dataset in the Oncomine database, with a total of 2022 leukemia samples and 74 normal controls (Figure1). SIRT1was significantly up regulated in CLL compared with normal PBMC as well as other leukemia types. We found that SIRT inhibitors EX-527 and sirtinol impair cell growth (IC50 50-100 microM for EX-527 and 10-20 microM for sirtinol), cause apoptosis (>2-fold increase in apoptosis in cell lines JVM-3 and MEC-2), induce ROS production (up to 90% increase in mean fluorescence intensity (MFI) with EX-527 and sirtinol), loss of mitochondrial membrane potential, (MFI from 4 to <1 after treatment with SIRT inhibitors) and increase alpha-tubulin acetylation in primary CLL cells and cell lines. Using shRNA knock down of SIRT1 and SIRT2 in JVM-3 and MEC-2 cell lines, we showed that expression of both proteins is crucial for the survival of these cells. Furthermore, studies in nutrient deprived conditions suggest a role of SIRT in metabolism in CLL. Conclusion: These findings suggest that CLL cells are characterized by increased expression and function of SIRT1 and SIRT2, both directly inhibited by SIRT inhibitors. SIRT1 and SIRT2 inhibition using specific inhibitors could be a novel therapeutic approach for the treatment of CLL and other SIRT expressing hematologic malignancies. Figure 1. Figure 1. Disclosures Gordon: Northwestern University: Employment; Dr Leo I. Gordon: Patents & Royalties: Patent for gold nanoparticles pending.

Mitochondrial Regulation of Calcium in the Avian Cochlear Nucleus

1997 ◽  
Vol 78 (4) ◽  
pp. 1928-1934 ◽  
Author(s):  
Sam P. Mostafapour ◽  
Edward A. Lachica ◽  
Edwin W Rubel
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Cochlear Nucleus ◽  
Mitochondrial Membrane ◽  
Atp Synthesis ◽  
Cytosolic Calcium ◽  
Mitochondrial Regulation ◽  
Mitochondrial Number ◽  
And Function

Mostafapour, Sam P., Edward A. Lachica, and Edwin W Rubel. Mitochondrial regulation of calcium in the avian choclear nucleus. J. Neurophysiol. 78: 1928–1934, 1997. The role of mitochondria and the endoplasmic reticulum in buffering [Ca2+]i in response to imposed calcium loads in neurons of the chick cochlear nucleus, nucleus magnocellularis (NM), was examined. Intracellular calcium concentrations were measured using fluorometric videomicroscopy. After depolarization with 125 mM KCl, NM neurons demonstrate an increase in [Ca2+]i that returns to near-basal levels within 6 min. Addition of the protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP) dissipated the mitochondrial membrane potential, as evidenced by increased fluorescence when cells were loaded with rhodamine-123. Two micromolar CCCP had minimal effect on baseline [Ca2+]i. However, 2 or 10 μM CCCP interfered with the ability of NM cells to buffer [Ca2+]i in response to KCl depolarization without significantly affecting peak [Ca2+]i. Oligomycin also interfered with postdepolarization regulation of [Ca2+]i, but blocked late (7–8 min postdepolarization) increases in [Ca2+]i caused by CCCP. Thapsigargin had no effect on baseline, peak, or postdepolarization [Ca2+]i in NM cells. These results suggest that normal mitochondrial membrane potential and ATP synthesis play an important role in buffering [Ca2+]i in response to imposed calcium loads in NM neurons. Furthermore, the endoplasmic reticulum does not appear to play a significant role in either of these processes. Thus increases in mitochondrial number and function noted in NM cells after deafferentation may represent an adaptive response to an increased cytosolic calcium load.

scholarly journals SERS uncovers the link between conformation of cytochrome c heme and mitochondrial membrane potential

2021 ◽  
Author(s):  
Nadezda A. Brazhe ◽  
Evelina I. Nikelshparg ◽  
Adil A. Baizhumanov ◽  
Vera G. Grivennikova ◽  
Anna A. Semenova ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Conformational Changes ◽  
Mitochondrial Membrane ◽  
Atp Synthesis ◽  
Surface Enhanced Raman Spectroscopy ◽  
Complex Iii ◽  
Label Free ◽  
Inner Mitochondrial Membrane ◽  
The Impact

AbstractCytochrome c is an essential component of the electron transport chain (ETC), which regulates respiratory chain activity, oxygen consumption, and ATP synthesis. But the impact of conformational changes in cytochrome c on its function is not understood for lack of access to these changes in intact mitochondria. Here we describe a label-free tool that identifies conformational changes in cytochrome c heme and elucidates their function. We verify that molecule bond vibrations assessed by surface-enhanced Raman spectroscopy (SERS) is a reliable indicator of the planar heme configuration during activation of ETC and decrease in inner mitochondrial membrane potential. The planar conformation of cytochrome c heme enables its optimal orientation towards the heme of cytochrome c1 in complex III. This ensures a faster electron transfer, which is important during ETC speed-ups and acceleration of ATP synthesis. The ability of our tool to track mitochondrial function opens wide perspectives on cell bioenergetics.

scholarly journals Enhanced mitochondrial membrane potential and ATP synthesis by photobiomodulation increases viability of the auditory cell line after gentamicin-induced intrinsic apoptosis

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
So-Young Chang ◽  
Min Young Lee ◽  
Phil-Sang Chung ◽  
Sehwan Kim ◽  
Bernard Choi ◽  
...  
Keyword(s):  
Hearing Loss ◽  
Membrane Potential ◽  
Cell Viability ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Atp Synthesis ◽  
Therapeutic Effects ◽  
Intrinsic Apoptosis ◽  
Downstream Process ◽  
Intracellular Mechanism

AbstractPhotobiomodulation (PBM) has been suggested to have a therapeutic effect on irreversible hearing loss induced by aminoglycosides, including gentamicin (GM). However, its intracellular mechanism(s) in GM-induced ototoxicity remain poorly understood. In the present study, we investigated the effect of PBM in GM-induced ototoxicity in auditory cells. We tried to characterize the downstream process by PBM, and the process that triggered the increased cell viability of auditory cells. As a result, the effects of PBM against GM-induced ototoxicity by increasing ATP levels and mitochondrial membrane potential was confirmed. These results suggest a theory to explain the therapeutic effects and support the use of PBM for aminoglycoside-induced hearing loss.

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