Optical computed tomography for spatially isotropic four-dimensional imaging of live single cells.

Quantitative three-dimensional (3D) computed tomography (CT) imaging of living single cells enables orientation-independent morphometric analysis of the intricacies of cellular physiology. Since its invention, x-ray CT has become indispensable in the clinic for diagnostic and prognostic purposes due to its quantitative absorption-based imaging in true 3D that allows objects of interest to be viewed and measured from any orientation. However, x-ray CT has not been useful at the level of single cells because there is insufficient contrast to form an image. Recently, optical CT has been developed successfully for fixed cells, but this technology called Cell-CT is incompatible with live-cell imaging due to the use of stains, such as hematoxylin, that are not compatible with cell viability. We present a novel development of optical CT for quantitative, multispectral functional 4D (three spatial + one spectral dimension) imaging of living single cells. The method applied to immune system cells offers truly isotropic 3D spatial resolution and enables time-resolved imaging studies of cells suspended in aqueous medium. Using live-cell optical CT, we found a heterogeneous response to mitochondrial fission inhibition in mouse macrophages and differential basal remodeling of small (0.1 to 1 fl) and large (1 to 20 fl) nuclear and mitochondrial structures on a 20- to 30-s time scale in human myelogenous leukemia cells. Because of its robust 3D measurement capabilities, live-cell optical CT represents a powerful new tool in the biomedical research field.

Cytopathological image analysis using deep-learning networks in microfluidic microscopy.

Cytopathologic testing is one of the most critical steps in the diagnosis of diseases, including cancer. However, the task is laborious and demands skill. Associated high cost and low throughput drew considerable interest in automating the testing process. Several neural network architectures were designed to provide human expertise to machines. In this paper, we explore and propose the feasibility of using deep-learning networks for cytopathologic analysis by performing the classification of three important unlabeled, unstained leukemia cell lines (K562, MOLT, and HL60). The cell images used in the classification are captured using a low-cost, high-throughput cell imaging technique: microfluidics-based imaging flow cytometry. We demonstrate that without any conventional fine segmentation followed by explicit feature extraction, the proposed deep-learning algorithms effectively classify the coarsely localized cell lines. We show that the designed deep belief network as well as the deeply pretrained convolutional neural network outperform the conventionally used decision systems and are important in the medical domain, where the availability of labeled data is limited for training. We hope that our work enables the development of a clinically significant high-throughput microfluidic microscopy-based tool for disease screening/triaging, especially in resource-limited settings.

Magnetic Resonance Imaging of Mitochondrial Dysfunction and Metabolic Activity, Accompanied by Overproduction of Superoxide.

This study shows that a mitochondria-penetrating nitroxide probe (mito-TEMPO) allows detection of superoxide and visualization of mitochondrial dysfunction in living cells due to the effect of T1 shortening in MRI. Mitochondrial dysfunction was induced by treatment of cells with rotenone and 2-methoxyestradiol (2-ME/Rot). The MRI measurements were performed on 7T MRI. The 2-ME/Rot-treated cells were characterized by overproduction of superoxide, which was confirmed by a conventional dihydroethidium test. In the presence of mito-TEMPO, the intensity of MRI signal in 2-ME/Rot-treated cells was ∼30-40% higher, in comparison with that in untreated cells or culture media. In model (cell-free) systems, we observed that superoxide, but not hydrogen peroxide, increased the intensity of T1-weighted MRI signal of mito-TEMPO. Moreover, the superoxide restores the T1-weighted MRI contrast of mito-TEMPOH, a noncontrast (diamagnetic) analogue of mito-TEMPO. This was also confirmed by using EPR spectroscopy. The results demonstrate that superoxide radical is involved in the enhancement of T1-weighted MRI contrast in living cells, in the absence and presence of mito-TEMPO. This report gives a direction for discovering new opportunities for functional MRI, for detection of metabolic activity, accompanied by overproduction of superoxide, as well as by disturbance of the balance between superoxide and hydrogen peroxide, a very important approach to clarify the fine molecular mechanisms in the regulation of many pathologies. The visualization of mitochondrial activity in real-time can be crucial to clarify the molecular mechanism of the functional MRI in its commonly accepted definition, as a method for detection of neurovascular coupling.

MicroRNA-320a acts as a tumor suppressor by targeting BCR/ABL oncogene in chronic myeloid leukemia.

Accumulating evidences demonstrated that the induction of epithelial-mesenchymal transition (EMT) and aberrant expression of microRNAs (miRNAs) are associated with tumorigenesis, tumor progression, metastasis and relapse in cancers, including chronic myeloid leukemia (CML). We found that miR-320a expression was reduced in K562 and in CML cancer stem cells. Moreover, we found that miR-320a inhibited K562 cell migration, invasion, proliferation and promoted apoptosis by targeting BCR/ABL oncogene. As an upstream regulator of BCR/ABL, miR-320a directly targets BCR/ABL. The enhanced expression of miR-320a inhibited the phosphorylation of PI3K, AKT and NF-κB; however, the expression of phosphorylated PI3K, AKT and NF-κB were restored by the overexpression of BCR/ABL. In K562, infected with miR-320a or transfected with SiBCR/ABL, the protein levels of fibronectin, vimentin, and N-cadherin were decreased, but the expression of E-cadherin was increased. The expression of mesenchymal markers in miR-320a-expressing cells was restored to normal levels by the restoration of BCR/ABL expression. Generally speaking, miR-320a acts as a novel tumor suppressor gene in CML and miR-320a can decrease migratory, invasive, proliferative and apoptotic behaviors, as well as CML EMT, by attenuating the expression of BCR/ABL oncogene.

Inhibition effects of lamellarin D on human leukemia K562 cell proliferation and underlying mechanisms.

Lamellarin D (LamD) is a marine alkaloid with a pronounced cytotoxicity against a large panel of cancer cells, affecting cell growth and inducing apoptosis. However, the molecular mechanisms of action of this compound are poorly understood. In this study, the anticancer efficacy of LamD was investigated in human leukemia K562 cells. The results showed suppressed cell proliferation and induction of G0/G1-phase arrest,while expression of CDK1, and activity of smad3 and smad5 were reduced, but that of p27, p53 and STGC3 was increased. LamD induced cell apoptosis through activation of caspases-8/-3, inhibition of survivin and Bcl-2, suggesting that this compound may also act through a caspase-independent pathway. Moreover, LamD inhibited the secretion of TGF-ß, IL-1ß, IL-6, IL-8 and other inflammatory cytokines and the transcriptional activity of transcription factor NF-κB in human leukemia K562 cells. Taken together, our results suggest that LamD-mediated inhibition of leukemia cell proliferation may be related to the induction of apoptosis and the regulation of cell cycle, tumor- related gene expression and cytokine expression, which may provide a new way of thinking for the treatment leukemia.

Imaging proliferation in human leukemia-tumor bearing mice with (18)F-FLT: Comparison with (18)F-FDG PET.

Leukemia threatens human life due to its uncontrolled proliferative malignancy. 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) has been suggested as a new positron emission tomography (PET) tracer for imaging tumor proliferation. The aim of the study was to investigate the usefulness of (18)F-FLT PET for imaging human leukemia-tumor bearing mice, compared with fluorine-18-fluorodesoxyglucose ((18)F-FDG PET). In vitro the experiments of (18)F-FLT and (18)F-FDG uptake were performed in K562 cell lines at various time points and radioactive tracer uptake was measured in a gamma counter. (18)F-FLT and (18)F-FDG PET imaging were performed both in the same mouse when eight tumor-bearing mice models of human chronic myeloid leukemia were established successfully by injecting K562 cells. Regions of interest were drawn over the tumor, the crossed normal tissue was regarded as background and the ratio of tumor to non-tumor counts (T/NT) in tissues was calculated. A higher uptake of (18)F-FLT (15min, 5.73±0.05%; 30min, 5.90±0.06%; 60min, 6.16±0.19%; 120min, 6.32±0.08%) than that of (18)F-FDG (15min, 1.05±0.10%; 30min, 1.11±0.14%; 60min, 1.14±0.37%; 120 min, 1.36±0.25%) was observed in K562 cells in the tracer uptake experiment. Ratios of T/NT of (18)F-FLT PET (0.5h, 5.39±0.42; 1h, 4.88±0.43; 2h, 3.81±0.38) were higher than those of (18)F-FDG PET/CT (0.5h, 0.34±0.12; 1h, 0.21±0.06; 2h, 0.13±0.05) after injection. Both uptake and T/NT differences of (18)F-FLT versus (18)F-FDG were significant (P>0.05). In conclusion, (18)F-FLT and (18)F-FDG quantitative and semi-quantitative uptake measurements resulting from cell lines and PET imaging respectively suggested a promising potential of (18)F-FLT for metabolic imaging of human chronic myeloid leukemia.

Involvement of oxidative stress in taxol-induced apoptosis in chronic myelogenous leukemia K562 cells.

It is now well accepted that taxol exhibits cytotoxicity and antitumor activity in many human tumors through microtubule stabilization and induction of G2/M cell cycle arrest with final extensive cell apoptosis. Since many anti-cancer agents exert their cytotoxic effects through reactive oxygen species (ROS), we were interested to evaluate whether oxidative stress is involved in taxol-induced cytotoxicity among human leukemia K562 cells. Our results showed that induction of apoptosis was associated with generation of ROS and glutathione (GSH) depletion. The increase in ROS production and apoptosis were both suppressed by antioxidant N-acetyl-l-cysteine (NAC). Moreover, taxol caused an increase in c-Jun NH(2)-terminal kinase (JNK) and p38 activities, two of the well known mediators of the stress activation pathways. Attenuation of JNK expression in the presence of NAC might indicate the modulation of the level of JNK activity by ROS. Furthermore, our data indicated that Bcl-2α was down-regulated in taxol-treated cells and its expression was modulated by ROS and JNK activity. The activities of caspase-9 and -3 were also increased upon treatment with taxol; however, pre-treatment of cells with NAC or JNK inhibitor (SP600125) impeded taxol-mediated caspase activation and apoptosis in K562 cells, suggesting that JNK acts upstream of the caspases. Taken together, these results indicate that taxol induces apoptosis in chronic myelogenous leukemia cells by inducing intracellular oxidative stress and JNK activation pathway.

Induction of anion exchanger-1 translation and its opposite roles in the carcinogenesis of gastric cancer cells and differentiation of K562 cells.

Anion exchanger-1 (AE1), an erythroid-specific membrane protein, mediates the Cl(-)/HCO(-)(3) exchange across the plasma membrane and regulates intracellular pH. We have found that AE1 was unexpectedly expressed in gastric cancer cells and participated in the tumorigenesis of the cancer. Here, we focus on the induction of AE1 expression and its role in gastric carcinogenesis as well as in the differentiation of K562 cells. The results show that expression of AE1 is not related to genetic mutation or the mRNA level, but rather, that it is modulated by miR-24. miR-24 decreases the expression of AE1 through binding to the 3'UTR of AE1 mRNA. Transfection of an miR-24 into gastric cancer cells reduced the elevation of the AE1 protein, which resulted in return of AE1-sequestrated p16 to the nucleus, thereby inhibiting proliferation of the cells. Furthermore, the miR-24 inhibitor cooperated with hemin to induce the expression of AE1 in K562 cells and differentiation of the cells, which is consistent with results obtained from the cells cultured at pH 7.6 or from forced stable expression of AE1. These findings establish a novel regulation of miR-24-related AE1 expression in gastric carcinogenesis and erythropoiesis.

Inhibition of imatinib-mediated apoptosis by the caspase-cleaved form of the tyrosine kinase Lyn in chronic myelogenous leukemia cells.

Once cleaved by caspases, the Lyn tyrosine kinase (LynDeltaN) is relocalized from the plasma membrane to the cytoplasm of apoptotic cells, but the function of such a cleavage is incompletely understood. We evaluated the effect of LynDeltaN overexpression on imatinib sensitivity of the chronic myelogenous leukemia (CML) cell line K562. Therefore, we generated stable cells that express plasmids encoding LynDeltaN or its catalytically inactive counterpart LynDeltaNKD. We established that Lyn is cleaved in imatinib-treated parental K562 cells in a caspase-dependent manner. Lyn cleavage also occurred following BCR-ABL silencing by specific short hairpin RNA (sh-RNA). Imatinib-induced apoptosis was abrogated in LynDeltaN-overexpressing cells, but not in cells overexpressing its inactive counterpart. Conversely, the overexpression of LynDeltaN failed to affect the differentiation of K562 cells. Importantly, the protective effect of LynDeltaN was suppressed by two inhibitors of Lyn activity. LynDeltaN also inhibits imatinib-mediated caspase-3 activation in the small proportion of nilotinib-resistant K562 cells overexpressing Lyn that can engage an apoptotic program upon imatinib stimulation. Finally, Lyn knockdown by sh-RNA altered neither imatinib-mediated apoptosis nor differentiation. Taken together, our data show that the caspase-cleaved form of Lyn exerts a negative feedback on imatinib-mediated CML cell apoptosis that is entirely dependent on its kinase activity and likely on the BCR-ABL pathway.

Upregulation of stem cell genes in multidrug resistant K562 leukemia cells.

The transmembrane transporter P-glycoprotein (P-gp) encoded by ABCB1, is one major cause for multidrug resistance (MDR). We compared the genomic profile and gene expression pattern of the P-gp positive K562VCR cells with parental P-gp negative K562wt cells. In K562VCR array CGH revealed amplification of ABCB1, ABCB4, ABCB5 and SEMA3D, whereas expression microarrays demonstrated upregulation of stem cell genes (e.g. KIT and HOXB4), anti-apoptotic genes (e.g. IGF1R and CCNG1), and downregulation of pro-apoptotic genes (e.g. CASP4, 6 and 7). Thus, K562VCR cells disclose stem cell characteristics including a range of drug resistance mechanisms possibly attained as a stem cell program switched on en bloc.

Heat shock protein 70 (HSP70) induces cytotoxicity of T-helper cells.

Heat shock protein 70 (HSP70) has gained plenty of attention because of its adjuvant capability to induce CD8(+) cytotoxic T lymphocyte and CD4(+) T-helper cell responses. We investigated the behavior of T-cell subsets stimulated with endotoxin-free HSP70 with respect to proliferation, cytokine expression, cytotoxicity against allogeneic B-lymphoblastoid cell line and K562 cells, as well as target-independent cytotoxicity. CD4(+) cells exhibited a strong increase in proliferation after stimulation with HSP70 (29%). In the presence of targets, a 35-fold up-regulation of granzyme B was observed after stimulation of CD4(+) T cells with HSP70 in combination with interleukin-7 (IL-7)/IL-12/IL-15. The target cell-independent secretion of granzyme B by CD4(+) cells was greatly augmented after stimulation with HSP70 plus IL-2 or IL-7/IL-12/IL-15. In this study, we showed that HSP70 is capable of inducing a cytotoxic response of T-helper cells in the absence of lipopolysaccharide. The granzyme B secretion and cytolytic activity of T-helper cells are induced in a target-independent way, whereas the cytotoxic activity of CD3(+) and CD8(+) T cells can be further enhanced in the presence of target cells. Our data provide novel insights into the role of extracellular HSP70 on T-cell immune response concerning the induction of target-independent T-helper cell cytotoxicity.

Nuclear organization of PML bodies in leukaemic and multiple myeloma cells.

The nuclear arrangement of promyelocytic leukaemia nuclear bodies (PML NBs) was studied in vitro after the cell treatment by clinically used agents such as all-trans retinoic acid (RA) in human leukaemia and cytostatics or gamma radiation in multiple myeloma cells. In addition, the influence of phorbol ester (PMA) on PML NBs formation was analyzed. A reduced number of PML bodies, which led to relocation of PML NBs closer to the nuclear interior, mostly accompanied RA- and PMA-induced differentiation. Centrally located PML NBs were associated with transcriptional protein RNAP II and SC35 regions, which support importance of PML NBs in RNA processing that mostly proceeds within the nuclear interior. Conversely, the quantity of PML NBs was increased after cytostatic treatment, which caused re-distribution of PML NBs closer to the nuclear envelope. Here we showed correlations between the number of PML NBs and average Centre-to-PML distances. Moreover, a number of cells in S phase, especially during differentiation, influenced number of PML NBs. Studying the proteins involved in PML compartment, such as c-MYC, cell-type specific association of c-MYC and the PML NBs was observed in selected leukaemic cells undergoing differentiation, which was accompanied by c-MYC down-regulation.

Substances from the medicinal mushroom Daedalea gibbosa inhibit kinase activity of native and T315I mutated Bcr-Abl.

Human chronic myelogenous leukemia (CML) is a malignancy of pluripotent hematopoietic cells characterized by a distinctive cytogenetic abnormality resulting in the creation of a p210 Bcr-Abl fusion protein with abnormal tyrosine kinase activity. Recently, a selective Abl kinase inhibitor, Imatinib mesylate, was introduced as a first line therapy for CML. Despite the initial response, CML patients develop a resistantance to Imatinib, which is mediated mainly by point mutations within the Abl protein. Herein, we describe the identification of mycelium organic extracts of Daedalea gibbosa with selective anti-proliferating and apoptosis-inducing activities against K562 cells and other laboratory model of CML. Using activity-guided purification, we isolated an active fraction, F6, which inhibits in vitro kinase activity of recombinant Abl. The active fraction significantly inhibits the autophosphorylation of native and mutated Bcr-Abl, which are resistant to Imatinib treatment including the T315I mutation. Using a colony-forming assay, we demonstrated that the active fraction is effective in inhibiting the colony formation of the Ba/F3 cell line harboring either native Bcr-Abl or its mutations, including the T315I mutation. Our data illustrated the potential of natural products in cancer therapeutics.

Global DNA hypomethylation, rather than reactive oxygen species (ROS), a potential facilitator of cadmium-stimulated K562 cell proliferation.

Cell proliferation plays a critical role in the process of cadmium (Cd) carcinogenesis. Although both induction of reactive oxygen species (ROS) and alteration of DNA methylation are involved in Cd-stimulated cell proliferation, the detailed mechanism of Cd-stimulated cell proliferation remains poorly understood. In this study, K562 cells pre-treated with N-acetylcysteine (NAC) or methionine (Meth) were exposed to Cd to investigate the potential contribution of ROS and global DNA methylation pathways in Cd-induced cell proliferation. The results showed that Cd-stimulated cell proliferation, increased ROS and DNA damage levels, and induced global DNA hypomethylation. The increases of ROS and DNA damage levels were attenuated by pre-treatment with NAC. Cd-stimulated cell proliferation did not appear to be suppressed through eliminating ROS by NAC. However, methionine was shown to prevent Cd-induced global DNA hypomethylation and Cd-stimulated cell proliferation. Our results suggest that global DNA hypomethylation, rather than ROS, is a potential facilitator of Cd-stimulated K562 cell proliferation.

Ex vivo detection of primary leukemia cells resistant to granule cytotoxin-induced cell death: a rapid isolation method to study granzyme-B-mediated cell death.

Cytotoxic T lymphocytes and natural killer cells (CTL/NK) induce cell death in leukemia cells by the granzyme B (grB)-dependent granule cytotoxin (GC) pathway. Resistance to GC may be involved in immune evasion of leukemia cells. The delivery of active grB into the cytoplasma is dependent on the presence of perforin (PFN) and grB complexes. We developed a rapid method for the isolation of GC to investigate GC-mediated cell death in primary leukemia cells. We isolated GC containing grB, grB complexes and PFN by detergent free hypotonic lysis of the human NK cell leukemia line YT. The GC induce grB-mediated, caspase-dependent apoptosis in live cells. The human leukemia cell lines KG-1, U937, K562 (myeloid leukemia), Jurkat, Daudi, and BV173 (lymphoblastic leukemia) treated with GC internalized grB and underwent cell death. In primary leukemia cells analyzed ex vivo, we found GC-resistant leukemia cells in three out of seven patients with acute myeloid leukemia and one out of six patients with acute lymphoblastic leukemia. We conclude that our method is fast (approximately 1 h) and yields active GC that induce grB-dependent cell death. Furthermore, resistance to GC can be observed in acute leukemias and may be an important mechanism contributing to leukemia cell immune evasion.

Synthesis and antiproliferative activities of isoindigo and azaisoindigo derivatives.

In the course of structure-activity relationship studies, diversely substituted 1-(beta- d-acetylatedglucopyranosyl)isoindigo derivatives were prepared from indolines. New 7'-azaisoindigo analogues were also synthesized by coupling a glycosylated isatine and a 7-azaindolin-2-one derivative. Compounds containing a 7'-azaisoindigo framework have never been described before. To get an insight into the substitution pattern required for the best biological potencies, their antiproliferative activities were evaluated toward a human buccal carcinoma cell line (KB) and two human myeloid leukaemia cell lines (K562, HL60).

On the factors modulating the effect of statins on malignant cell proliferation.

HuCC, EHEB, K562, and Raji cells were incubated with 3 hydrophobic statins (atorvastatin, lovastatin, and simvastatin) and one hydrophilic statin (pravastatin). The 4 statins inhibited HuCC cell proliferation; however, none of them affected their apoptosis. EHEB and K562 cell proliferation was inhibited by the hydrophobic statins, but not by pravastatin. Raji cell proliferation was not affected by any of the 4 statins. The hydrophobic statins solely enhanced early apoptosis of the hematological cell lines. The results point out that the antiproliferative effect of statins on tumor cells and their effect on the apoptotic rate depends on their dosage, physiochemical property, and the type of the malignant cells.

Biocompatible conductive architecture of carbon nanofiber-doped chitosan prepared with controllable electrodeposition for cytosensing.

A novel architecture was designed by combining the biocompatibility of chitosan (CS) and excellent conductivity of carbon nanofiber (CNF). The controllable electrodeposition of soluble CNF-doped CS colloidal solution formed a robust CNF-CS nanocomposite film with good biocompatibility for the immobilization and cytosensing of K562 cells on an electrode. The formed architecture was characterized using scanning electron microscopic, infrared spectrum, contact angle, and thermogravimetric analyses. The adhesion of K562 cells on the nanocomposite film-modified electrode could be followed with electrochemical impedance spectroscopy and cyclic voltammetry. The presence of CNF facilitated the electrochemical behavior of K562 cells. The impedance of electronic transduction was related to the amount of the adhered cells, producing a highly sensitive impedance sensor for K562 cells ranging from 5 x 10(3) to 5.0 x 10(7) cells mL-1 with a limit of detection of 1 x 10(3) cells mL-1. This work suggested a strategy to prepare a biocompatible and conductive interface for immobilization and electrochemical detection of cells and opened a way for the application of CNF in cytosensing.

Cyclosporin A potentiates the cytotoxic effects of methyl methanesulphonate in HL-60 and K562 cells.

Methyl methanesulphonate (MMS) is a DNA damaging agent, which induces oxidative stress, ATP depletion, and consequently, cell death, in HL-60 and K562 cells. The cell death induced by MMS predominantly exhibited the morphological and biochemical hallmarks of necrosis. A minor population of dying cells exhibited apoptotic hallmarks, especially in K562 cell cultures. Cyclosporin A (CsA) was used to modulate the MMS-induced cell death. Our results indicated that CsA did not prevent cells from dying, but changed the mode of death from necrotic to apoptotic. Surprisingly, CsA enhanced oxidative stress and increased the overall number of dead cells. Based on these results, we conclude that the modulatory effect of CsA on MMS-induced cell death might arise from an interference by CsA with mitochondrial metabolism, rather than from inhibition of the MMS efflux mediated by P-glycoprotein.