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Real-time deformability cytometry allows the continuous mechanical characterization of cells with high throughput and is applied to distinguish cell-cycle phases, track differentiated cells and profile cell populations in whole blood. We introduce real-time deformability cytometry (RT-DC) for continuous cell mechanical characterization of large populations (>100,000 cells) with analysis rates greater than 100 cells/s. RT-DC is sensitive to cytoskeletal alterations and can distinguish cell-cycle phases, track stem cell differentiation into distinct lineages and identify cell populations in whole blood by their mechanical fingerprints. This technique adds a new marker-free dimension to flow cytometry with diverse applications in biology, biotechnology and medicine.

While the molecular and biophysical mechanisms underlying cell protrusion on two-dimensional substrates are well understood, our knowledge of the actin structures driving protrusion in three-dimensional environments is poor, despite relevance to inflammation, development and cancer. Here we report that, during chemotactic migration through microchannels with 5 μm × 5 μm cross-sections, HL60 neutrophil-like cells assemble an actin-rich slab filling the whole channel cross-section at their front. This leading edge comprises two distinct F-actin networks: an adherent network that polymerizes perpendicular to cell-wall interfaces and a ‘free’ network that grows from the free membrane at the cell front. Each network is polymerized by a distinct nucleator and, due to their geometrical arrangement, the networks interact mechanically. On the basis of our experimental data, we propose that, during interstitial migration, medial growth of the adherent network compresses the free network preventing its retrograde movement and enabling new polymerization to be converted into forward protrusion. Much of our understanding of the role of actin in cell migration is based on studies of cells moving across two-dimensional surfaces. Wilson et al. show that cells crawling in three dimensions through a narrow channel form two functionally distinct actin networks at the leading edge.

Background: Digalloyl-resveratrol (di-GA) is a synthetic compound aimed to combine the biological effects of the plant polyhydroxy phenols gallic acid and resveratrol, which are both radical scavengers and cyclooxygenase inhibitors exhibiting anticancer activity. Their broad spectrum of activities may probably be due to adjacent free hydroxyl groups. Methods: Protein activation and expression were analysed by western blotting, deoxyribonucleoside triphosphate levels by HPLC, ribonucleotide reductase activity by 14 C-cytidine incorporation into nascent DNA and cell-cycle distribution by FACS. Apoptosis was measured by Hoechst 33258/propidium iodide double staining of nuclear chromatin and the formation of gaps into the lymphendothelial barrier in a three-dimensional co-culture model consisting of MCF-7 tumour cell spheroids and human lymphendothelial monolayers. Results: In HL-60 leukaemia cells, di-GA activated caspase 3 and dose-dependently induced apoptosis. It further inhibited cell-cycle progression in the G1 phase by four different mechanisms: rapid downregulation of cyclin D1, induction of Chk2 with simultaneous downregulation of Cdc25A, induction of the Cdk-inhibitor p21 Cip/Waf and inhibition of ribonucleotide reductase activity resulting in reduced dCTP and dTTP levels. Furthermore, di-GA inhibited the generation of lymphendothelial gaps by cancer cell spheroid-secreted lipoxygenase metabolites. Lymphendothelial gaps, adjacent to tumour bulks, can be considered as gates facilitating metastatic spread. Conclusion: These data show that di-GA exhibits three distinct anticancer activities: induction of apoptosis, cell-cycle arrest and disruption of cancer cell-induced lymphendothelial disintegration.

In vitro evidence for immune activating effect of specific AGE structures retained in uremia. Advanced glycation end-products (AGEs) have been identified to be accumulated in blood and tissues of patients with end-stage renal disease (ESRD). AGEs have been shown to modulate immune competent cell activities and in this way they may contribute to the progression of atherosclerosis. All studies in this context have been performed, however, with generated mix of glycation compounds, and not with structures similar to those encountered in uremia. In the present study, the immunologic effect of specific AGE compounds, known to be retained in uremia, has been evaluated. Four albumin preparations, modified chemically at lysine or arginine residues, respectively, to contain N-ε-carboxymethyllysine (CML albumin), N-ε-carboxyethyllysine (CEL albumin), glyoxal-induced imidazolinones (Arg I albumin) or methylglyoxal-induced imidazolinones (Arg II albumin) were applied. Their effect on chemiluminescence production, CD14 expression, and the DNA synthesis of calcitriol-differentiated HL-60 (monocyte/macrophage phenotype) was studied. The phorbol 12-myristate 13-acetate (PMA)-stimulated chemiluminescence production of the calcitriol differentiated HL-60 cells was enhanced in the presence of CEL albumin (44.1 ± 18.5 vs. 64.7 ± 28.1 counts 103/30 min) (P < 0.05), Arg I albumin (46.4 ± 18.8 vs. 66.1 ± 32.6 counts 103/30 min) (P < 0.05) and CML albumin (41.9 ± 25.5 vs. 60.9 ± 5.5 counts 103/30 min) (P = 0.0625) pointing to an increase in free radical production. The latter AGE compounds also significantly increased the calcitriol-induced CD14 expression on HL-60 cells (1675 ± 796 vs. 2075 ± 1044; 768 ± 143 vs. 890 ± 150; 647 ± 63 vs. 716 ± 69 mean fluorescence intensity) (P < 0.05, respectively) pointing to an increase in expression of the lipopolysaccharide (LPS) receptor. Finally, the DNA synthesis of the calcitriol-differentiated HL-60 cells was enhanced in the presence of Arg I albumin [34.5 ± 4.6 vs. 27.7 ± 9.7% 5-bromo-2′-deoxyuridine (BrdU)-positive cells] (P < 0.05) resulting in an increased cell proliferation. Genuine AGE compounds, as they are encountered in the uremic condition, activate leukocyte response, and hence could play a role in uremia related atherogenesis.

During the last years remission rates of more than 72% for arsenic(III)-oxide (As 2 O 3 ) treatment in relapsed or refractory acute promyelocytic leukemia have been published. As 2 O 3 is under clinical investigation for therapy of leukemia and solid tumors. Due to the chemical affinity of arsenic and antimony, we analyzed the potency of antimony(III)-oxide (Sb 2 O 3 ) to exert As 2 O 3 -like effects. Based on the same molar concentrations, lower efficacy in apoptosis induction and caspase-independent decrease of mitochondrial membrane potential was observed for Sb 2 O 3 . No difference in sensitivity to As 2 O 3 or Sb 2 O 3 was detected in CEM cells when compared to their multiple drug resistant derivatives. Apoptosis was induced by combining sub-apoptotic concentrations of Sb 2 O 3 or As 2 O 3 with sub-apoptotic concentrations of dl -buthionine-[ S , R ]-sulfoximine (BSO). Other modulators of the cellular redox system showed this effect to a lower extent and enhancement was not consistent for the different cell lines tested. Caspase inhibitors protected cell lines from Sb 2 O 3 - and As 2 O 3 -induced apoptosis. When BSO was added, the inhibitors lost their protective ability. The ability of modulators of the cellular redox system in clinically applicable concentrations to enhance the apoptotic effects of the two oxides in a synergistic way may be helpful to reduce their toxicity by optimizing their dose.

Oxidizing species (OS), produced by photosensitization or derived from cytotoxic agents, activate apoptotic pathways. We investigated whether two different OS, formed at the same subcellular sites, have equivalent ability to initiate apoptosis in HL-60 cells. Our previous work showed that absorption of visible light by rose bengal (RB) produces singlet oxygen exclusively, whereas absorption of ultraviolet A produces RB-derived radicals in addition to singlet oxygen. Singlet oxygen, but not the RB-derived radicals, induced nuclear condensation and DNA fragmentation into nucleosome-size fragments in a dose dependent manner. In contrast, the RB-derived radicals caused greater lipid oxidation than singlet oxygen. These results indicate that different OS, produced at the same subcellular sites, do not have the same ability to induce apoptosis and that the ability of an OS to initiate lipid oxidation does not necessarily correlate with its ability to induce apoptosis.

In the present study, we investigated the mechanism of CD44 ligation with the anti-CD44 monoclonal antibody A3D8 to inhibit the proliferation of human acute myeloid leukemia (AML) cells. The effects of A3D8 on myeloid cells were associated with specific disruption of cell cycle events and induction of G0/G1 arrest. Induction of G0/G1 arrest was accompanied by an increase in the expression of p21, attenuation of pRb phosphorylation and associated with decreased Cdk2 and Cdk4 kinase activities. Since c-Jun is an important regulator of proliferation and cell cycle progression, we analysed its role in A3D8-mediated growth arrest. We observed that A3D8 treatment of AML patient blasts and HL60/U937 cells led to the downregulation of c-Jun expression at mRNA and protein level. Transient transfection studies showed the inhibition of c- jun promoter activity by A3D8, involving both AP-1 sites. Furthermore, A3D8 treatment caused a decrease in JNK protein expression and a decrease in the level of phosphorylated c-Jun. Ectopic overexpression of c-Jun in HL60 cells was able to induce proliferation and prevent the antiproliferative effects of A3D8. In summary, these data identify an important functional role of c-Jun in the induction of cell cycle arrest and proliferation arrest of myeloid leukemia cells because of the ligation of the cell surface adhesion receptor CD44 by anti-CD44 antibody. Moreover, targeting of G1 regulatory proteins and the resulting induction of G1 arrest by A3D8 may provide new insights into antiproliferative and differentiation therapy of AML.

Microbial extracellular glycolipids, succinoyl trehalose lipid (STL), and mannosylerythritol lipid (MEL) inhibited the growth of a human promyelocytic leukemia cell line, HL60, and induced their morphological changes. The results of specific and nonspecific leukocyte esterase activities showed that STL induced monocytotic differentiation while MEL induced granulocytic differentiation. STL and MEL markedly increased common differentiation‐associated characteristics in monocytes and granulocytes, such as nitroblue tetrazolium (NBT) reducing ability, expression of Fc receptors, and phagocytic activities in HL60 cells, respectively. Neither sugar moieties nor fatty acids in the free form, the individual components of STL and MEL, were effective at inducing the differentiation of HL60 cells. The induction of differentiation was not due to surface activities of STL and MEL on the basis of the complete ineffectiveness of the analogues tested. The composition of cell surface glycosphingolipids (GSL) changed such that the GM3/LacCer ratio increased in STL‐treated cells, whereas it decreased in MEL‐treated cells. HL60 cells treated with STL and MEL exhibited a significant decrease in the activity of the intracellular phospholipid‐ and Ca2+‐dependent protein kinase (protein kinase C). Furthermore, the serine/threonine phosphorylations in intact HL60 cells were clearly inhibited by the presence of GM3 and MEL, but not by LacCer and STL. These results suggest that the differentiation‐inducing activity of STL and MEL is not due to a simple detergent‐like effect but due to a specific action on the plasma membrane. The inhibitory effect of STL on protein kinase activity was through increasing GM3, but MEL had a direct inhibitory effect.

Molecular mechanisms of apoptosis have been extensively studied, but little is known about non‐apoptotic cell death. To study the mechanism of non‐apoptotic cell death, we searched for non‐apoptotic cell death inducers for U937 cells, which are highly sensitive to apoptosis induction by various stimuli. We found that 8‐nitrocaffeine and its analog, which are candidate radiosensitizers for cancer therapy, induced exclusively caspase‐independent necrotic cell death in cell lines such as U937, HL‐60, K562 and Jurkat. The 8‐nitrocaffeine‐induced necrotic cell death was mediated by reactive oxygen species (ROS) because (i) ROS were produced in the 8‐nitrocaffeine‐treated cells, (ii) ROS scavengers inhibited the caspase‐independent necrotic cell death induced by 8‐nitrocaffeine, and (iii) the necrotic cell death was completely suppressed in hypoxic cells. Cells selected for resistance to nitrocaffeine showed cross resistance to CH‐11, an anti‐Fas antibody, suggesting that the necrotic process plays an important role in Fas‐mediated cell death in this cell line. Since cancer cells are often derived from a selected population of cells resistant to apoptosis, inducers of necrotic cell death could be beneficial to kill cancer cells that have acquired resistance to apoptosis‐induction therapy.

Human promyelocytic leukemia HL-60 cells are well known to differentiate into granulocytes or monocytes in the presence of some agents such as DMSO or PMA, respectively. Differentiated HL-60 cells become resistant to some apoptotic stimuli including anticancer drugs or irradiation though undifferentiated cells significantly respond to these stimuli. TRAIL (TNF-related apoptosis-inducing ligand) which is also known as Apo2 ligand (Apo2L), a new member of TNF family, can induce apoptosis in some tumor cells but not in many normal cells. We show here that apoptosis is well induced in HL-60 cells by TRAIL, but susceptibility to TRAIL is reduced during granulocytic differentiation by DMSO. We also suggest some possible mechanisms by which granulocytic differentiated cells become resistant to TRAIL-induced apoptosis. First, in granulocytic differentiated cells, expression of antagonistic decoy receptors for TRAIL (TRAIL-R3/TRID/DcR1/LIT and TRAIL-R4/TRUNDD/DcR2) were enhanced. In addition, expression of Toso, a cell surface apoptosis regulator, seemed to block activation of caspase-8 by TRAIL via enhanced expression of FLIPL in granulocytic differentiated cells. These findings suggest that differentiated cells are resistant using plural mechanisms against various apoptosis-inducing stimuli rather than undifferentiated cells.