Studying Signaling Pathway Activation in TRAIL-Resistant Macrophage-Like Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is a malignant neoplasm characterized by extremely low curability and survival. The inflammatory microenvironment and maturation (differentiation) of AML cells induced by it contribute to the evasion of these cells from effectors of antitumor immunity. One of the key molecular effectors of immune surveillance, the cytokine TRAIL, is considered a promising platform for developing selective anticancer drugs.

Pro-Inflammatory Activation Suppresses TRAIL-induced Apoptosis of Acute Myeloid Leukemia Cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) is a promising agent for treatment of AML due to its specific apoptosis-inducing effect on tumor cells but not normal cells. However, emergence of resistance to TRAIL in the AML cells limits its potential as an antileukemic agent. Previously, we revealed increase in the resistance of the human AML THP-1 cells to the TRAIL-induced death during their LPS-dependent proinflammatory activation and in the in vitro model of LPS-independent proinflammatory activation - in a long-term high-density cell culture.

Multimodal nonlinear-optical imaging of nucleoli.

Multimodal nonlinear microscopy combining third-harmonic generation (THG) with two- and three-photon-excited fluorescence (2PEF and 3PEF) is shown to provide a powerful resource for high-fidelity imaging of nucleoli and nucleolar proteins. We demonstrate that, with a suitably tailored genetically encoded fluorescent stain, the 2PEF/3PEF readout from specific nucleolar proteins can be reliably detected against the extranucleolar 2PEF/3PEF signal, enabling high-contrast imaging of the key nucleolar ribosome biogenesis components, such as fibrillarin. THG is shown to provide a versatile readout for unstained nucleolus imaging in a vast class of biological systems as different as neurons in brain slices and cultured HeLa cells.

Drug Screening with Genetically Encoded Fluorescent Sensors: Today and Tomorrow.

Genetically-encoded fluorescent sensors have been actively developed over the last few decades and used in live imaging and drug screening. Real-time monitoring of drug action in a specific cellular compartment, organ, or tissue type; the ability to screen at the single-cell resolution; and the elimination of false-positive results caused by low drug bioavailability that is not detected by in vitro testing methods are a few of the obvious benefits of using genetically-encoded fluorescent sensors in drug screening. In combination with high-throughput screening (HTS), some genetically-encoded fluorescent sensors may provide high reproducibility and robustness to assays.

Enhanced-contrast two-photon optogenetic pH sensing and pH-resolved brain imaging.

We present experiments on cell cultures and brain slices that demonstrate two-photon optogenetic pH sensing and pH-resolved brain imaging using a laser driver whose spectrum is carefully tailored to provide the maximum contrast of a ratiometric two-photon fluorescence readout from a high-brightness genetically encoded yellow-fluorescent-protein-based sensor, SypHer3s. Two spectrally isolated components of this laser field are set to induce two-photon-excited fluorescence (2PEF) by driving SypHer3s through one of two excitation pathways-via either the protonated or deprotonated states of its chromophore. With the spectrum of the laser field accurately adjusted for a maximum contrast of these two 2PEF signals, the ratio of their intensities is shown to provide a remarkably broad dynamic range for pH measurements, enabling high-contrast optogenetic deep-brain pH sensing and pH-resolved 2PEF imaging within a vast class of biological systems, ranging from cell cultures to the living brain.

Genetically Encoded Tools for Research of Cell Signaling and Metabolism under Brain Hypoxia.

Hypoxia is characterized by low oxygen content in the tissues. The central nervous system (CNS) is highly vulnerable to a lack of oxygen. Prolonged hypoxia leads to the death of brain cells, which underlies the development of many pathological conditions.

Therapeutic Effect of the Mitochondria-Targeted Antioxidant SkQ1 on the Culture Model of Multiple Sclerosis.

Multiple sclerosis (MS) is a heterogeneous autoimmune disease of unknown etiology characterized by inflammation, demyelination, and axonal degeneration that affects both the white and gray matter of CNS. Recent large-scale epidemiological and genomic studies identified several genetic and environmental risk factors for the disease. Among them are environmental factors of infectious origin, possibly causing MS, which include Epstein-Barr virus infection, reactivation of some endogenous retrovirus groups, and infection by pathogenic bacteria (mycobacteria, , and ).

Mitochondria-targeted Antioxidants as a Prospective Therapeutic Strategy for Multiple Sclerosis.

Background: Multiple sclerosis (MS) is one of the most widespread chronic neurological diseases that manifests itself by progressive demyelination in the central nervous system. The study of MS pathogenesis begins with the onset of the relapsing-remitting phase of the disease, which becomes apparent due to microglia activation, neuroinflammation and demyelination/ remyelination in the white matter. The following progressive phase is accompanied by severe neurological symptoms when demyelination and neurodegeneration are spread to both gray and white matter.

[Brugada Syndrome: Possibilities of the Use of Noninvasive Electrocardiographic Mapping. A Case Report].

Patient P., 50 years old, male, with type I Brugada syndrome was examined. The patient had aborted sudden death event (2006) in his clinical history, ICD Gem III VR was implanted in 2006, ICDLumax DR was reimplanted in 2012.

[Diagnosis and treatment for Paget's breast cancer: state of the art].

Breast cancer is the most common cancer in women. According to WHO experts in the world each year there are revealed from 800,000 up to 1 million new cases of breast cancer. In the structure of malignant tumors morbidity among female population, in Russia in 2012 breast cancer ranked the first place (20.

Radioprotective effects of mitochondria-targeted antioxidant SkQR1.

We show here that mitochondria-targeted antioxidant composed of plastoquinone conjugated through hydrocarbon linker with cationic rhodamine 19 (SkQR1) protected against nuclear DNA damage induced by gamma radiation in K562 erythroleukemia cells. We also demonstrate that SkQR1 prevented the early (1 h postirradiation) accumulation of phosphorylated histone H2AX (γ-H2AX) an indicator of DNA double-strand break formation, as well as the radiation-induced increase in chromosomal aberrations. These data suggested that nuclear DNA damage induced by gamma radiation may be mediated by mitochondrial reactive oxygen species (ROS) production.

[Mitochondria-targeted antioxidant SkQR1 selectively protects MDR-negative cells against ionizing radiation].

Radioprotection appeared to be an important problem of today due to atom energetic development and utilization of radiation material in the industry, science and medicine. It has been shown that mitochondrial targeted antioxidant SkQR1 could attenuate radiation injury of human erythroleukemia K562 cells. Pretreatment with SkQR1 before irradiation decreased DNA double strand breaks formation, diminished the number of chromosomal aberrations and suppressed delayed ROS production.

In search of novel highly active mitochondria-targeted antioxidants: thymoquinone and its cationic derivatives.

Since the times of the Bible, an extract of black cumin seeds was used as a medicine to treat many human pathologies. Thymoquinone (2-demethylplastoquinone derivative) was identified as an active antioxidant component of this extract. Recently, it was shown that conjugates of plastoquinone and penetrating cations are potent mitochondria-targeted antioxidants effective in treating a large number of age-related pathologies.

[Multidrug resistance P-glycoprotein inhibits the antiapoptotic action of mitochondria-targeted antioxidant SkQR1].

Mitochondria-targeted antioxidants of the SkQR1 family, being accumulated in energized mitochondria, protect cells from oxidative stress by increasing the level of reduced glutathione and decreasing the cell-damaging effect induced by hydrogen peroxide. Using various human transformed cell lines and SkQR1 (a fluorescent member of the SkQ family), we show that SkQRI is ejected from chemotherapy-resistant cells by P-glycoprotein - one of the main transport proteins determining multidrug resistance typical for many neoplastic cells. It is also shown that SkQR1 ejection is neutralized by P-glycoprotein inhibitors (verapamil and pluronic L61).

[Multidrug resistance p-glycoprotein inhibits the antiapoptotic action of mitochondria-targeted antioxidant SkQR1].

Mitochondria-targeted antioxidants of the SkQRI family, being accumulated in energized mitochondria, protect cells from oxidative stress by increasing the level of reduced glutathione and decreasing the cell-damaging effect induced by hydrogen peroxide. Using various human transformed cell lines and SkQR1 (a fluorescent member of the SkQ family), we show that SkQR1 is ejected from chemotherapy-resistant cells by P-glycoprotein--one of the main transport proteins determining multidrug resistance typical for many neoplastic cells. It is also shown that SkQR1 ejection is neutralized by P-glycoprotein inhibitors (verapamil and pluronic L61).

Mitochondria-targeted antioxidant SkQR1 selectively protects MDR (Pgp 170)-negative cells against oxidative stress.

A conjugate of plastoquinone with decylrhodamine 19 (SkQR1) selectively accumulates in mitochondria of normal and tumor cells. SkQR1 protected the cellular pool of reduced glutathione under oxidative stress. Overexpression of P-glycoprotein (Pgp 170) multidrug resistance pump strongly suppresses accumulation of SkQR1.

Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 3. Inhibitory effect of SkQ1 on tumor development from p53-deficient cells.

It was proposed that increased level of mitochondrial reactive oxygen species (ROS), mediating execution of the aging program of an organism, could also be critical for neoplastic transformation and tumorigenesis. This proposal was addressed using new mitochondria-targeted antioxidant SkQ1 (10-(6'-plastoquinonyl) decyltriphenylphosphonium) that scavenges ROS in mitochondria at nanomolar concentrations. We found that diet supplementation with SkQ1 (5 nmol/kg per day) suppressed spontaneous development of tumors (predominantly lymphomas) in p53(-/-) mice.

Hydrogen peroxide produced inside mitochondria takes part in cell-to-cell transmission of apoptotic signal.

In monolayer of HeLa cells treated with tumor necrosis factor (TNF), apoptotic cells formed clusters indicating possible transmission of apoptotic signal via the culture media. To investigate this phenomenon, a simple method of enabling two cell cultures to interact has been employed. Two coverslips were placed side by side in a Petri dish, one coverslip covered with apoptogen-treated cells (the inducer) and another with non-treated cells (the recipient).

Marginal blebbing during the early stages of TNF-induced apoptosis indicates alteration in actomyosin contractility.

Dynamics of alterations of cell surface topography during TNF-induced apoptosis of HeLa cells was examined by phase-contrast videomicroscopy and immunomorphological analysis. The final stage of apoptosis accompanied by cell rounding and general blebbing of the cell surface became after 4-6 h of incubation but much earlier, after 1.5-3 h, essentially flattened lamellipodia at the active edges transformed into the small blebs that were continuously extended and retracted during the next 1-2 h.

Oligomycin, inhibitor of the F0 part of H+-ATP-synthase, suppresses the TNF-induced apoptosis.

The release of cytochrome c from the intermembrane space of mitochondria into the cytosol is one of the critical events in apoptotic cell death. In the present study, it is shown that release of cytochrome c and apoptosis induced by tumor necrosis factor alpha (TNF) in HeLa cells can be inhibited by (i) overexpression of an oncoprotein Bcl-2, (ii) Cyclosporin A, an inhibitor of the mitochondrial permeability transition pore (PTP) or (iii) oligomycin, an inhibitor of H+- ATP-synthase. Staurosporine-induced apoptosis is sensitive to Bcl-2 but insensitive to Cyclosporin A and oligomycin.

[Cytoskeletal mechanisms of heterocellular contact formation].

We studied the interaction of two main cell types, epitheliocytes and fibroblasts, in a mixed culture. Heterotypic cells had a different cytoskeleton organization and expressed different cell adhesion molecules, cadherins. In spite of this, when the cells contacted in the mixed cultures, a heterophilic contact was formed and the actin cytoskeleton of an epitheliocyte at the site of contact was reorganized: the marginal actin bundle was decomposed and actin structures were formed in its place, that were typical for the fibroblast lamella.

[Mode of epithelium-fibroblast interaction in mixed heterotypic cell cultures].

The interaction between epithelium (dog kidney epithelium MDCJ/clone 20) and fibroblasts (diploid human fibroblasts M19 and AG-1523) was studied in mixed heterotypic cell cultures. The mode of cell interaction depends on the manner of their collision. At collision of the epithelium lamella and the lateral side of fibroblast, the lamella was seen to creep under the lateral side to force back the fibroblast.