[P66shc action on resistance of colon carcinoma RKO cells to oxidative stress].

P66shc protein is an alternative transcript product of SHC1 gene. While two other isoforms (p52shc and p46shc) have adaptor function in RAS signaling pathway, p66shc regulates reactive oxygen species (ROS) level. P66shc genome knockout significantly extends lifespan in mice. Though p66shc was determined to translocate into mitochondria and led to increase in intracellular ROS, the mechanism by which the protein take part in signaling pathways that regulates resistance to cellular stresses remains poorly studied. P66shc has an important role in carcinogenesis and its increased expression correlates with poor prognosis in colon cancer. In this work we have applied RNA interference using lentiviral constructions that express short hairpin RNA (shRNA) against N-terminal CH2 domain of p66shc isoform. Using this approach p66 but not p52 and p46 SHC1 isoform expression was selectively suppressed in colon carcinoma RKO cells. RKO cells with p66shc knockdown have shown to be more resistant to oxidative stress induced by hydrogen peroxide or serum starvation. Fragmentation of mitochondria that depends on mitochondrial ROS accumulation during oxidative stress was significantly decreased in this cells. The data obtained are in agreement with hypothesis that p66shc participates in ROS accumulation in mitochondria and by this means promotes induction of apoptosis.

[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). In experiments on K562 cells, it was found that the subline sensitive to chemotherapy is protected by SkQRI from apoptotic action of hydrogen peroxide. Protection of the resistant subline occurs only after inhibition of P-glycoprotein.

[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). In experiments on K562 cells, it was found that the subline sensitive to chemotherapy is protected by SkQR1 from apoptotic action of hydrogen peroxide. Protection of the resistant subline occurs only after inhibition of P-glycoprotein.

[Preparation and characterization of mouse embryonic fibroblasts with K72W mutation in somatic cytochrome C gene].

Mouse embryonic fibroblasts (MEF) with point mutation in somatic cytochrome C gene were generated and characterized. It was shown that substitution of lysine for tryptophan in position 72 (K72W) decreased the proapoptotic functions of cytochrome C in response to staurosporin treatment without disrupting its respiratory functions. The presence of this mutation did not affect the pattern of cytochrome C gene expression or its localization inside the cell. These cell cultures therefore represent an interesting model for study apoptotic signaling and physiological functions of cytochrome C.

Effects of the inhibitors of dynamics of cytoskeletal structures on the development of apoptosis induced by the tumor necrosis factor.

Changes in cytoskeletal structures have been investigated during apoptosis of epithelial HeLa cells induced by tumor necrosis factor-alpha (TNF-alpha). Shape and surface cell activity were investigated by time-lapse video microscopy, and changes of the cytoskeletal structure were studied by immune fluorescent microscopy. Addition of TNF-alpha to HeLa cell culture caused early disruption of the actin cytoskeleton and vinculin-containing focal contacts, keratin filaments, and microtubules. Rounding of cells, general blebbing, and nuclear fragmentation were observed at the terminal apoptotic stages. Actomyosin complex inhibitors, H7 and HA1077, suppressed blebbing (but not cell rounding) and activated the development of apoptosis. The latter suggests that in contrast to blebbing the general rounding does not depend on increased contractility of actomyosin cortex. These cytoskeletal inhibitors accelerated the development of apoptosis of HeLa cells and increased sensitivity of HeLa-Bcl-2 cells (transfected with DNA encoding antiapoptotic protein Bcl-2) to TNF-induced apoptosis. Damage of cytoskeletal structures significantly attenuated antiapoptotic activity of Bcl-2 in the HeLa-Bcl-2 cells. It is suggested that the stimulation of apoptosis by cytoskeletal inhibitors may be attributed to the altered distribution of cell organelles, especially, mitochondria.

[Effect of protein inhibitors of mitochondrial ATPase in intact rat thymocytes and Ehrlich ascites carcinoma cells]. / Deistvie belka-ingibitora mitokhondrial'noi ATPazy v intaktnykh timotsitakh krysy i kletkakh astsitnoi kartsinomy érlikha.

It has been found that inhibition of mitochondrial ATPase in living thymocytes and Ehrlich ascites carcinoma (EAC) cells after incubation of cells with uncoupler, rotenone or cumene hydroperoxide, depends, in a large measure, on the inhibitor protein (IF1) action. Maximum inhibition (up to 70% of the oligomycin-sensitive ATPase activity) was found in the presence of the uncoupler in the incubation medium. Even when IF1 action was maximum, the residual ATPase activity caused marked ATP depletion in thymocytes, while in EAC cells other ATP-consuming processes prevailed. No inactive ATPase-IF1 complexes were found in intact thymocytes and EAC cells. The extent of inhibition of mitochondrial ATPase under oxidative stress was higher in thymocytes than in EAC cells and depended on cumene hydroperoxide concentration and duration of cell cellular ATP depletion. It is suggested that under certain experimental conditions IF1 can prevent cell death by slowing down the hydrolysis of cellular ATP.