[Response of HeLa cells to mitomycine C. III. The analysis of nucleoli of mother and daughter cells].

The comparative analysis of the number of nucleoli in cells of the established HeLa-M line was carried out before and after exposure to mitomycin C in a concentration of 10 µg/ml for 2 h. Using time-lapse microscopy, nucleoli in mother and their respective daughter cells were computed. It has been shown that the average number of nucleoli per cell is generally higher in daughter cells than in mother cells, and a standard deviation, on the contrary, decreases. An average number of nucleoli in daughter cells, whose mother cells had been treated with mitomycin C, was higher than in corresponding cells of control group. The separate analysis has been performed for the cells having from 1 to 4 nucleoli. Nonrandom complete coincidence of the number of nucleoli in mather and daughter cells has been typicaly shown for about 1/7 of the total cell population. Mitomycin C reduces this value of about 1.5 times.

[Colocalization of nucleoli in cell nuclei of HeLa line].

The pattern of localization of nucleoli relative to each other and to cell nucleus was studied in M-HeLa cell line. For this puspose, the following morphometric parameters were introduced. For the two-nucleolar cells: 1) the ratio of the nucleus long axis to the length of a segment between the centers of the nucleoli, and 2) the angle between the segment connecting the centers of the nucleoli and a longitudinal axis of cell nucleus. For the three-nucleolar cells: the ratio perimeter of the nucleus to perimeter of a triangle with vertexes in the centre of nucleoli. We have shown that the values of these parameters are individual for each cell but their values remain constant for the cell in spite of the changes in cell shape. These results allow us to conclude that, on the one hand, the nucleoli colocalization is individual for each cell, and, on the other hand, location of nucleoli in relation to nucleus is not changed during interphase. Thereby, the distance between nucleoli increases proportionally with nucleus growth.

[Morphology of NCTC cells upon a contact with type I collagen added to culture medium].

The morphometric characteristics of NCTC cells upon their contact with type I collagen added to culture medium were studied. The cells were plated on plastic in the colony form. In a day after seeding, the culture medium was changed for the same medium complemented with 0.1% type I collagen. And the cells were incubated in this medium for 30 min more. Then the cells were washed out of collagen. Using time-lapse microscopy, the cell state at a colony edge was registered during 7 h. The area, spreading, and polarization of the cells were evaluated. It is shown that the contact with collagen did not affect the cell area, decreased cell spreading and sharply reduced a portion of polarized cells. These results probably demonstrate sensitivity of NCTC cells to the presence of type I collagen in culture medium and suggest that the cell response involves inhibition of long filopodia formation.

[The functional characteristics oF TRPV5 and TRPV6 channels in normal and transformed human blood lymphocytes].

Calcium signaling and Ca(2+)-conducting channels are involved in the development of immune response, cell proliferation, growth and differentiation of lymphocytes. In this paper the calcium channels TRPV5 and TRPV6 (transient receptor potential vanilloid channels) were studied in the plasma membrane of T cell line Jurkat and normal human blood lymphocytes. The channels were activated by removing Ca2+ and Mg2+ from surrounding solution, characterized by inward rectification, and were inactivated by the effective blocker of TRPV5 and TRPV6, ruthenium red. Channel activity was significantly higher in Jurkat cells, than in normal human lymphocytes. Quantitative PCR analysis revealed higher levels of mRNA genes encoding channels TRPV5 and TRPV6 in the proliferation cells compared with resting lymphocytes. In general these data showed that TRPV5/TRPV6 in human lymphocytes are functionally active, and their activity is associated with proliferative status of blood cells.

[Response of HeLa cells to mitomycine C. II. Morphometry of the cells].

HeLa-M cells were analyzed after the 2h incubation in the medium with mitomycin C (10 µg/ml). It has been shown that a part of the cells contacted with the cytostatic agent passes mitosis normally, but the daughter cells no longer divide. During the observation period (2 days), the area of the cells increased linearly reaching twice the size of intact cells. Thereby spreading of the cells contacted with mitomycine decreased, and their polarisation was not changed. Along with the increasing cell size, the nucleus size is also increased indicated de novo protein synthesis. Analysis of nucleoli revealed a small decrease of their area during the first hours after the contact with mitomycine followed by an increase of the area. Finally, the nucleolus area exceeded that of control cells, which can only happen if rRNA was synthesized. On the other hand, DNA cross-linking by mitomycine should inhibit transcription because the mechanism of transcription is assumed to involve local melting of DNA within its minor groove. This contradiction can be overcome by assuming that transcription occurs in a DNA major groove without separation of the chains.

[Molecular and functional identification of sodium channels in K562 cells].

Modulations of ion channel activity underlie rapid changes in membrane transport of cations in various non-excitable cells. Previously, in smooth muscle cells, macrophages, lymphocytes, carcinoma and leukemia cell lines, non-voltage-gated sodium (NVGS) channels have been found. The activity of NVGS channels was shown to be critically dependent on the organization of actin cytoskeleton. The molecular identity of NVGS channels remains unclear. The present work is focused on molecular and functional identification of NVGS channels in human myeloid leukemia K562 cells. Degenerin/epithelial Na+ channels (DEG/ENaC) could be considered as a possible molecular correlates. Using RT-PCR, expression of alpha-, beta-, gamma-hENaC subunits in K562 cells was detected. Various modes of the patch-clamp method were employed to examine functional properties of sodium channels, specifically, to test the effect of amiloride on single channel and integral currents. Biophysical characteristics of NVSG channels were close to ENaC; the channels have unitary conductance 12 pS (145 mM Na+) and were impermeable for divalent cations (Ca2+ and Mg2+). We found that amiloride did not inhibit NVGS channels. Importantly, no amiloride-blockable sodium current was detected in the plasma membrane of K562 cells. Taken together, our observations suggest that amiloride-insensitive sodium channels in K562 cells belong to the ENaC family.

[The estimation of similarity in characteristics of the post-mitotic daughter L-929 cells during their migration along the substrate].

Using time-lapse microscopy, spreading of the post-mitotic daughter cells has been studied. The work was performed on non-synchronized cells of established L-929 cell line. The study was aimed to characterize the morphology of the cells as they move along the substrate and to determine whether the area of the migrating cells changes nonrandom. Two new parameters have been proposed for comparison of cell morphology: the identity indicator (II) and the synchronism indicator (SI). Time-dependent changes in the area in pairs of cells were measured to calculate these parameters. The first indicator shows the degree of coincidence between the absolute values of the area in the pair of the cells, whereas the second indicator shows synchronism of the changes in the cell areas and does not depend on their absolute values. The lower are the indicators, the higher is the similarity in the time-dependent changes in the areas of cell pairs studied. The indicators were shown to be approximately 1.5-fold lower for the pairs of the post-mitotic daughter cells than those for any other pair of the cells. The results indicate a nonrandom pattern of change in the morphology of the cells during their movement along the substrate.

[Dynamics of spreading of cells of L-929 line after the mitosis].

Using time-lapse microscopy, the changes in L-929 cells shape were analyzed during a cell cycle. During this time the cells were established to pass through three spreading stages. The highest rate of the cell spreading was observed during the first 1.5 h of mitosis. In this period, the cell area increases approximately 3-3.5 times following sigmoid dependence. After a short plateau the augmentation of the cell area starts also as a sigmoid dependence. This period is longer (up to 6 h after the beginning of cell division) with an additional 1.5-fold augmentation of the cells size. Next, the augmentation of the cells area goes linearly up to the beginning of the following mitosis. After the mother L-929 cell division, the daughter cells remained to be bridged together in the fission furrow site almost in 100% cases. The structure known as an intercellular bridge is related to a late telophase. In this connected state the L-cells are spreading and migrating up to 2.13 +/- 0.06 h where upon they are separated. Transition of the daughter cells from a round shape to the spread one occurring with the simultaneous maintenance of the intercellular bridge during a strictly determined time allows us to consider this phenomenon as independent and not relating to mitosis. We suggest naming this junction between the daughter cells as the "posttelophase intercellular bridge".

[Analysis of the cell cycle duration of cells of permanent line L-929].

The direct measurement of the cell cycle duration in L-929 cells was performed using time-lapse photography. The cell cycle duration was 15.77 +/- 0.08 h with a standard deviation of 1.54 +/- 0.06 h. The experimental value fit to a normal distribution with a correlation coefficient 0.999. High homogeneity of this parameter and a wide range of variability of the karyotype (58-66 chromosomes) indicate that there is no correlation between these characteristics of L-929 cells. It is also shown that the difference between cell cycle durations of daughter cells tent to zero and fits by an exponent.

[The average cell size as a factor reflecting the interaction of the CHO cells during process of proliferation].

Changes in the cell area during cultivation of the CHO line cells were studied using time-lapse technique (start of registration in one day after cell plating). It was established that the size of the daughter cells after mitosis remains lees than the size of the mother cell for a long time (up to 6 h). Nevertheless, the average cell area of the whole population is constant throughout the observation period (up to 18 h). We assume that this phenomenon could be a result of interaction among dividing and not-dividing cells. The experimental data confirming this conclusion are presented.

[Mechanism of epithelial sodium channel (ENaC) regulation by cortactin: involvement of dynamin].

We have recently shown that epithelial sodium channels (ENaC) are regulated by the actin-binding protein cortactin via the Arp2/3 protein complex. However, it has been also demonstrated that GTPase, dynamin, which is known to regulate clathrin-mediated endocytosis, can as well initiate signaling cascades regulated by cortactin. This study was designed to investigate the involvement of dynamin into cortactin-mediated regulation of ENaC. Initially, a recently described inhibitor of dynamin, dynasore, was used. However, use of this inhibitor seemed to be inappropriate due to discovered side effects. F. i., treatment of mpkCCD(c14) cells monolayers with dynasore (in concentrations of 10 and 100 microM) resulted in a decrease in ENaC-mediated transepithelial currents. Besides, the same concentrations of dynasore caused reduced currents in CHO cells transfected with ENaC subunits. Therefore, the data demonstrated that dynasore down regulates both native and overexpressed channel's activity and is not suitable for studies of a role of dynamin in the clathrin-mediated endocytosis of ENaC. This effect is most likely caused either by dynasore's toxic effect upon the cells or by enhanced endocytosis of ENaC-activating proteins. In the following experiments designed to study the role of dynamin different plasmids encoding mutant forms of dynamin and cortactin were used. Dominant negative dynamin K44A transfected into CHO cells together with ENaC subunits significantly increased amiloride-sensitive current density compared to cells transfected with ENaC subunits only (control); additional transfection of cortactin in this system resulted in current density restitution back to the control level. Moreover, ENaC overexpression with the SH3 domain of cortactin, which is responsible for dynamin binding, caused a decrease if ENaC current. Thus, we have shown in this study that cortactin can mediate ENaC activity not only via the Arp2/3 complex, but apart from that dynamin and related processes also might be involved into ENaC regulation by cortactin.

[The position of cleavage furrow in cultivated cells on the example of lines L-929 and CHO].

The aim of the study carried out on cells of lines L-929 (NCTC a clone 929) and CHO was to examine whether the position of cleavage furrow is random or not. CHO cells were seeded in the traditional way (evenly across the surface of a Petri dish), and L-cell were put as colonies to monitor the migration of cells emanating from them. The time-lapse filming (imaging) was used for registration of behavior of cells. Analyzing the obtained images of cells, the angle between the axis of polarization of a cell and its cleavage furrow was measured. Besides, the angle between an axis of cell polarization or its cleavage furrow and the horizontal axis of image field was measured. It was shown that position of CHO cells in a dish plane was random as well as the value of the angle between the axis of polarization of these cells and their cleavage furrows. The L-929 cells migrating from colony were orientated so, that their polarization axis was directed to the colony center, and the cleavage furrow was perpendicular with this axis. Assumptions about nonrandom position of both cultivated cells during mitosis and their cleavage furrows were made.

[Functional properties of sodium channels in cholesterol-depleted K562 cells].

The level of cellular cholesterol is known to determine functional compartmentalization of membrane lipids into ordered microdomains (rafts). Lipid rafts are assumed to play an essential role in the interactions between cell membrane and cortical cytoskeleton. As we have shown earlier, the activity of non-voltage-gated sodium channels in K562 human leukaemia cells is critically dependent on actin cytoskeleton organization. In the present paper, functional properties of sodium channels in K562 cells were examined after cholesterol-depleting treatment using methyl-beta-cyclodextrin (MbCD), selective acceptor of sterols. Single currents through sodium channels were recorded in cell-attached and inside-out mode experiments with the use of patch clamp technique. After incubation with MbCD (2.5 or 5.0 mM), an activation of sodium channels in response to cytochalasin B or D was observed in membrane fragments as well as in native cells. Characteristics of the channels in cholesterol-depleted K562 cells were similar to those in control; unitary conductance was 12 pS. Inside-out experiments with the use of globular actin have indicated that filament assembly on cytoplasmic membrane side causes an inactivation of sodium channels. These data imply that there is no association of sodium channels with cholesterol-rich membrane microdomains in K562 cells. Possible mechanisms underlying an interplay between plasma membrane and cortical cytoskeleton are discussed.

[TRPV5 and TRPV6 calcium channels in human T cells].

The recent cloning of the special calcium channels TRPV5 and TRPV6 (transient receptor potential vanilloid channels) provided the molecular base for the studying of new candidate of calcium influx in non-excitable cells. Using RT-PCR technique we obtained endogenous expression of the mRNAs trpv5 and trpv6 in lymphoblast leukemia Jurkat cells and in human blood primary T lymphocytes. Additionally, Western blot analysis showed TRPV5 proteins in both the whole lysate and in the crude membrane preparations from Jurkat cells and normal T lymphocytes. The using of the immunoprecipitation revealed TRPV6 proteins in Jurkat cells, whereas in normal T lymphocytes TRPV6 was not detected. The expression pattern and the selective Ca2+ permeation properties of TRPV5 and TRPV6 channels indicate an important role of these channels in the Ca2+ homeostasis and probably in malignant transformation of blood cells.

[Inhibiting and stimulating effect of amiloride on potential-dependent cation channels in K562 cells]. / Ingibiruiushchee i stimuliruiushchee deistvie amilorida na potentsialnezavisimye kationnye kanaly v kletkakh K562.

Non-voltage-gated ion channels play an essential role in cellular signalling and ionic homeostasis in nonexcitable cells. The patch clamp method in cell-attached configuration was used to search for the effects of amiloride and gadolinium (Gd3+) exerted on two types of voltage-insensitive cationic channels in plasma membrane of human leukemia K562 cells: Na-selective channels activated by actin disassembly, and mechanosensitive channels. Here we demonstrate that amiloride in high concentrations (1 mM) caused a full inhibition of mechanosensitive channels in K562 cells similarly to Gd3+ effect in micromolecular concentration range. Na-selective channels controlled by actin dynamics were shown to be unaffected by Gd3+ similarly as by amiloride. We also found that application of amiloride to the extracellular surface of membrane patch resulted in a significant increase in the activity of sodium channels. This unexpected stimulatory effect of amiloride may represent an unknown mechanism of activation of non-voltage-gated sodium channels. The data show an essential difference of the activation and blockage of these types of cation-selective channels.

[Store-operated cationic channels in human myeloid leukaemia K562 cells]. / Depozavisimye kationnye kanaly v kletkakh mieloidnoi leikemii cheloveka K562.

Using the whole-cell patch clamp technique, single channels operated by intracellular Ca(2+)-store depletion were first revealed in human myeloid leukaemia cells K562. A single store-operated channel could be detected in divalent-free extracellular solutions with Na+ as a permeant ion, and intracellular solutions with strong Ca(2+)-helating agent with some delay after whole-cell formation. Addition of inositol-1,4,5-triphosphate to the pipette solution resulted in a significant decrease of this latency. These channels had a conductance of 29 pS, and were inhibited by low concentration of external Ca2+. Our results enable us to assume that the revealed channels are calcium release-activated calcium channels, operated by Ca2+ depletion of endoplasmic reticulum.

[Activation of mechanosensitive ion channels in plasma membrane of K562 cells]. / Aktivatsiia mekhanochuvstvitel'nykh ionnykh kanalov v plazmaticheskoi membrane kletok K562.

Patch clamp method in cell-attached configuration was used to search for mechanogated ion channels in plasma membrane of human myeloid leukemia K562 cells. A reversible activation of transmembrane currents in response to negative pressure applied to membrane patch was observed. Four types of mechanosensitive channels were identified in K562 cells: two main types were characterized with conductance values of 16 and 25 pS; while two others, showing higher conductance values (about 35 and 50 pS), were rarely met. In terms of gating, all channels described here could be assigned to the stretch-activated type. No inactivation of mechanosensitive channels at the sustained stimulation was observed. The activation of mechanosensitive channels in K562 cells was not dependent upon the presence of bivalent cations in the extracellular solution.

[Functional characteristic and molecular topology of voltage independent sodium channels]. / Funktsional'naia kharakteristika i molekuliarnaia topologiia potentsialnezavisimykh natrievykh kanalov.

The channel proteins so far known are transmembrane oligomers arranged in a manner that the polar residues are lining the central ion-conducting hydrophilic pore. In the last decade, electrophysiology and molecular biology studies revealed the principal similarity in the functional properties and membrane topology within a large family of sodium-conducting channels. Amiloride-sensitive channels are expressed in the apical membranes of renal epithelia. Moreover, in different mammalian cells non-voltage-gated sodium-selective channels have been recently found. According to molecular cloning of the respective DNAs and amino acid sequence analysis, epithelial channel subunits, degenerins and some other channel proteins display a significant homology in the regions forming two presumable transmembrane domains. This paper reviews some relevant data and current opinions of the superfamily of sodium-conducting cation channels.

[Functional properties and cytoskeletal-dependent regulation of sodium channels in leukemia cell membranes]. / Funktsional'nye svoistva i tsitoskeletzavisimaia reguliatsiia natrievykh kanalov v plazmaticheskoi membrane leikoznykh kletok.

The paper is devoted to membrane mechanisms of sodium influx from the extracellular medium to the cytoplasm in nonexcitable cells. With the use of patch clamp technique, the activity of non-voltage-gated ionic channels in plasma membrane of human leukemia K562 cells was examined. We have identified two types of Na-permeable channels characterized by unitary conductance of 12 pS and differing in their selectivity among monovalent cations. A relative permeability value PNa/PK was estimated for both types referred to as channels of high (HS, PNa/PK = 10) and low (LS, PNa/PK = 3) selectivity, resp. Both the channels were impermeable to bivalent cations (Ca2+, Ba2+), not blocked by tetradotoxin. Their sensitivity to amiloride was extremely low. Cytochalasin D treatment of cells resulted in a significant increase in the activity of LS Na-conducting channels. Application of exogenous gelsolin to the cytoplasmic surface of inside-out membrane patch at free Ca2+ level of 1 mkM induced a similar effect of sodium channel activation; the subsequent addition of actin reduced the channel activity up to the background level. Our results show that the cortical F-actin network plays an important role in regulating the novel family of sodium channels in nonexcitable cells. It could be assumed that the actin disassembly causes a rise in LS channel activity, whereas the actin assembly induces inactivation of the channels.