ADAPTOR PROTEIN Ruk/CIN85REGULATES REDOX BALANCE IN 4T1MOUSE BREAST CANCER CELLS EXPOSED TO PLASMIN(OGEN).

BACKGROUND: Cell surface plasmin is involved in tumor growth and metastatic dissemination by regulating cancer cells adhesion, migration and invasion. Plasmin-induced cell detachment is accompanied by an increased rate of reactive oxygen species (ROS) generation and cell death. However, cancer cells acquire the ability to develop adaptive mechanisms to resist ROS-mediated apoptosis. AIM: To establish the role of adaptor protein Ruk/CIN85 in the control of viability and redox balance in breast adenocarcinoma cells exposed to plasmin(ogen). MATERIALS AND METHODS: Mouse 4T1 cells with the stable overexpression of adaptor protein Ruk/CIN85 (RukUp subline) and corresponding control (Mock subline) were treated with Glu-plasminogen (1-100 nM). Plasminogen to plasmin conversion was monitored spectrophotometrically by cleavage of the specific chromogenic substrate S2251. Specific uPA inhibitor BC11 was used to verify the uPA-mediated mechanism of plasminogen pericellular activation by 4T1 cells. Cell survival rate was assessed by MTT-test and cell proliferation was estimated by colony formation assay. Enzymatic activities of catalase, glutathione peroxidase, superoxide dismutase, as well as hydrogen peroxide (H2O2) levels were measured by spectrophotomertric and fluorometric assays. The intracellular ROS generation was monitored by flow cytometry using H2DCF-DA fluorescent probe. RESULTS: Plasminogen was shown to be converted into an active proteinase plasmin on the surface of carcinoma cells in uPA-dependent manner. Plasmin(ogen) suppressed proliferation and affected survival of both studied 4T1 sublines. However, RukUp cells displayed higher resistance to plasmin(ogen)-induced cytotoxicity than Mock cells. Plasmin(ogen) promoted significant elevation in ROS generation rate in cells with the basal level of Ruk/CIN85 expression. In contrast, RukUp cells appear to be more effective in counteracting prooxidant changes due to the activation of some enzymes of the glutathione system, in particular glutathione peroxidase, and a concomitant decrease of H2O2 accumulation. CONCLUSION: Adaptor protein Ruk/CIN85 is involved in the regulation of redox homeostasis in cancer cells to maintain levels of ROS, thus promoting redox adaptation in cancer cells exposed to plasmin(ogen). Thus, Ruk/CIN85 may represent one of the relevant targets in order to diminish the resistance of cancer cells to ROS-mediated apoptosis.

Photocytotoxic effect of C60 fullerene against L1210 leukemic cells is accompanied by enhanced nitric oxide production and p38 MAPK activation.

AIM: To estimate the combined action of C60 fullerene and light irradiation on viability of L1210 leukemic cells, nitric oxide (NO) generation, p38 mitogen-activated protein kinase (MAPK) activity and cell cycle distribution. METHODS: Cell viability was assessed by MTT test. Light-emitting diode lamp (λ = 410-700 nm, 2.45 J/cm(2) ) was used for C60 fullerene photoexcitation. Nitrite level and NO-synthase activity were measured by Griess reaction and by conversion of L-arginine to L-citrulline, respectively. p38 MAPK activity was assessed by Western blot analysis. Cell cycle distribution was determined by flow cytometry. RESULTS: It was shown that light irradiation of C60 fullerene-treated L1210 cells was accompanied by 55% decrease of their viability at 48 h of culture. Nitrite level measured as an index of reactive NO generation was increased at the early period after C60 fullerene photoexcitation due to activation of both constitutive and inducible NO-synthase isoforms. The simultaneous activation of p38 MAPK was detected. Accumulation of L1210 cells in sub-G1 phase of cell cycle was observed after C60 fullerene photoexcitation. CONCLUSION: Photoexcited C60 fullerene exerts cytotoxic effect, at least in part, through triggering production of reactive NO species and activation of p38 kinase apoptotic pathways in L1210 leukemic cells.

Transcriptional regulation of NOX genes express ion in human breast adenocarcinoma MCF-7 cells is modulated by adaptor protein Ruk/CIN 85.

NADPH oxidases are key components of redox-dependent signaling networks involved in the control of cancer cell proliferation, survival and invasion. The data have been accumulated that demonstrate specific expression patterns and levels of NADPH oxidase homologues (NOXs) and accessory genes in human cancer cell lines and primary tumors as well as modulation of these parameters by extracellular cues. Our previous studies revealed that ROS production by human colorectal adenocarcinoma HT-29 cells is positively correlated with adaptor protein Ruk/CIN85 expression while increased levels of Ruk/CIN85 in weakly invasive human breast adenocarcinoma MC F-7 cells contribute to their malignant phenotype through the constitutive activation of Src/Akt pathway. In this study, to investigate whether overexpression of Ruk/CIN85 in MC F-7 cells can influence transcriptional regulation of NOXs genes, the subclones of MCF-7 cells with different levels of Ruk/CIN85 were screened for NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1 and DUOX2 as well as for regulatory subunit p22Phox mRNA contents by quantitative RT-PCR (qPCR). Systemic multidirectional changes in mRNA levels for NOX1, NOX2, NOX5, DUOX2 and p22Phox were revealed in Ruk/CIN85 overexpressing cells in comparison to control WT cells. Knocking down of Ruk/CIN85 using technology of RNA-interference resulted in the reversion of these changes. Further studies are necessary to elucidate, by which molecular mechanisms Ruk/CIN85 could affect transcriptional regulation of NOXs genes.

Multiple molecular forms of adaptor protein Ruk/CIN85 specifically associate with different subcellular compartments in human breast adenocarcinoma MCF-7 cells.

Ruk/CIN85 is a receptor-proximal 'signalling' adaptor that possesses three SH3 domains, Pro- and Ser-rich regions and C-terminal coiled-coil domain. It employs distinct domains and motifs to act as a transducer platform in intracellular signaling. Based on cDNA analysis, various isoforms of Ruk/CIN85 with different combination of protein-protein interaction domains as well as additional Ruk/CIN85 forms that are the products of post-translational modifications have been demonstrated. Nevertheless, there is no precise information regarding both the subcellular distribution and the role of Ruk/CIN85 multiple molecular forms in cellular responses. Using MCF-7 human breast adenocarcinoma cells and cell fractionation technique, specific association of Ruk/CIN85 molecular forms with different subcellular compartments was demonstrated. Induction of apoptosis of MCF-7 cells by doxorubicin treatment or by serum deprivation resulted in the system changes of Ruk/CIN85 molecular forms intracellular localization as well as their ratio. The data obtained provide a new insight into potential physiological significance of Ruk/CIN85 molecular forms in the regulation of various cellular functions.

Photoactivated fullerene C60 induces store-operated Ca2 entry and cytochrome c release in Jurkat cells.

The values of endoplasmic reticulum Ca(2+)-pool and store-operated Ca2+ entry (SOCE) were estimated in rat thymocytes and Jurkat cells loaded with indo-1 and treated with thapsigargin. It was shown that the relative value of SOCE in thymocytes was substantially lower than in Jurkat cells. Significant increase of SOCE in Jurkat cells preincubated with 10(-5) M C60 and exposed to uv/visible light irradiation was detected at 1-3 h after exposure. At this time FCCP-induced Ca(2+)-release from mitochondria was shown to be reduced, while cytochrome c level into the cytoplasm of Jurkat cells, detected by Western blot analysis, to be increased. It is supposed that Ca2+ flux remodulation induced by photoexcited fullerene C60 in Jurkat cells might be involved in the initiation of signalling events leading to cell apoptosis.

Apoptosis photoinduction by C60 fullerene in human leukemic T cells.

The viability of normal (Wistar rat thymocytes) and transformed (human leukemia Jurkat cells) T cells after UV/Vis irradiation in the presence of pristine C60 fullerene was studied. The data obtained have shown that C60 fullerene exhibits cytotoxic effect against transformed T lymphocytes when combined with UV/Vis irradiation using mercury-vapor lamp (320-600 nm). C60 fullerene photocytotoxicity was not detected in thymocytes. C60-dependent photoinduced apoptosis of Jurkat cells was confirmed by DNA fragmentation and caspase-3 activation. No substantial increase of caspase-3 activation was observed in thymocytes treated with C60 fullerene plus irradiation, while antileukemic agent cytosine arabinoside was shown to induce caspase-3 activation both in Jurkat cells and thymocytes. The data obtained may be useful for development of photosensitizers for photodynamic therapy with selective action on leukemia cells.

[Changes in functional state of the low-molecular G-proteins of platelets in type 1 diabetes mellitus]. / Zminy funktsional'noho stanu nyz'komolekuliarnykh G-bilkiv trombotdytiv za tsukrovoho diabetu 1-ho typu.

Platelets from healthy donors and insuline dependent patients with type 1 diabetes mellitus were examined for proteins specifically interacting in vitro with GST-fused constitutively active (Val12) forms of small GTPases of Rac, Rho and Cdc42. Differential changes in pattern of proteins which bind to these G-proteins in diabetic platelets have been revealed. Obtained results suggest that signalling pathways mediated by Rho GTPases are altered under type I diabetes mellitus. Such changes of actin cytoskeleton regulation may contribute to the higher level of platelet aggregation, which prove to be the etiological background of the late diabetes complications.

Adaptor protein Ruk1 forms protein-protein complexes with endonuclease activity in HEK293 cells.

The structural and functional organization of the adaptor protein Ruk(1) is characterized by the presence of three SH3-domains at the N-terminus followed by Pro- and Ser-rich sequences and a C-terminal coiled-coil region. Multiple modules in the Ruk(1) structure involved in protein-protein interactions can provide for formation of ligand clusters with varied properties and subcellular location. To study the nature and biological role of such complexes, the recombinant protein Ruk(1) with a Glu-epitope at the C-terminus (Ruk(1) Glu-tagged) was purified from transfected HEK293 cells by affinity chromatography on protein G-Sepharose with covalently conjugated anti-Glu-tag antibodies. By SDS polyacrylamide gel electrophoresis with subsequent staining with silver, a set of minor bands in addition to the 85-kD Ruk(1) Glu-tagged was detected in the purified preparation of the recombinant protein. Proteins with affinity for nucleic acids were also revealed in the Ruk(1) Glu-tagged preparation by retardation of electrophoretic mobility of 32P-labeled oligodeoxyribonucleotides in gel. The Ruk(1) Glu-tagged preparation was also shown to hydrolyze both deoxyribonucleotides and plasmid DNA. ZnCl2 and heparin inhibited the DNAse activity. These findings suggest the presence of DNases associated with the Ruk(1) protein in HEK293 cells. Such complexes were isolated from lysates of HEK293 cells by chromatography on heparin-Sepharose. By elution with 0.5 and 1.0 M NaCl, two fractions with DNase activity and containing proteins with molecular weights of 83, 80, and 72 kD were obtained. The reaction was inhibited by ZnCl2 and heparin, and previous precipitation of Ruk-related proteins with anti-Ruk antibodies resulted in the exhaustion of nuclease activity. By immunoblotting with anti-Ruk antibodies, 83-kD protein immunologically related to the Ruk(1) protein was identified in the fractions. It was concluded that the adaptor protein Ruk(1) forms complexes with endonucleases in HEK293 cells.

[Method of detection of protein kinase activity in polyacrylamide gel using two-dimensional protein separating system]. / Metod vyznachennia proteïnkinaznoï aktyvnosti v poliakrylamidnomu heli z vykorystanniam systemy dvovymirnoho rozdilennia bilkiv.

Method for detection of protein kinase activity in polyacrylamide gel have been developed. After separation of proteins by isoelectric focusing in non-denaturing condition, gel was incubated in a reaction buffer containing [gamma-32P]ATP. 32P-labeled proteins were separated by subsequent SDS/PAGE electrophoresis in second dimension. The proposed method was used for detection of protein kinase activity in human blood serum and triton X-100 soluble proteins of heads of Drosophila melanogaster.

[Study of expression of desoxyribonucleases in mammalian cells by a protein renaturation method in polyacrylamide gel]. / Doslidzhennia ekspresiï dezoksyrybonukleaz u klitynakh ssavtsiv metodom renaturatsiï bilkiv u poliakrylamidnomu heli.

Analysis of enzymatic activity in polyacrylamide gel is based on highly effective separation of proteins by SDS-electrophoresis with their subsequent renaturation and detection of enzymatic activity. This method was used to study an expression of DNAases in culturing of cells HEK293, NIH 3T3, U937. We have found that in HEK293 cells the nucleases with molecular weights 47 and 45 kDa were expressed. The localization of DNAases in the cell nuclei was shown as well. Induction of apoptosis in HEK293 cells increase the level of p47 DNAase and causes the expression of novel 50 kDa DNAase. We suggested that those discovered DNAases could take part in apoptotic DNA degradation.

[Involvement of regulating the phosphatidylinositol 3-kinase signaling pathway in the process of herbimycin A-induced erythroid cell differentiation in the K562 erythroleukemia cell line]. / Vyvchennia rehuliatsiï fosfatydylinozytol 3-kinaznoho syhnal'noho shliakhu v protsesi herbimitsyn A-indukovanoï erytroïdnoï dyferentsiatsiï klityn erytroleikemiï liniï K562.

The changes in protein-protein interactions mediated by SH3 domain of the regulatory p85 alpha subunit of phosphatidylinositol 3-kinase (Pl 3-kinase) in the course of herbimycin A-induced erythroid differentiation of the human erythroleukemia cell line K562 have been analyzed. Binding assay was performed in vitro using a recombinant form of SH3 domain of p85 alpha, conjugated with glutathione-S-transferase. pTyr-containing 210, 116, 52 and 46 kDa proteins, binding of which are modulated in differentiated cells, were identified and binding dynamics was analysed. The obtained data on binding the specific pTyr-containing proteins with regulatory subunit of Pl 3-kinase testify about the coordinated control of Pl 3-kinase signalling pathway in the course of herbimycin A-induced K562 cells differentiation.

Negative regulation of PI 3-kinase by Ruk, a novel adaptor protein.

Class I(A) phosphatidylinositol 3-kinase (PI 3-kinase) is a key component of important intracellular signalling cascades. We have identified an adaptor protein, Ruk(l), which forms complexes with the PI 3-kinase holoenzyme in vitro and in vivo. This interaction involves the proline-rich region of Ruk and the SH3 domain of the p85 alpha regulatory subunit of the class I(A) PI 3-kinase. In contrast to many other adaptor proteins that activate PI 3-kinase, interaction with Ruk(l) substantially inhibits the lipid kinase activity of the enzyme. Overexpression of Ruk(l) in cultured primary neurons induces apoptosis, an effect that could be reversed by co-expression of constitutively activated forms of the p110 alpha catalytic subunit of PI 3-kinase or its downstream effector PKB/Akt. Our data provide evidence for the existence of a negative regulator of the PI 3-kinase signalling pathway that is essential for maintaining cellular homeostasis. Structural similarities between Ruk, CIN85 and CD2AP/CMS suggest that these proteins form a novel family of adaptor molecules that are involved in various intracellular signalling pathways.

[Features of fibronectin-dependent activation of ribosomal protein S6 kinase (S6K1 and S6K2)]. / Osoblyvosti fibronektynzalezhnoï aktivatsiï kinaz rybosomnoho bilka S6 (S6K1 i S6K2).

Integrin family of adhesion receptors play an important role in organizing the actin cytoskeleton and in signal transduction from the extracellular matrix. The previous studies have shown that exposure of fibroblast cells to extracellular matrix proteins activates ribosomal S6 kinase 1 (S6K1) pathway in a ligand dependent manner. Recently, a new, highly homologous ribosomal S6 kinase, termed S6K2, was identified. It has 70% amino acid identity in the overall sequence with S6K1, and the potential phosphorylation sites of S6K1 are conserved in S6K2. However, the N- and C-terminal domains of S6K2 are quite different from those of S6K1. In this study we have examined dynamics of fibronectin-induced activation of these two kinases, transiently expressed in human HEK 293 cells. Differences between profiles of activation of S6K1 and S6K2 were observed in the early period of fibronectin stimulation. Fibronectin-induced changes in S6K2 activity were closely correlated with phosphorylation at Ser423, which is homologues to Ser 434 of S6K1. Although we didn't observe considerable changes in phosphorylation of S6K1 at Ser434, suggesting potential differences in the regulation of these homologous kinases upon fibronectin stimulation.

[Change in the level of sphingosine in rat liver nuclei and cells during oncogene superexpression induced by cycloheximide]. / Izmenenie urovnia sfingozina v iadrakh i kletkakh pecheni krys pri indutsirovannoi superékspressii onkogenov tsiklogeksimidom.

Changes in the sphingosine content in rat liver cells and nuclei have been studied with reference to the level of nuclear oncogene expression, induced by cycloheximide (0.1, 0.5 and 3.0 mg/kg). It has been found that only the sublethal (3 mg/kg) dose of cycloheximide which induces the superexpression of c-fos and c-myc oncogenes can promote sphingosine accumulation in the cell. At the moment of enhanced expression of nuclear oncogenes, the maximum content of free sphingosine exceeds the control level 1.5- and 3-fold in the cell and in the nuclei, respectively. The difference in the sphingosine accumulation patterns in the cell and in the nuclei testifies to the fact that sphingomyelin metabolism is more active in the nuclei than in the cell. Sphingosine accumulation in the nuclei is characterized by coordination of sphingomyelinase activity and changes in the sphingomyelin content. A comparative analysis of activities of enzymes of sphingomyelin (sphingomyelinase) and phosphatidyl inositol (phosphatidyl inositol kinase) cycles indicates that in the nuclei the activation of the sphingomyelin cycle forestalls the cycloheximide-induced activation of the phosphatidyl inositol cycle and the maximal accumulation of nuclear oncogene mRNAs. A model of activation of oncogene expression with participation of sphingosine inhibiting protein kinase C and activating casein kinase II, the key enzymes of the signal transduction system of cell proliferation and differentiation, is proposed.

[Epidermal growth factor stimulates protein kinase activity, specific for ribosomal protein S6 in loach embryo blastoderm]. / Epidermalnyi faktor rosta stimuliruet proteinkinaznuiu aktivnost', spetsifichnuiu k ribosomnomu belku S6, v blastoderme zarodyshei v'iuna.

The activity of a protein kinase specific to ribosomal protein S6 was determined in early loach embryos in basal conditions and after their treatment with epidermal growth factor (EGF). The cytosol of loach blastoderms isolated at the early gastrula stage possessed a high level of protein kinase activity catalysing incorporation of 0. 33 pmol.min-1.mg-1 of Pi into exogenous S6 protein of rat liver ribosomal 40S subunit. The treatment of embryos for 30 min with EGF (10 ng/ml) added to the incubation medium caused an 25% increase of total S6-kinase activity in cytosol compared with its counterpart in non-stimulated embryos. After chromatography of loach embryos cytosol on DE-52 three fractions possessing S6-kinase activity were revealed, which were eluted with 10 microM cAMP (I), 150 mM NaCl (II) and 300 mM NaCl (III), respectively. After treatment of blastoderms with EGF in the described conditions the enzymatic activity 2-fold decreased in fraction I, increased in fraction II 4-fold and remained practically unchanged in fraction III. The mitogen-stimulated kinase, apart from S6 protein, phosphorylated also casein and but not histone H1.