Biomedical subjects

E S Kornilova

Publications and source records attributed to E S Kornilova.

At least 19 recordsLinked to original sources

PubMed2005

Nocodazole inhibits macronuclear infection with Holospora obtusa in Paramecium caudatum.

Holospora obtusa is a Gram-negative bacterium inhabiting the macronucleus of the ciliate Paramecium caudatum. Experimental infection with H. obtusa was carried out under nocodazole treatment. Nocodazole has been shown to cause disassembly of the cytoplasmic microtubules radiating from the cytopharynx and postoral fibers in P. caudatum. Treatment with this drug did not prevent the ingestion of both prey bacteria and H. obtusa, but it reduced the phagosome number and affected cyclosis. In situ hybridization revealed infectious forms of this endobiont very close to the macronucleus, but never inside it. These results indicate that disassembly of microtubules does not impair transportation of the infectious forms of H. obtusa in the cytoplasm, but that it completely blocks the invasion of the nucleus by the bacteria.

Actin Cytoskeleton↗

PubMed2004

[Effect of synthetic proteasomal inhibitor MG132 on dynamics of EGF-receptor complexes endocytosis in A431 cells].

The effect of proteasomal activity suppression induced by MG132, a synthetic proteasomal inhibitor of EGF-receptor complexes endocytosis in human epidermoid carcinoma A431 cell line, was studied. Using subcellular fractionation in 17% Percoll gradient, it was demonstrated that the addition of MG132 to the cells 15 min following stimulation of EGF endocytosis resulted in a slight accumulation of 125I-EGF in early endosomes, and in much more significant accumulation of the labeled growth factor in late endosomes/lysosomes, as compared to untreated cells. The release of 125I-EGF degradation products into the incubation medium was significantly (3-12-fold) inhibited in the presence of MG132. At the same time biochemical analysis has demonstrated that the EGF receptor itself is not a direct target of proteasomes, since it is revealed as a full-length protein with native mol. mass (170 kDa) in fractions of early and late endosomes and lysosomes. Possible mechanisms of the MG132 effect on intracellular processing of EGF-receptor complexes are discussed.

Cell Fractionation↗

PubMed2003

[The role of phosphatidylinositol-3-kinases P85/P110 and hVPS34 in endocytosis of EGF-receptor complexes].

The previous data (Zheleznova et al., 2001) did not enable the authors to conclude which particular wortmannin sensitive PI-3-kinase--p85/p110 (I class PI-3-K) or hVPS34 (III class PI-3-K)--may be involved in the regulation of EGF-receptor endocytosis. In the present work, we have shown that upon stimulation of EGF-receptor endocytosis additional structures stained with antibody against p85 appear in A431 cells, but the p85-positive compartment never co-localized with EGF-receptor-containing compartments either in control or in wortmannin-treated cells. At the same time, wortmannin treatment prevented association of hVPS34 with endosomal membranes. We have also found that early endosomal markers--Rab5 and EEA1 (membrane association of the latter depends on Rab5 and hVPS34)--co-localized with EGF-receptor in the juxtranuclear region during late stages of endocytosis, both in control and upon wortmannin treatment. These observations favor our suggestions that the transition of EGF-receptors from early to late endosomes may occur directly in this juxtranuclear region and be tightly associated with the formation of so called multivesicular bodies (MVB), which are late endosomes per se. We suggest that wortmannin may have no effect on early EEA1-dependent stage of the receptor endocytosis but blocks a transition of EGF-receptor complexes into the late endosomes by inhibiting activity of hVPS34 and removing it from membranes. The hVPS34 product PI-3-K, according to the known data, is involved in the formation of internal vesicles of MVB. Accumulation of EGF-receptors in these vesicles is believed to be necessary for the receptor degradation.

Androstadienes↗

PubMed2003

[Cbl--a polyfunctional regulator of cellular processes].

C-Cb1 protein is a protooncogene product that was initially identified as part of a murine retrovirus transforming protein. C-Cb1 is ubiquitously expressed in cells of different origin. A number of isoforms subsequently identified in vertebrates and invertebrates allows to consider the existence of a family of Cb1 proteins. These proteins contain a set of sequences providing interactions with a wide range of receptor and nonreceptor tyrosine kinases and signaling proteins with SH2- and SH3-domains (for example, EGF and PDGF receptors, Src-kinases, PI-3-kinase p85, Crk, GRB2, Vav, etc.). Cb1 proteins possess also multiple tyrosine, residues, which undergo phosphorylation upon stimulation of several surface receptors. These properties permit Cb1 to take part in many protein-protein interactions as an adaptor, which forms multimolecular signaling complexes, and coordinates the activity of its components. C-Cb1 and its mutant transforming forms can act as both positive and negative regulators of many signaling pathways. Negative action of C-Cb1 on signals stimulated by receptor tyrosine kinases is thought to result from accelerated receptor degradation caused by Cb1. This ability is attributed mostly to ubiquitin-ligase activity of Cb1 proteins, since the latest research evidence suggests that ubiquitination may be a signal of not only proteasomal, but also lysosomal degradation. Thus, Cb1 manifests itself as a many-sided protein working both as an adaptor and a regulator of endocytic trafficking. In spite of numerous studies in this area, the regulation of Cb1 functions, interrelations between these functions, physiological significance of Cb1-mediated interactions, and the place of Cb1 proteins in signaling coordinating still remain obscure. In the present review, an attempt is made to summarize the recent data, with special reference to Cb1 functioning as a regulator of tyrosine kinase receptor endocytosis.

Adaptor Proteins, Vesicular Transport↗

PubMed2002

[Effect of a hypertonic sucrose solution and 5-(N,N-hexamethylene)-amiloride on receptor-mediated and liquid-phase endocytosis].

Hypertonic sucrose and amiloride or derivatives of the latter are commonly used to selectively inhibit clathrin-dependent (usually considered as a synonim of receptor-mediated endocytosis) or clathrin-independent endocytosis. Though these approaches are widely used in experimental practice, their limitations have not been studied in detail. In the present work, an attempt was made to evaluate possible side effects and selectivity of these agents towards the type of endocytosis. It was found that the incubation of A431 cells in 0.4 M sucrose resulted in a decrease in both intracellular accumulation and surface binding of the RME marker 125I-EGF. However, while the binding drops by about 3 times, the internalization of EGF in low concentrations was inhibited by more than 30 times, and that of high EGF concentrations by 5-10 times. It may evidence that at high EGF concentrations about 10-20% of ligand-receptor complexes can enter the cells through clathrin-independent pathway. Nevertheless, these results cannot be interpreted so unambigously, because we found that at the incubation longer than 30 min a significant portion of cells became dead or damaged, yielding about 50% of the whole population. By immunofluorescent assay, 5-(N,N-hexametylen)-amiloride commonly used to inhibit fluid phase endocytosis was demonstrated to reduce the staining of fluorescein-containing pinocytic vesicles, but it did not inhibit totally the entering of this marker. Under simultaneous stimulation of fluorescein and EGF endocytosis in the presence of the amiloride derivative, such a residual fluorescence shifted with time to the juxtranuclear region, which is characteristic of the late steps of RME. We suppose that a significant portion of extracellular fluid phase can be included in clathrin-dependent vesicles, whose formation is not disturbed by amiloride. It means that conclusions about the degree of pinocytosis inhibition are to be corrected, taking into account the constitutive clathrin-dependent endocytosis.

Amiloride↗

PubMed2002

[Effect of an vacuolar proton pump inhibitor bafilomycin A1 on the intracellular processing of markers for receptor-mediated and liquid phase endocytosis].

By means of subcellular fractionation in density Percoll gradients, immunoblotting and immunofluorescense, the effect of BafA1 on endocytosis of EGF-receptor complexes and horse radish peroxidase (HRP) in A431, HER14 and HC11 cell lines was studied. It was shown that the pretreatment of all used cell lines with BafA1 completely inhibited EGF degradation, but did not interfere with the delivery of significant portion of EGF-receptor complexes to late endosomes and lysosomes and transition of the receptor to juxtranuclear region. At the same time, BafA1 was found to dramatically inhibit the delivery of fluid phase marker HRP to late endosomes of A431 cells. The BafA1 effect on endocytosis of high concentrations of EGF was similar to that on HRP endocytosis. Regulatory mechanisms of early-to-late endosomal compartment transition are discussed.

3T3 Cells↗

PubMed2002

[The route of a bacterium Holospora in the cell of Paramecium (Ciliophora, Protista) from phagosome to the nucleus].

Problems encountered at the initial stages of stable symbiotic system formation are discussed in the review. The most studied models for interaction between pathogenic bacteria and metazoan cells are compared with a similar system including Paramecium (a ciliatte)--Holospora (a bacterium). Literary and our own data on the infection of P. caudatum with specific endocytobionts inhabiting the nuclear apparatus (H. obtusa in the macronucleus), and H. undulata (in the micronucleus) are analysed with respect to the modern understanding of the intracellular vesicle trafficking.

Animals↗

PubMed2001

[Effect of wortmannin on endocytosis of epidermal growth factor receptors].

Using subcellular fractionation of human carcinoma A431 cells in Percoll gradient it was shown that P13-kinase (P13-K) inhibitor wortmannin blocked the transition of the EGF-receptor complexes from the early to the late endosomes. Under conditions when the receptor TK-dependent sorting system is mainly involved, i.e. at low EGF concentrations, the efficiency of sorting was seen to fall 5-10-folls in the presence of wortmannin compared to the control. At high EGF concentrations of the toxin inhibitory effect was no more than 30%. Immunofluorescent analysis has demonstrated that wortmannin treatment led to a juxtranuclear localization of EGF-receptors, which is presumably characteristic of the late endosomes. However, this localization became obvious even in 15 min following endocytosis stimulation, when EGF-receptors, according to the Percoll data, were associated mainly with the early endosomes. A possible role of phosphatidylinositol metabolism products in endocytosis regulation is discussed in addition to the structural and functional organization of the early endosomal compartments. A conclusion is made that P13-K may be a component of the EGF receptor-specific sorting system.

Androstadienes↗

PubMed2001

[The role of Src-kinase in the regulation of endocytosis of EGF-receptor complexes. I. Dynamics of EGF internalization, recycling, sorting, and degradation during inhibition of Src-kinase activity].

In the present work, the role of Src-kinase in regulation of different stages of EGF-receptor endocytosis was studied. We used murine fibroblasts with knockout of Src gene and CGP77675, and the inhibitor of Src-family kinases. The absence of Src protein in the cells did not lead to any changes in the rates of 125I-EGF internalization or recycling and caused only slight decrease in the rate of its degradation. At the same time, treatment of the wild type cells with the inhibitor resulted in a small decrease in internalization rate and an increase in recycling. The influence of the inhibitor on 125I-EGF degradation was also more pronounced. But even in this case, the effects were no more than 30% of control values. CGP77675 extended the same effect upon cells of HER14 and HC11 lines. Subcellular fractionation of these cells in Percoll gradient has also demonstrated a slight inhibition of 125I-EGF sorting from early to late endosomes. The more pronounced effect of the Src-family kinase inhibitor on the EGF endocytosis, compared to that of the absence of a single Src protein, suggests a compensating mechanism of the Src-family kinases. A conclusion is made that in spite of a slight influence on practically all stages of intracellular pathway of EGF-receptor complexes, Src-kinases are obviously not the key regulators of their endocytosis.

Animals↗

PubMed2001

[The role of Src-kinase in the regulation of endocytosis of EGF-receptor complexes. Distribution of clathrin after stimulation of EGR endocytosis in various cell lines during inhibition of Src-kinase activity].

A distribution of EGF receptor and clathrin during EGF endocytosis in A431, HER14, WT and PURO cell lines was studied by indirect immunofluorescence. Though the initial distribution of EGF-receptors on A431 and HER14 cells was somewhat different, the late stages of endocytosis proceeded equally and were marked by formation of bright spots in the juxtanuclear region characteristic of the late endosomes. The Src-family kinase inhibitor CGP77675 had no influence on the dynamics of receptor endocytosis at the immunofluorescent level in both cell lines. Stimulation of EGF-receptor endocytosis in A431 cells did not also result in any redistribution of clathrin in the areas where the majority of EGF-receptors are localized, i.e. in the lateral plasma membrane both in the control cells and under CGP77675 treatment. Clathrin in A431, WT and PURO cells demonstrated even a punctuated pattern throughout the cytoplasm with some accumulation in the juxtanuclear region. This distribution depended neither on the absence or presence of Src activity nor on EGF addition. The data obtained indicate that 1) EGF-receptors do not serve as the initiation sites during clathrin coated pit assembly; 2) Src-kinase activation does not result in significant clathrin redistribution in the plasma membrane, and its influence on EGF endocytosis can be considered as a secondary effect.

Animals↗

PubMed1999

[Association of MAP kinases with the endoplasmic reticulum in NIH3T3 (HER14) and A431 cells].

Intracellular distribution of p42/p44 MAP-kinases in HER14 and A431 cell lines was investigated. Using subcellular fractionation and immunofluorescence approaches we have shown that in quiescent cells of both types MAP-kinases are associated with endoplasmic reticulum. Moreover, ER-localized MAP-kinases were shown to exist only in a nonphosphorylated form. In HER14 cells the epidermal growth factor (EGF) elevates the level of the ER-associated MAP-kinases. In contrast, exposure of A431 cells to EGF leads to a significant decrease in the observed association. The physiological role of this association is discussed.

3T3 Cells↗

PubMed1999

[Endocytosis of EGF-receptor complexes at various cell cycle stages].

Parameters of EGF-receptor complex endocytosis have been studied in the early and late G1 phase and in mitosis. As a model, mouse mammary epithelial cells HC11 were used, whose growth depends on EGF presence in the medium. The Scatchard analysis has demonstrated that the surface receptors are represented by two receptor populations: 4800 high affinity (KD = 10(-11) M) receptors, and 73,000 low affinity (KD = 4.10(-9) M) receptors. Incubation of cells with the growth factor (5 ng/ml) resulted in a decrease in 125I-EGF binding, with its level being low until entering the S-phase. Under these conditions, receptors disposed on the plasma membrane presented a homogeneous population (KD = 8.10(-11) M, 14,000 receptors per cell). No reliable difference was revealed between the EGF-receptor complexes, internalized in early and late G1 phases, in respect to the internalization rate, level of recycling, degradation, and dynamics of compartmentalization. However, endocytosis of EGF-receptor complexes was found to be completely blocked in mitosis at the stage of internalization.

Animals↗

PubMed1998

[EGF-dependent epithelial cells of the mammary cell line HC11 demonstrate a high degree of synchronized cell cycle during stimulation with epidermal growth factor].

The most popular object for studying endocytosis of EGF-receptor complexes, human epidermoid carcinoma A431, was shown to answer to EGF in high concentration (100 ng/ml) by growth inhibition, being indifferent to lower (0.1-1 ng/ml) concentrations. At the same time, cells NIH 3T3, expressing human EGF receptor (HER14), and epithelial mammary cells HC11 increased 14C-thymidine incorporation into DNA after EGF addition. However, for HER14 cells stimulatory effect of EGF was twice weaker than that induced by serum, whereas the effect of EGF on 14C-thymidine incorporation in DNA of cells HC11 was approximately 5 times stronger compared to serum. Therefore, cells HC11 may be referred to as EGF-dependent. Cell cycle analysis by fluorimetry showed that more than 90% of serum-starved HER14 and HC11 were in G0/G1. Within 19-20 h after stimulation by EGF 70-90% of HC11 cells and only 30-40% of HER14 cells were in S-phase. EGF removing from culture medium earlier than 9-11 h after stimulation blocked entering of HC11 cells into S-phase, whereas such EGF-dependent period was not found for cells HER14. Thus, synchronization of progression through early stages of cell cycle, stimulated by EGF and the presence of well defined "early" (EGF-dependent) and "late" (EGF-independent) phases, make cells HC11 convenient object for studying physiological role of EGF receptor complexes endocytosis.

Animals↗

PubMed1998

[Effect of nocodazole on endocytosis of epidermal growth factor receptor].

During endocytosis EGF-receptor complexes are transported from early peripheral endosomes to late juxtranuclear-located endosomes to be then degraded in lysosomes. It is suggested that such a spatial organization of endosomal compartments is maintained by microtubule system and is necessary for lysosomal degradation of endocytosed cargo. In the present work, a study was made of the influence of Nocodazole, a microtubule depolymerizing agent, on endocytosis of fluid phase marker HRP and EGF entering the cell via receptor-mediated endocytosis. By subcellular fractionation in Percoll gradient it was shown that Nocodazole did not affect HRP internalization but stimulated its accumulation in a fraction sedimented together with late endosomes, thus preventing HRP delivery to lysosomes. On the contrary, Nocodazole exerted no influence on dynamics of compartmentalization and lysosomal degradation of EGF-receptor complexes. Moreover, no alterations were found in the functioning of a so well-known EGF-stimulated signal transduction pathway as MAP-kinase cascade. At the same time microtubule depolymerization dramatically changed the morphology of endosomal compartments abolishing juxtranuclear localization of late endosomes. Our data suggest that translocation of EGF-receptor complexes is not necessary for their normal lysosomal processing. Rab7, traditionally considered as a marker of late endosomes, has been found to demonstrate in Nocodazole-treated cells, in contrast to the control, a low extent of co-localization with endosomal structures. It could be supposed that the role of Rab7 is not so much to mediate early-to-late endosome transition as to maintain spatial organization of endosomal apparatus by mediating endosome-cytoskeleton interactions.

3T3 Cells↗

PubMed1998

[Studies of small GTPase Rab7 association with endosomes of cells expressing normal and mutant forms of epidermal growth factor receptors].

A study was made of an association of small GTPase Rab7, commonly considered as a marker of late endosomes, with endosomal compartments of cells expressing EGF receptor with different ability to be sorted for degradative pathway. It was found that in cells HER14, expressing normal EGF receptor, Rab7 was associated with both early and late endosomes and the extent of association correlated with the number of EGF-receptor complexes in the specific endosomal fraction. Cels with a receptor, lacking major sites of autophosphorylation by deletion of 126 C-terminal residues (CD126), demonstrated a low efficiency of EGF-receptor sorting to late endosomes and decreased association dynamics with Rab7. Interaction of Rab7 with endosomes of cells expressing kinase negative receptors (K721) was found to be minimal. At the same time, in cells Cd126 and K721 with a low sorting efficiency Rab7 was mainly associated with early endosomes. These data favor Rab7 involvement in mediating early-to-late endosomal transition.

3T3 Cells↗

PubMed1996

[The association of a protein regulator of intracellular Rab7 membrane transport with endosomes containing an epidermal growth factor receptor with activated and inactivated tyrosine kinase].

By double indirect immunofluorescent microscopy, Rab7, traditionally considered as a late endosomal marker, has been demonstrated to colocalize with an internalized epidermal growth factor receptor (EGFR) possessing active and inactive tyrosine kinase (TK). The epidermal growth factor (EGF), which induces TK activity of EGFR, and monoclonal antibody Mab 108, which does not, have been exploited as ligands to stimulate endocytosis of EGFR. Colocalization between EGFR and Rab7 has been detected at both early (10 min) and delayed (60 and 120 min) endocytosis of EGFR, while it turned out to be much more obvious at the later ones. A comparison between EGFR-mediated and peroxidase fluid-phase endocytoses has revealed that Rab7 failed to be recruited on endosomal structures, containing peroxidase, even after 180 min endocytosis. Subcellular fractionation of endosomes containing 125I-EGF and 125I-Mab 108 in Percoll density gradients, in parallel with the analysis of ligand degradation, have verified the efficient transition of EGF-receptor complexes into the late endosomes and retention of Mab 108-receptor complexes within the early (sorting) endosomes. Taken together, the data cotained suggest that endogenous Rab7 is able to be recruited not only on late but also on maturating sorting EGFR-containing endosomes, thus mediating sorting along the EGFR endocytotic pathway.

Animals↗

PubMed1996

[The dependence of the type of endocytosis of EGF receptor complexes on the basal level of tyrosine kinase activity in the epidermal growth factor receptor].

Two types of EGF-mediated endocytosis have been identified in A431 cells by the method of subcellular fractionation in Percoll density gradients. One ("slow") type of endocytosis is characterized by 125I-EGF retention in the fraction of light endosomes, while the other ("fast") type demonstrates efficient 125I-EGF transition into heavy endosomes and lysosomes. 32P-ATP phosphorylation assay of Percoll fractions, followed by alkaline treatment on the gels, has demonstrated that "slow" cells reveal an increased basal level of EGF-receptor tyrosine kinase (TK) activity compared to the "fast" cells. Pretreatment of "fast" cells with Mn2+, which was shown to induce TK stimulation without EGF (Mohammadi [correction of Muhammedi et al., 1993), caused a dramatic decrease in 125I-EGF transition to heavy endosomes and lysosomes. Analysis of tyrosine phosphorylation of the receptor, being performed in Mn(2+)-pretreated A431 cells, has confirmed the significant increase of 32P-incorporation into unoccupied EGFR. Taken together, our data suggest that sorting of internalized EGF-receptor complexes depends on the basal TK activity level of EGFR.

Animals↗

PubMed1995

EGF receptor and p185erbB-2-specific single-chain antibody toxins differ in their cell-killing activity on tumor cells expressing both receptor proteins.

Many human tumors over-express erbB-2 and EGF receptors. The membrane localization of these receptor tyrosine kinases make them appropriate targets for directed tumor therapy. We have used recombinant DNA technology to produce single-chain antibody exotoxin A (scFv-ETA) fusion proteins which specifically bind the erbB-2 and EGF receptors. The scFv portion is composed of the heavy- and light-chain variable domains of monoclonal antibodies which recognize the extracellular portion of each receptor. We have previously described the anti-tumor activity of the bacterially produced scFv(FRP5)-ETA directed to the erbB-2 receptor. In this paper we describe the characteristics of scFv(225)-ETA, a protein which binds the EGF receptor. The bacterially produced recombinant protein binds to the receptor with high affinity and inhibits the in vitro growth of the EGF receptor over-expressing tumor cell lines A431 and MDA-MB468. Combination treatment with scFv-(FRP5)-ETA and scFv(225)-ETA led to an additive inhibitory effect on the in vitro growth of A431 cells. SKBR3 cells expressing low levels of EGF receptor but high levels of p185erbB-2 were not affected by scFv(225)-ETA treatment but were sensitive to scFv(FRP5)-ETA. Stimulation of SKBR3 cells and HCII RI#11 mouse mammary epithelial cells expressing the human erbB-2 with EGF led to an increase in scFv(FRP5)-ETA activity, showing that the EGF-induced activation of erbB-2 can potentiate the action of the erbB-2-directed toxin. Treatment of athymic nude mice with scFv(FRP5)-ETA and the combination of both scFv-ETA proteins led to the transient arrest of growth of established A431 tumors. scFv(225)-ETA treatment alone was the most effective, leading to tumor shrinkage during the course of treatment, whereas treatment with the parental monoclonal antibody 225 led to retarded tumor growth.

ADP Ribose Transferases↗