ABSTRACT
For the first time it has been shown that RNase activity is induced under the influence of EGF on epidermoid carcinoma cell line A431. Proteasomes from EGF-treated A431 cells destabilize the 3'-untranslated regions of non-muscle beta actin mRNA, creating a specific cleavage pattern. In addition, these particles have been shown to specifically cleave Alu-containing informational RNA. The enzymatic activity under study has been shown to be dependent on phosphorylation of proteasomal subunits and specifically and selectively regulated by Ca and Mg ions. Proteasome involvement in the coordinated control of stability of specific messenger RNA molecules is suggested. The endoribonuclease activity of 26S proteasomes can constitute a link between EGF signaling pathways and RNA stability.
Subject(s)
Endoribonucleases/metabolism , Epidermal Growth Factor/pharmacology , Peptide Hydrolases/drug effects , Proteasome Endopeptidase Complex , RNA, Messenger/metabolism , 3' Untranslated Regions/metabolism , Actins/genetics , Aluminum , Calcium Chloride , Cell Line, Tumor , Humans , Magnesium Chloride , Peptide Hydrolases/metabolism , Phosphorylation , RNA, Messenger/chemistry , Signal TransductionABSTRACT
A comparative study was made of reactive oxygen species (ROS) in rat embryo fibroblasts and their transformants. Primary rat embryo fibroblasts (REF), REF transformed by the complementing oncogenes E1A plus cHa-ras (cell line E1A + Ras), and REF transformed by E1A plus E1B-19 kDa (cell line E1A + E1B) were studied. ROS generation was measured with microfluorometric assay using fluorescent probe 2',7'-dichlorofluorescin diacetate. It has been shown that the block of REF and E1A + 1B cells in the G1/S under serum-starved conditions (0.5% serum) for 24-48 h was paralleled by a decrease in ROS generation. Activation of serum-starved REF and E1A + 1B cells with 10% serum resulted in reactivation of cell cycle and gradual increase in ROS generation. The maximum intracellular level of ROS correlated in time with the phase of DNA synthesis. Serum-starved E1A + Ras cells were not stopped in the G1/S and ROS production of these cells was not dependent on serum growth factors. The prolonged cultivation of E1A + Ras cells in the medium with low serum content (0.5%) caused a sharp increase in ROS generation, which was accompanied by apoptotic death.
Subject(s)
Endoribonucleases/metabolism , Epidermal Growth Factor/genetics , Ribonucleoproteins, Small Nuclear/metabolism , 3' Untranslated Regions , Epidermal Growth Factor/metabolism , Humans , Molecular Weight , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , Signal Transduction , Tumor Cells, CulturedABSTRACT
Our analysis detected in 26S proteasomes of human A-431 cells a strong endoribonuclease activity, degrading cytoplasmic high-molecular-mass RNA, particularly, specific mRNAs. Enzymatic nature of this activity has been confirmed, and the optimal conditions studied. This endonuclease activity of proteasomes has not been earlier observed. Proteasome involvement in the stability control of specific messenger RNA molecules is suggested, and proteasome participation in the coordinated control of various stages of gene expression is discussed.
Subject(s)
Endoribonucleases/metabolism , Peptide Hydrolases/metabolism , Proteasome Endopeptidase Complex , Humans , Tumor Cells, CulturedABSTRACT
Here we demonstrate that the epidermal growth factor (EGF) induces association of prosomes (20S-proteasomes) with its receptor in A-431 cells. Additionally, ligand-dependent association of ribonucleoprotein particles (alpha-RNP), containing small ALU-like RNA, with the EGF receptor was demonstrated. A suggestion has been put forward on the involvement of prosomes and alpha-RNP in the EGF signal transmission to different stages of gene expression.
Subject(s)
Cysteine Endopeptidases/metabolism , ErbB Receptors/metabolism , Multienzyme Complexes/metabolism , Ribonucleoproteins/metabolism , Gene Expression Regulation, Enzymologic/physiology , Humans , Particle Size , Proteasome Endopeptidase Complex , Tumor Cells, CulturedABSTRACT
By electron microscopy using immunocytochemical reactions on actin and EGF-R with mild glycerinization it became possible to reveal a thick layer of actin filaments under the apical membrane within the first minutes of the impact of EGF on A431 cells. Keratin fibrils being located near the nucleus are transported apart from the membrane. It is supposed that this process is connected with retardation of capping on principles of competition.
Subject(s)
Actins/metabolism , Epidermal Growth Factor/pharmacology , Keratins/metabolism , Biological Transport , Cell Membrane/metabolism , Cell Nucleus/metabolism , Epidermal Growth Factor/metabolism , ErbB Receptors/antagonists & inhibitors , Glycerol , Humans , Immunohistochemistry , Protein Binding , Tumor Cells, CulturedABSTRACT
The human chromosomes were obtained from lymphoblastoid cell line BOLD with normal karyotype, and separated on the basis of their size by velocity sedimentation at 190 g in sucrose gradient. Different chromosomal fractions were stained by applying fluorochromes with different DNA-base specificity: Hoechst 33258 and Olivomycine and were analysed using dual laser cell-sorter ATC-3000 (Brucker). Velocity sedimentation allowed to enrich certain fractions by individual chromosomes and thus speed up the sorting rate from 90 up to 200 chromosomes per second.
Subject(s)
Cell Fractionation/methods , Chromosomes, Human/ultrastructure , Flow Cytometry/methods , Cell Separation , Centrifugation, Density Gradient/instrumentation , Centrifugation, Density Gradient/methods , Flow Cytometry/instrumentation , Humans , Lasers , MetaphaseABSTRACT
By the use of rhodamine-phalloidin, the distribution of actin in A-431 cells during the action of epidermal growth factor (EGF) has been studied. Changes in the pattern of staining are observed in 30-60 s after addition of the EGF. Microvilli and wrinkles are created on the cell surface. Following a 5-10 min action of EGF, rhodamine-phalloidin stained intensely ruffles and cell borders. After 60 min, the ruffling of cell surface disappeared, and actin was seen concentrating on the cell borders only. Electron microscopy of the EGF-treated A-431 cells lysed by Triton X-100 also revealed some vigorous fibrillar bunches on the cell edges.
Subject(s)
Cytoskeleton/drug effects , Epidermal Growth Factor/pharmacology , Actins/drug effects , Actins/metabolism , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/ultrastructure , Cell Line , Cytochalasin B/pharmacology , Cytoskeleton/metabolism , Cytoskeleton/ultrastructure , Humans , Microscopy, Electron, Scanning , Microscopy, Fluorescence , Staining and Labeling/methods , Time Factors , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/metabolism , Tumor Cells, Cultured/ultrastructure , Vinblastine/pharmacologyABSTRACT
As was demonstrated elsewhere (L. V. Teslenko et al., 1987), the epidermal growth factor (EGF) can recycle after internalization by A431 cells in membrane-bound state. In the present study, direct evidence on recycling of EGF-receptor complexes is presented using a covalently crosslinking reagent. The recycling was shown to occur via peripheral endosomes as well as through para-Golgi endosomes. It was found that among EGF degradation inhibitors tested only primaquine (300 microM) was able to decrease significantly the rate of recycling. The lowering of the temperature to 17 degrees C led to blocking the EGF degradation as well as to inhibiting the recycling. The data obtained suggested that the recycling of EGF-receptor complexes is relatively independent of their degradation.
Subject(s)
ErbB Receptors/metabolism , Animals , Cell Compartmentation , Cell Line , Epidermal Growth Factor/metabolism , Humans , Iodine Radioisotopes , Mice , Organelles/metabolism , Rhodamines , Temperature , Tumor Cells, CulturedABSTRACT
Certain changes in human carcinoma A-431 are found by scanning electron microscopy. The early cell response to growth factor (after 10 minutes) involves a disappearance of microvilli, an appearance of ruffles and rounded cells, along with a decrease in cell attachment to the substrate. The cell surface changes correlate with the state of cytoskeleton elements: the material stained with iron hematoxylin is accumulated in ruffle formation sites. Retractional fibrils filled with the cytoskeleton material result from a decrease in the cell area.
Subject(s)
Cytoskeleton/drug effects , Epidermal Growth Factor/pharmacology , Cell Membrane/drug effects , Cell Membrane/ultrastructure , Cytoskeleton/ultrastructure , Humans , Microscopy, Electron, Scanning , Microvilli/drug effects , Microvilli/ultrastructure , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/ultrastructureABSTRACT
The endocytosis and intracellular fate of epidermal growth factor (EGF) were studied in A431 cells. After 15-20 min of internalization at 37 degrees C, rhodamine-labeled EGF (EGF-Rh) accumulated into large juxtanuclear compartment consisting of closely related vesicles. This structure was shown to be localized in the para-Golgi region. Fluorescein-labeled transferrin (Tr-FITC) was observed in the same region when added to the cells simultaneously with EGF-Rh. Using microscope spectrofluorometer, we determined that the Tr-FITC-containing para-Golgi structures have a pH of 6.1 +/- 0.3 while lysosomes containing dextran-fluorescein have a pH of 5.0 +/- 0.2. To study the dynamics of EGF-receptor dissociation during endocytosis a mild detergent treatment of living cells was used for extraction of an intracellular receptor-unbound EGF. During the first hour of internalization at 37 degrees C, neither significant dissociation of EGF-receptor complexes nor EGF degradation was observed. After 3 h of endocytosis, the percentage of unbound EGF increased to 55% of the total internalized EGF. These results suggest that EGF remains associated with receptors during endocytosis in A431 cells until it is transferred to lysosomes where the pH of the EGF microenvironment is dropped to 5. A prolonged presence of EGF-receptor complexes in the para-Golgi region might be of importance in mitotic signaling.
Subject(s)
Endosomes/metabolism , Epidermal Growth Factor/metabolism , ErbB Receptors/metabolism , Hydrogen-Ion Concentration , Cell Compartmentation , Cell Line , Detergents , Endocytosis , Golgi Apparatus/metabolism , Humans , Lysosomes/metabolismABSTRACT
The fate of epidermal growth factor (EGF) after internalization by A431 cells was studied. First, cells containing 125I-EGF-receptor complexes in endosomes were obtained. Subsequent incubation of the cells at 37 degrees C resulted in the recycling of 125I-EGF from endosomes to the cell surface in the receptor-bound state and the gradual release of recycled ligand into the medium. The excess of unlabeled EGF blocked both rebinding and re-internalization of recycled 125I-EGF to produce enhanced accumulation of ligand in the medium. The rate of recycling was shown to be much higher than that of EGF degradation.
Subject(s)
Carcinoma, Squamous Cell/metabolism , Epidermal Growth Factor/metabolism , Animals , Cell Fractionation , Cell Line , Cell Membrane/metabolism , Centrifugation, Density Gradient , Cytoplasm/metabolism , Endocytosis , ErbB Receptors/metabolism , Humans , Kinetics , MiceABSTRACT
Our previous paper (Rodionov et al., 1985) reported production of monoclonal antibodies RN-17 reacting in cultured fibroblasts with a protein having a molecular weight of 100 kD. Immunofluorescence and immunoelectron microscopy showed that this protein was a component of microtubules, intermediate filaments and coated vesicles. We challenged a possibility whether these coated vesicles containing the 100 kD protein may take part in the receptor-mediated endocytosis. alpha 2-Macroglobulin conjugated with fluorescein isothiocyanate or 20 nm colloidal gold particles was used as a marker of the receptor-mediated endocytosis. Mouse embryo fibroblasts or Swiss 3T3 cells were incubated with labeled alpha 2 M, fixed and "stained" with DN-17 antibody, and the distribution of alpha 2 M and 100 kD protein was examined within the same cells. In both cell lines the endocytic vesicles contained 100 kD protein and alpha 2 M. Therefore 100 kD protein is a component of endocytic vesicles. Probably this protein mediates microtubule-dependent transport of endocytic vesicles in the cells.
Subject(s)
Cytoskeletal Proteins/analysis , Endocytosis , Endosomes/ultrastructure , Animals , Antibodies, Monoclonal , Cells, Cultured , Fluorescent Antibody Technique , Mice , Microscopy, Electron , Molecular Weight , Staining and Labeling/methods , alpha-Macroglobulins/analysisABSTRACT
Internalization process of the fluoresceinisothiocyanate-labeled alpha 2-macroglobulin (MG-F) in Swiss 3T3 cells has been studied. The presence of internalized MG-F, formation of endocytotic vesicles and endosomal pH value were estimated visually on living monolayer cells with TV microscope, on the base of supervidicon LI-702. The use of the TV method allowed to obtain a series of photographs of cell images without any significant dye fading. A brief characteristics of microprocess control system is presented. The influence of various chemical agents on MG-F internalization and endosomal pH value was investigated. It was shown that dansylcadaverine (150-200 microM) and monensin (10-20 microM) inhibited MG-F internalization. Methylamine, chloroquine and monensin quickly enhanced the endosomal pH value, while other amines did not, for example dansylcadaverine.
Subject(s)
Endocytosis , alpha-Macroglobulins/metabolism , Animals , Cells, Cultured , Endosomes/metabolism , Fluorescein-5-isothiocyanate , Fluoresceins , Fluorescent Dyes , Humans , Hydrogen-Ion Concentration , Mice , Microscopy, Fluorescence/instrumentation , Television/instrumentation , ThiocyanatesSubject(s)
Endocytosis , Epidermal Growth Factor/metabolism , Golgi Apparatus/metabolism , Transferrin/metabolism , Cell Compartmentation , Cell Line , ErbB Receptors , Humans , Hydrogen-Ion Concentration , Receptors, Cell Surface/metabolism , Receptors, Transferrin , Skin Neoplasms/metabolism , Skin Neoplasms/ultrastructureABSTRACT
The dependence of L-alanine uptake by 3T6 and CHO-K1 cells on Na+ electrochemical gradient has been studied. The Na+ chemical gradient was changed by a short-term (partial or complete) replacement of Na+ for choline. The membrane potential change was achieved by addition of potassium ionophore--valinomycin (10 microM) into the medium. It is determined that the value of Km for alanine uptake by 3T6 cells increases from 2 mM, with 140 mM Na+ in the medium, up to 30 mM, if the replacement of Na+ for choline is complete. Similar results are obtained for CHO cells. The membrane potential increase under the influence of valinomycin leads to the increase in the value of Vmax of the uptake. The data obtained are interpreted on the basis of the well known scheme of Na+ alanine complex transfer, where Na+ increases the affinity of the carrier to the amino acid, and the membrane potential increases the carrier mobility.
Subject(s)
Alanine/metabolism , Cells, Cultured/metabolism , Sodium/metabolism , Animals , Biological Transport/drug effects , Cell Line , Choline/metabolism , Dose-Response Relationship, Drug , Membrane Potentials/drug effects , Valinomycin/pharmacologyABSTRACT
The activities of Val-tRNA synthetase and Ala-tRNA synthetase in the intact rabbit M. soleus are considerably higher than those in the lateral portion of the intact M. gastrocnemius. The rates of tRNA aminoacylation by the enzymes from both denervated muscles were levelled out 35 days after sciatic nerve section. Insulin injections or diabetes did not significantly influence the aminoacyl-tRNA formation catalyzed by aminoacyl-tRNA synthetases (ARS-ases) from intact muscles. Insulin substantially increased (while diabetes decreased) the activity of both enzymes in the denervated muscles. The results obtained show that the deprivation of striated muscles of trophic nervous influence results in an increase of their sensitivity to the effect of insulin on the activity of aminoacyl-tRNA-synthetases.
Subject(s)
Alanine-tRNA Ligase/metabolism , Amino Acyl-tRNA Synthetases/metabolism , Insulin/pharmacology , Muscle Denervation , Muscles/enzymology , Valine-tRNA Ligase/metabolism , Animals , Diabetes Mellitus, Experimental/enzymology , Muscles/drug effects , Organ Specificity , RabbitsABSTRACT
The stability of alanyl-tRNA synthetase and valyl-tRNA synthetase from the intact and denervated for 30 days rabbit skeletal muscles towards the inactivating effect of heat (42 degrees for 10 min) has been studied. The activity of the enzymes was measured in the supernatant fraction obtained by centrifugation of muscle homogenates at 105, 000 g for 1 h. The stability of both alanyl- and valyl-tRNA synthetases from the denervated m. soleus and m. gastrocnemius was shown to be considerably decreased as compared with the enzymes from the intact muscles.
Subject(s)
Alanine-tRNA Ligase , Amino Acyl-tRNA Synthetases , Muscle Denervation , Muscles/enzymology , Valine-tRNA Ligase , Animals , Hot Temperature , RabbitsABSTRACT
Activities of alanyl- and valyl tRNA synthetases were considerably higher in rabbit muscle soleus than in muscle gastrocnemius. Repeated injections of insulin into normal rabbits as well as impairment of animals by experimental alloxan diabetes within two weeks did not alter significantly the activity of the synthetases in red and mixed muscles.
