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1.
A sensitive method for measuring cell surface and secreted protease activity utilizing 3H-labelled casein is described. The method is based upon proteolytic degradation of the casein substrate into trichloracetic acid soluble 3H-labelled peptides. Utilizing the radioassay we found that all cultured cell lines examined contain cell surface proteolytic activity which is not secreted into the media. The protease activity was found to be due to protease(s) other than plasminogen activator or plasmin. A comparison of surface protease activity of normal and transformed mouse epidermal cells indicated that the transformed cells contained approximately 3--1 times more proteolytic activity than the normal cells. Surface protease activity was also correlated with the doubling times of various cultured cells. The results indicated that cultured cells with doubling times of greater than three days possess less surface protease activity than cells with shorter doubling times. In order to determine changes in the levels of surface protease activity during the cell cycle several cell lines were synchronized. In synchronized rabbit aortic fibroblasts, mouse transformed epidermal cells and human melanoma cells, a marked increase in surface protease activity was observed during or before mitosis. The protease levels decreased following mitosis. The results suggest that in culture, cell surface protease(s) may be important factor in regulating the rate of cell growth.  相似文献   

2.
Surface protease activity and secreted protease activity has been determined on several cell lines utilizing 3H-acetyl casein as substrate. When normal rabbit aortic smooth muscle cells and fibroblasts stopped proliferating at high cell density a decrease in surface protease activity was observed. No decrease in surface protease activity or in the rate of cell proliferation was observed in either transformed melanoma or epidermal cells. The decrease of surface protease activity was related to a decrease in cell proliferation.  相似文献   

3.
Clonal mouse skeletal muscle cells which differentiate in culture and from synpases with neuronal cells were found to secrete high levels of protease activity as measured with an 125I-fibrin assay. The secreted proteolytic activity was more than 90% dependent upon the presence of plasminogen in the medium, and had a pH optimum at 7 to 8. This activity was not inhibited by n-ethylmaleimide, pepstatin, EDTA, or EGTA. At millimolar concentrations, greater than 90% inhibition was obtained with either soybean typsin inhibitor, epsilon aminocaproic acid, Trasylol, or leupeptin. Almost complete inhibition occured with 1 mM diisopropylfluorophosphate suggesting the presence of a serine residue at the catalytic site. In contrast to the high levels of secreted activity, a lower steady-state level of cell-associated protease activity was detected in cell lysates. The high level of plasminogen activator secreted into the medium of cultured muscle cells suggests a role for such extracellular protease activity in myogenesis during development and remodeling following muscle injury. Such information may be useful in understanding the initial degeneration of neuromusclar contacts in experimental and pathologic denervation.  相似文献   

4.
Surface charge and cell volume of synchronized mouse lymphoblasts (L5178-Y)   总被引:1,自引:0,他引:1  
Cellular electrophoretic mobility, and therefore, surface charge/ unit area, was found to be constant throughout the cell cycle of synchronized L5178-Y mouse lymphoblasts, remaining at – 1.21 μ sec?1 V?1 cm. Measured cell volumes of these synchronized cells increased linearly over the cell cycle and at mitosis each cell divided into two cells, each cell having half of the parent volume. An hypothesis is presented which explains the presence of a mobility peak at mitosis in cells which normally are grown attaching to a glass surface on the basis of differential morphological changes during mitosis; cells such as the L5178-Ys, which do not attach to glass normally, do not undergo such morphological changes at mitosis and, therefore, would not demonstrate a mobility peak at that time.  相似文献   

5.
An assay measuring the release of TCA soluble radioactive peptides from 3H acetylated casein or hemoglobin has been used to demonstrate that human peripheral blood lymphocytes contain a number of proteases, including cathepsin D, a neutral serine protease(s) inhibited by DFP and TLCK and probably a thiol protease(s) as well. We have also found a neutral protease activity bound to the surface of the lymphocyte, but not secreted into the medium which is not inhibited by TLCK. TLCK inhibits blast transformation to PHA under conditions that do not profoundly affect protein synthesis and inhibits the total extractable proteolytic activity of lymphocytes by approximately 25%. Lymphocytes contain one or more proteases that may play a role in blast transformation and other lymphocyte functions.  相似文献   

6.
We have investigated methionine adenosyltransferase activity (MAT) in extracts of a variety of normal and malignant human tissues and cultured cell lines. MAT activity assayed from 17 different cultured cell lines varied to a great extent. Ramos (human, Burkitt's lymphoma) and EL4 (mouse, T cell lymphoma) cell showed MAT activity near 300 pmol/mg per min. Daudi (human, Burkitt's lymphoma) and almost all monolayer cells had MAT activity below 100 pmol/mg per min. Human peripheral blood lymphocytes had MAT activity of 36 pmol/mg permin. The MAT activity of the cell lines can be related to doubling time: cell lines with short doubling times have much higher MAT activity than other cell lines. A large variation in MAT activity in different human tissues was observed. In autopsy samples MAT activity was highest in the brain and in the colon. Malignant tissue samples gave much higher MAT activity than normal tissues. Lung cancer (carcinoma squamocellulare pulmonis) had MAT activity of 30.7 pmol/mg per min, while in normal lung it was 2.4 pmol/mg per min.  相似文献   

7.
The proteolytic activity of High-Five insect cell culture supernatants was analysed using substrate gel electrophoresis (zymography). During growth in serum-free media, High-Five cells constitutively expressed and secreted proteases that were active on casein gel but not on gelatin or bovine serum albumin gels. Two main protease bands were visible at about 41–42 kDa and 32–33 kDa. By addition of various protease inhibitors in the incubation buffer, the proteases were identified as metalloproteases as complete and specific inhibition of the proteolytic activities was only obtained by 1,10-phenanthroline.  相似文献   

8.
Using 32P-labeled phosphocasein or phosphohistones as exogenous substrates it was possible to detect a phosphoprotein phosphate activity on the outer surface of intact normal and transformed 3T3 fibroblasts. Incubation of monolayers of intact cells in buffered salt solution with the radioactively labeled substrate resulted in the release of alkali-labile 32P counts into the surrounding medium. The reaction was: (a) linear with time (at least up to 20 min); (b) proportional to the cell density; (c) dependent on the temperature and pH of the incubation medium; (d) stimulated by K+; and (e) inhibited by sodium fluoride, inorganic pyrophosphate, zinc chloride and relatively impermeant sulfhydryl reagents. Less than 2% of the externally located phosphoprotein phosphatase activity was detectable in pooled cell-free washings of the intact cell monolayer. Phosphocasein did not cause any detectable leakage of intracellular lactate dehydrogenase or soluble phosphoprotein phosphatase activity into the external medium; incubation of the cells with phosphohistones, on the other hand, resulted in appreaciable leakage of both these cytoplasmic activities. Neoplastic transformation was associated with a nearly two-fold decrease in the activity of the surface phosphoprotein phosphatase. Addition of serum to either non-transformed 3T3 or spontaneously transformed 3T6 cells resulted in a rapid and remarkable drop in the cell surface dephosphorylating activity. Acrylamide gel electrophoresis of the dephosphorylated casein or histone substrate revealed no proteolytic degradation or change in electrophoretic mobility. The intact cells showed no damage upon microscopic examination as a result of exposure to phosphocasein or phosphohistones.  相似文献   

9.
The proteolytic activity of E.coli measured using 125I-labelled αS1 casein as substrate, is mainly localised in the outer membrane and is due to an intrinsic outer membrane protein which can be solubilized by deoxycholate. This enzyme exhibits maximum activity at pH 7,5 in Tris-HCl buffer, is resistant to thermal denaturation with a half-life of 28 min. at 90°C in deoxycholate-NaCl buffer and is inhibited by ethylene-diamine tetraacetate, high concentrations of p-aminobenzamidine, tosyl-L-lysine chloromethyl ketone, tosyl-L-phenylalaninechloromethyl ketone and by two inhibitors of the processing of the secreted protein precursors, procaine and phenehylalcohol. Whole cells do not exhibit proteolytic activity, nevertheless, some is unmasked when the outer membrane is permeabilized by Tris or ethylenediamine tetraacetate or when vesicles are sonicated. This suggests that the protease is on the inner side of the outer membrane. Because the protease is different from the soluble proteases described in E.coli, and especially from proteases I,II and III, it has been called protease IV.  相似文献   

10.
The cell membrane potential of cultured Chinese hamster cells is known to increase at the start of the S phase. The putative role of the cell membrane potential as a regulator of cell proliferation was examined by following the cell cycle traverse of synchronized Chinese hamster cells in the presence or absense of high exogenous levels of potassium. An increase in external potassium levels results in a depressed membrane potential and a reduced rate of cell proliferation. A potassium concentration of 115 mM was used in experiments with synchronized cells since at that level cell proliferation is almost completely halted, recovery of growth is rapid and complete, and the membrane potential is reduced to a level well below that normally found in cells in the G1 phase. A mitotic population was divided into four aliquots and plated in either control medium or medium containing 115 mM K+. Cells placed directly into high K+ medium were retarded in their exit from mitosis and displayed a delayed and abnormal entry into the S phase. If control medium was added after two hours, cell cycle traverse was normal, but delayed by two hours compared to control cells. If the mitotic cells were plated directly into control medium and two hours later were shifted to high K+ medium, the cells entered the S phase in the absence of the normally observed increase in membrane potential and proceeded to the next mitosis normally. It was concluded that the increase in membrane potential observed at the start of the S phase in isolated synchronized cells is not a requirement for the initiation of DNA synthesis. In addition, sensitivity to the high potassium regimen was found at two different times during the cell cycle. In one case, cells were impeded in their transit through mitosis. Such cells displayed an altered chromosome structure which may account for the partial mitotic block. In the second case, synchronized cells displayed a sensitivity to the high potassium regimen in early G1 which appeared to be separate from the block in mitosis and independent of a change in the membrane potential.  相似文献   

11.
Transforming growth factor beta1(TGF-beta1) is a stimulator of malignant progression in mouse skin carcinogenesis. TGF-beta1 exerts a differential effect on cultured nontumorigenic (MCA3D cell line) and transformed (PDV cell line) keratinocytes. Whereas MCA3D cells are growth arrested and committed to die in the presence of the factor, it induces a reversible epithelial-fibroblastic conversion in PDV cells. This conversion is associated in vivo with a squamous-spindle cell carcinoma transition. Here we have investigated the role of urokinase (uPA) during malignant progression of transformed epidermal keratinocytes. We show that the levels of uPA expression/secretion, and the uPA binding activity to the cell surface, correlate with the invasive and malignant potentials of mouse epidermal cell lines. TGF-beta1 enhanced uPA production, the number of uPA cell surface binding sites, and the expression of the plasminogen activator inhibitor PAI-1, in transformed PDV cells, but had no major effect on nontumorigenic MCA3D keratinocytes. Increased uPA production depended on the presence of the factor in the culture medium and occurred concomitantly to the stimulation of the migratory and invasive abilities of PDV cells. Synthetic peptides containing the amino terminal sequence of the mature mouse uPA inhibited the binding of uPA to the cell surface and decreased TGF-beta1-induced cell motility and invasiveness. These results demonstrate that the uPA system mediates at least part of the migratory and invasive phenotype induced by TGF-beta1 in transformed keratinocytes, and suggest a role for uPA on the changes that lead to the appearance of spindle carcinomas.  相似文献   

12.
The mathematical and statistical problem of estimating the mean and variance of cell cycle times from cinemicrographically observed durations until mitosis (cytokinesis at late anaphase), disintegration, collision (cells superimposing each other) or emigration (moving out of the field of vision) of randomly chosen individual cells in a population in balanced exponential growth is treated. The resulting formulae are simple and considerably reduce the average cell observation time. They are applied to two normal human foetal cell lines and their SV40-transformed counterparts. One result is that the latter seem to have longer cycle times in spite of their shorter doubling times. Another result is that the cell mobility seems somewhat increased in the transformed populations. This corroborates earlier findings, indicating that the extent of cell loss, rather than the length of cycle times, may play a decisive role for the (net) doubling time of cultivated cell populations.  相似文献   

13.
Two kinds of proteolytic enzyme, tentatively named acid protease A and B which showed a single peak on electrophoresis individually, were isolated from the crude enzyme powder obtained from the broth filtrate cultured with Asper gillus niger var. macrosporus. Acid protease B is similar too the fungal acid protease previously reported, bccause the enzyme exhibits optimum activity on milk casein at about pH 2.6 and 55°C when the incubation was done at pH 2.6. Acid protease A is a new proteolytic enzyme, because the enzyme exhibits optimum activity on milk casein at about 2.0 and 70°C or 60°C when the incubation was done at pH 2.6 or 1.5 respectively.  相似文献   

14.
One spontaneous and four N-methyl-N'-nitro-N-nitrosoguanidine-induced revertants of a mouse FM3A mutant, tsTF20, which has heat-labile DNA polymerase alpha activity and cannot grow at 39 degrees C, were isolated and characterized with respect to the thermolability of their DNA polymerase alpha activity, the intracellular level of enzyme activity, growth rate, cell cycle progression, and frequency of initiation of DNA replication at the origin of replicons. DNA polymerase alpha activity in the extracts from the revertant cells showed partial recovery of heat stability. The intracellular level of enzyme activity of the revertant cells was lower than that of wild-type cells even at 33 degrees C. The level of enzyme activity in the revertant cells decreased considerably after a temperature upshift to 39 degrees C, but the DNA synthesizing ability of these cells did not decrease as much as the level of enzyme activity. The growth rates of the wild-type and revertant lines were almost the same at 33 degrees C. At 39 degrees C, the rate for the wild-type increased considerably compared to that at 33 degrees C, while little difference in the growth rates of the revertant lines was observed at the two temperatures. Therefore, the doubling times of the revertant cells were relatively increased compared to those of wild-type cells cultured at the restrictive temperature. Flow microfluorometric analysis and cell cycle analysis to measure labeled mitosis revealed that the increase in the doubling time was due mainly to the increase in the duration of the S phase. Analysis of the center-to-center distance between replicons by DNA fiber autoradiography indicated that the frequency of replicon initiation per unit length DNA at a given time was reduced in the revertant cells growing at 39 degrees C.  相似文献   

15.
Soluble isoforms of the epidermal growth factor receptor (sEGFR) previously have been identified in the conditioned culture media (CCM) of the vulvar adenocarcinoma cell line, A431 and within exosomes of the keratinocyte cell line HaCaT. Here, we report that the extracellular domain (ECD) of EGFR is shed from the cell surface of human carcinoma cell lines that express 7 × 105 receptors/cell or more. We purified this proteolytic isoform of EGFR (PI-sEGFR) from the CCM of MDA-MB-468 breast cancer cells. The amino acid sequence of PI-sEGFR was determined by reverse-phase HPLC nano-electrospray tandem mass spectrometry of peptides generated by trypsin, chymotrypsin or GluC digestion. The PI-sEGFR protein is identical in amino acid sequence to the EGFR ECD. The release of PI-sEGFR from MDA-MB-468 cells is enhanced by phorbol 12-myristate 13-acetate, heat-inactivated fetal bovine serum, pervanadate, and EGFR ligands (i.e., EGF and TGF-α). In addition, 4-aminophenylmercuric acetate, an activator of metalloproteases, increased PI-sEGFR levels in the CCM of MDA-MB-468 cells. Inhibitors of metalloproteases decreased the constitutive shedding of EGFR while the PMA-induced shedding was inhibited by metalloprotease inhibitors, by the two serine protease inhibitors leupeptin and 3,4-dichloroisocoumarin (DCI), and by the aspartyl inhibitor pepstatin. These results suggest that PI-sEGFR arises by proteolytic cleavage of EGFR via a mechanism that is regulated by both PKC- and phosphorylation-dependent pathways. Our results further suggest that when proteolytic shedding of EGFR does occur, it is correlated with a highly malignant phenotype.  相似文献   

16.
The initial rates of deoxy-D-glucose transport by cultures of growing and density-inhibited mouse embryo cells and lines of mouse cells transformed spontaneously or after infection by murine leukemia virus or murine sarcoma virus were investigated as a function of the deoxyglucose concentration. The apparent Km for deoxyglucose transport was about the same for all types of cells (1–2 mM). The Vmax of secondary cultures of mouse embryo cells decreased from 6 nmoles/106 cells/minute for sparse cultures to less than 1 nmole/106 cells/minute for density-inhibited cultures. The Vmax was about the same whether estimated in monolayer culture or in suspensions of cells dispersed by treatment with trypsin. The Vmax for deoxyglucose transport by the established cells, whether transformed spontaneously or by virus infection, was 4 to 25 times higher than that for density-inhibited mouse embryo cells and was independent of the cell density of the cultures. Deoxyglucose transport was competitively inhibited by Cytochalasin B, Persantin, glucose and 3-O-methyl-D-glucose and the apparent Ki values of inhibition were similar for the mouse embryo cells and the various cell lines. Similarly, the sensitivity of the glucose transport systems to inactivation by p-chloromercuribenzoate was about the same for all types of cells. The results suggest that the glucose transport system of the normal mouse embryo cells and the cells of the various established lines is qualitatively the same, but that the number of functional transport sites differs for the various cell lines and decreases markedly in mouse embryo cells with an increase in cell density of the cultures.  相似文献   

17.
We studied intracellular activity of the plasminogen activator within the cell cycle of chemically synchronized normal and RSV-transformed chick fibrolasts in culture. Consideration has also been given to the relationship between the plasminogen activator activity and cycles of DNA synthesis or mitosis in cycling fibroblasts after viral infection. The plasminogen activator activity of the cell lysates was assayed on [125I]fibrin-coated Petri dishes and was expressed as the radioactivity released from the plates. Normal fibroblasts produced detectable levels of plasminogen activator in the S-phase and late G2-phase or mitosis of the cell cycle. In contrast, RSV-transformed cells produced high levels of this activator throughout the entire cell cycle although this activity fluctuated and reached a maximum in the G2-M periods. We also found that the level of plasminogen activator activity in the transformed fibroblasts is influenced by the cycles of DNA synthesis and that cell division is required for the appearance of plasminogen activator activity in the ‘de novo’ virus-infected cultures.  相似文献   

18.
The activity of histidase was studied in (1) epidermal tissue scraped from human infant foreskin, (2) fibroblast-like cells in monolayer serial culture from human foreskin, and (3) epithelial-like (epidermal) outgrowth from foreskin primary explants. Foreskin epidermal tissue without in vitro culture and epidermal outgrowth in primary culture from explants of foreskin showed equivalent mean levels of histidase activity, 5.22 × 10?3 and 5.01 × 10?3 μMoles urocanic acid produced per milligram protein per minute. Under the same assay conditions, there was no measurable histidase activity in cultured fibroblast-like cells from foreskin at various times after subculture. The Km for enzyme from human foreskin epidermal tissue ranged between 2 and 5 × 10?3 M histidine. Ability to demonstrate the presence or absence of this tissue-specific enzyme function in cultured cells suggests a useful means for studying differentiation, as well as a more precise way to identify epidermal origin of cultured cell types than morphological characteristics alone would permit.  相似文献   

19.
The movement and internalization of 3H-labelled cells and of inert polystyrene microspheres within multicellular spheroids has been examined through histological sectioning and autoradiography. EMT6 and RIF-1 spheroids were cultured in spinner flasks for approx. 2.5 weeks. At this time, 3H-labelled cells and/or microspheres were allowed to adhere to the spheroid surface. Microspheres, 3H-labelled RIF-1 monolayer cells and 3H-labelled EMT6 monolayer cells were observed to move centripetally as a wave into EMT6 spheroids. In contrast, 3H-labelled trypsinized RIF-1 and EMT6 spheroid cells became mixed with the other non-labelled spheroid cells in homotypic RIF-1 and EMT6 spheroids, respectively. Reduction of spheroid growth by maintaining the spheroids at room temperature and by treatment with 2500 rads irradiation did not prohibit the internalization of 3H-labelled EMT6 cells and microspheres in EMT6 spheroids.  相似文献   

20.
Malignantly transformed mouse fibroblasts synthesize and secrete large amounts of major excreted protein (MEP), a 39,000-dalton precursor to an acid protease (cathepsin L). To evaluate the possible role of this protease in the transformed phenotype, we transfected cloned genes for mouse or human MEP into mouse NIH 3T3 cells with an expression vector for the dominant, selectable human multidrug resistance (MDR1) gene. The cotransfected MEP sequences were efficiently coamplified and transcribed during stepwise selection for multidrug resistance in colchicine. The transfected NIH 3T3 cell lines containing amplified MEP sequences synthesized as much MEP as did Kirsten sarcoma virus-transformed NIH 3T3 cells. The MEP synthesized by cells transfected with the cloned mouse and human MEP genes was also secreted. Elevated synthesis and secretion of MEP by NIH 3T3 cells did not change the nontransformed phenotype of these cells.  相似文献   

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