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1.
Eur J Histochem ; 54(2): e14, 2010 Apr 13.
Article in English | MEDLINE | ID: mdl-20558337

ABSTRACT

The dynamic reorganization of the actin cytoskeleton is regulated by a number of actin binding proteins (ABPs). Four human colon adenocarcinoma cell lines - parental and three selected sublines, which differ in motility and metastatic potential, were used to investigate the expression level and subcellular localization of selected ABPs. Our interest was focused on cofilin and ezrin. These proteins are essential for cell migration and adhesion. The data received for the three more motile adenocarcinoma sublines (EB3, 3LNLN, 5W) were compared with those obtained for the parental LS180 adenocarcinoma cells and fibroblastic NRK cells. Quantitative densitometric analysis and confocal fluorescence microscopy were used to examine the expression levels and subcellular distribution of the selected ABPs. Our data show distinct increase in the level of cofilin in adenocarcinoma cells accompanied by the reduction of inactive phosphorylated form of cofilin. In more motile cells, cofilin was accumulated at cellular periphery in co-localization with actin filaments. Furthemore, we indicated translocation of ezrin towards the cell periphery within more motile cells in comparison with NRK and parental adenocarcinoma cells. In summary, our data indicate the correlation between migration ability of selected human colon adenocarcinoma sublines and subcellular distribution as well as the level of cofilin and ezrin. Therefore these proteins might be essential for the higher migratory activity of invasive tumor cells.


Subject(s)
Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Cofilin 1/metabolism , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Cytoskeletal Proteins/metabolism , Cell Adhesion , Cell Movement , Cofilin 1/analysis , Cytoskeletal Proteins/analysis , Humans , Immunohistochemistry , Neoplasm Metastasis , Tumor Cells, Cultured
2.
J Physiol Pharmacol ; 60 Suppl 4: 15-22, 2009 Oct.
Article in English | MEDLINE | ID: mdl-20083847

ABSTRACT

BACKGROUND: Lumican is a small leucine-rich proteoglycan (SLRP) of the extracellular matrix (ECM) involved in the control of melanoma growth and invasion. The aim of the present study was to analyse the role of lumican in the regulation of the development of lung metastasis. METHODS: B16F1 melanoma cells stably transfected with lumican expressing plasmid (Lum-B16F1) were injected to syngenic mice. The lung metastasis was compared to mice injected with mock-transfected B16F1 cells (Mock-B16F1). The expression of lumican, cyclin D1, apoptotic markers, vascular endothelium growth factor (VEGF) and Von Willebrand Factor (vWF) within lung metastasis nodules was investigated by immunohistochemistry. In parallel, cells cultured in presence of lumican were assayed for apoptosis and motility. RESULTS: We observed that the number and the size of lung metastasis nodules were significantly decreased in mice injected with Lum-B16F1 cells in comparison to Mock-B16F1 cells. This was associated with an increase of tumour cell apoptosis within metastasis nodules but the cell proliferation rate remained constant in the two mice groups. In contrast, the VEGF immunostaining and the number of blood vessels within the lung metastasis nodules were decreased in the lumican-expressing tumours. In vitro, a significant decrease of apoptotic markers in wild type B16F1 cells incubated with increasing amounts of lumican core protein was observed. In addition, pseudotubes formation on Matrigel(R) and the migratory capacity of endothelial cells was inhibited by lumican. Altogether, our results indicate that lumican decreases lung metastasis development not only by inducing tumour cell apoptosis but also by inhibiting angiogenesis.


Subject(s)
Antineoplastic Agents , Chondroitin Sulfate Proteoglycans/pharmacology , Keratan Sulfate/pharmacology , Lung Neoplasms/prevention & control , Lung Neoplasms/secondary , Melanoma, Experimental/drug therapy , Angiogenesis Inhibitors/pharmacology , Animals , Apoptosis/drug effects , Blotting, Western , Cell Movement/drug effects , Cells, Cultured , Endothelial Cells/metabolism , Female , Humans , Image Processing, Computer-Assisted , Lumican , Lung Neoplasms/pathology , Melanoma, Experimental/pathology , Mice , Mice, Inbred C57BL , Recombinant Proteins/biosynthesis , Recombinant Proteins/pharmacology , Vascular Endothelial Growth Factor A/biosynthesis
3.
J Physiol Pharmacol ; 57 Suppl 7: 111-23, 2006 Nov.
Article in English | MEDLINE | ID: mdl-17228099

ABSTRACT

Our studies were focused on the isolation and characterization of highly motile fraction of cells from hepatoma Morris 5123 population. Cells that underwent several migration cycles through Matrigel - coated filters were successfully cultured. The invasion index was determined by means of Matrigel invasion assay. Statically significant increase in the value of invasion factor for selected cells variant in comparison to the parental population was observed. The considerable changes in the cell shape were followed by the reorganization of the actin cytoskeleton structure including a dense subcortical congestion in the distribution of beta -actin isoform. The visualization of this protein in tumor cells was performed by immunostaining and scanning fluorescent confocal microscopy. The results were confirmed by densitometry analysis of Western blots. In addition, the increased state of actin polymerization in the cytoplasmic fraction of selected cells was determined as measured by filamentous to monomeric (F:G) actin ratio. Concluding, the selected fraction of hepatoma Morris 5123 cells with higher invasion capacity was characterized by rounded shape, remarkable increase of beta -actin level, its submembrane concentration as well as with the increased state of actin polymerization with respect to parental cells population.


Subject(s)
Actins/biosynthesis , Cytoskeleton/metabolism , Liver Neoplasms, Experimental/metabolism , Liver Neoplasms, Experimental/pathology , Animals , Cell Movement/physiology , Cell Separation/methods , Collagen , Cytoskeleton/pathology , Cytosol/metabolism , Drug Combinations , Laminin , Neoplasm Invasiveness , Proteoglycans , Rats , Tumor Cells, Cultured
4.
J Inorg Biochem ; 98(12): 2087-98, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15541498

ABSTRACT

The complexation processes of vanadium(III) with L-cysteinate and s-methyl-L-cysteinate ligands have been studied in aqueous solutions in the pH range 2-7 by the pH-potentiometric, UV-Vis absorption and CD spectroscopy methods. The equilibria model of complex formation, evaluated by SUPERQUAD program, so as careful inspection of spectroscopic data have allowed to determine the speciation and the coordination mode of vanadium(III) ion in the major species present in aqueous solutions. Relatively stable ML2 species of vanadium(III)-L-cysteinate system exists in aqueous solutions above pH 5. It was deduced from spectral data that the coordination sphere of vanadium(III) ion in V(Cys)2 is completed by oxygen, nitrogen and sulfur atoms of two L-cysteinate ligands. Solution of vanadium(III) with L-cysteine (pH approximately 7, L/M=20) was administrated to the culture medium of hepatoma Morris 5123 growing cells. Cytotoxic effect of this solution towards tumor cells was observed. The viability of these cells depended on the complex concentration. It was reduced by 70% at 100 microM of the vanadium species concentration in the culture medium. The death of cancer cells seems to be induced on apoptotic route. The statistically significant increase of total actin level and filamentous to monomeric actin ratios (F/G) were found in the cytoplasm of cells exposed to the vanadium(III)-L-cysteine complex. It was accompanied by the rearrangement of actin cytoskeleton architecture. These factors are important for migration and metastasis formation of the cancer cells.


Subject(s)
Actins/drug effects , Cysteine/chemistry , Organometallic Compounds/chemistry , Vanadium/chemistry , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Fractionation , Cell Survival , Cells, Cultured , Circular Dichroism , Cysteine/pharmacology , Cytosol/chemistry , Fluorescent Dyes , Hydrogen-Ion Concentration , Ligands , Liver Neoplasms, Experimental/drug therapy , Microscopy, Fluorescence , Organometallic Compounds/pharmacology , Phalloidine , Potentiometry , Rats , Spectrophotometry, Ultraviolet , Vanadium/pharmacology , Water/chemistry
5.
Acta Biochim Pol ; 48(4): 1051-60, 2001.
Article in English | MEDLINE | ID: mdl-11995967

ABSTRACT

Monomeric (G), total (T) and filamentous (F) actin and the state of actin polymerisation (F:G) were determined and actin filaments were visualized in hepatoma Morris 5123 cells cultured in the presence of methotrexate (MTX) at various concentration. The exposure of the cells to this drug resulted in a decrease of total and polymerised actin in cytoplasm and in some changes in actin filament organization. This coincided with a decrease of the cells' ability to migrate through Matrigel coated filters and with inhibition of tumour formation after reimplantation of the methotrexate treated cells to experimental rats.


Subject(s)
Actins/metabolism , Carcinoma, Hepatocellular/drug therapy , Methotrexate/pharmacology , Animals , Antimetabolites, Antineoplastic/pharmacology , Biocompatible Materials/chemistry , Cell Movement , Collagen/pharmacology , Cytoplasm/metabolism , Cytosol/metabolism , Dose-Response Relationship, Drug , Drug Combinations , Laminin/pharmacology , Microscopy, Fluorescence , Neoplasm Invasiveness , Proteoglycans/pharmacology , Rats , Tumor Cells, Cultured
6.
Biochim Biophys Acta ; 1451(1): 141-52, 1999 Aug 12.
Article in English | MEDLINE | ID: mdl-10446396

ABSTRACT

The aim of this study was to isolate and to characterize actin from the carp liver cytosol and to examine its ability to polymerize and interact with bovine pancreatic DNase I. Carp liver actin was isolated by ion-exchange chromatography, followed by gel filtration and a polymerization/depolymerization cycle or by affinity chromatography using DNase I immobilized to agarose. The purified carp liver actin was a cytoplasmic beta-actin isoform as verified by immunoblotting using isotype specific antibodies. Its isoelectric point (pI) was slightly higher than the pI of rabbit skeletal muscle alpha-actin. Polymerization of purified carp liver actin by 2 mM MgCl(2) or CaCl(2) was only obtained after addition of phalloidin or in the presence of 1 M potassium phosphate. Carp liver actin interacted with DNase I leading to the formation of a stable complex with concomitant inhibition of the DNA degrading activity of DNase I and its ability to polymerize. The estimated binding constant (K(b)) of carp liver actin to DNase I was calculated to be 1.85x10(8) M(-1) which is about 5-fold lower than the affinity of rabbit skeletal muscle alpha-actin to DNase I.


Subject(s)
Actins/isolation & purification , Deoxyribonuclease I/metabolism , Liver/metabolism , Actins/chemistry , Actins/metabolism , Animals , Carps , Chromatography, Affinity , Chromatography, Gel , Chromatography, Ion Exchange , Cytosol/metabolism , Deoxyribonuclease I/antagonists & inhibitors , Electrophoresis, Gel, Two-Dimensional , Immunoblotting , Polymers , Spectrometry, Fluorescence
7.
Acta Biochim Pol ; 46(4): 949-59, 1999.
Article in English | MEDLINE | ID: mdl-10824864

ABSTRACT

The hepatoma Morris 5123 tumor growth is accompanied by changes in actin content and polymerization (Malicka-Blaszkiewicz et al. (1995) Mat. Med. Pol., 27, 115-118; Nowak et al. (1995) J. Exp. Cancer Res. 14, 37-40). Presently actin isoforms from cytosol and cytoskeleton fractions were separated by SDS/PAGE and identified with antibodies directed against different actin isoforms. Actin isolated from the cytosol by affinity chromatography on DNase I bound to agarose shows the presence of only one protein spot on 2D gel electrophoresis corresponding to the mobility of the rabbit a skeletal muscle actin (Mr 43,000) and isoelectric point equal to 5.3. It interacts only with monoclonal anti beta actin isoform antibodies, posing the question of differential affinity of actin isoforms to DNase I.


Subject(s)
Actins/isolation & purification , Liver Neoplasms, Experimental/chemistry , Actins/immunology , Actins/metabolism , Animals , Antibodies, Monoclonal , Chromatography, Affinity , Cytoskeleton/chemistry , Cytosol/chemistry , Electrophoresis, Gel, Two-Dimensional , Female , Immunoblotting , Isoelectric Focusing , Isoelectric Point , Liver Neoplasms, Experimental/metabolism , Mice , Protein Isoforms/immunology , Protein Isoforms/isolation & purification , Protein Isoforms/metabolism , Rabbits , Rats , Rats, Inbred BUF
9.
Mater Med Pol ; 27(3): 115-8, 1995.
Article in English | MEDLINE | ID: mdl-8935149

ABSTRACT

Monomeric G-actin, total actin and filamentous F-actin were examined during the growth of experimental tumour hepatoma Morris 5123. Actin was measured by the inhibition of the standard DNase I from bovine pancreas. A remarkable increase in total actin, and F-actin content, as well as in the state of actin polymerization (measured by the F:G actin ratio) was shown in the cytosol of the tumour cells in the second week of the tumour growth, followed by a rapid decrease in the third week. Parallel observations were made in the cytosol of the liver and in the serum of the tumour bearing rats. The results were compared with the data obtained for control healthy rats. It was shown that hepatoma Morris tumour growth is accompanied by the changes in the actin content and polymerization, occurring also in the liver of the host animal. Only G-actin was found in the serum. It increased significantly in the second week of tumour growth as compared with the G-actin level in the serum of the control healthy rats.


Subject(s)
Actins/blood , Actins/metabolism , Liver Neoplasms, Experimental/blood , Liver Neoplasms, Experimental/metabolism , Animals , Cell Division/physiology , Male , Rats , Rats, Inbred BUF
10.
Int J Biochem ; 26(9): 1147-55, 1994 Sep.
Article in English | MEDLINE | ID: mdl-7988739

ABSTRACT

1. DNase-I-like activity occurs in the carp (Cyprinus carpio) liver cytosol (supernatant 105,000 g). 2. The enzyme resembles DNase I from bovine pancreas in respect to the molecular mass (approximately 31 kDa), pH (7.4) and ion requirements (Mg2+, Ca2+) and the ability to degrade native as well as denatured DNA. 3. As judged by comparison of DNase zymograms obtained after native- and SDS-PAGE, the enzyme occurs in the three molecular forms of similar molecular weight and different charges. 4. All these forms are inhibited by rabbit skeletal muscle actin as well as by endogenous actin isolated from the carp liver cytosol. 5. DNase from the carp liver cytosol does not interact with the antibodies directed against DNase I from bovine pancreas and against DNase I from the rat and bovine parotid glands.


Subject(s)
Actins/pharmacology , Deoxyribonuclease I/metabolism , Liver/enzymology , Muscle, Skeletal/metabolism , Animals , Antibodies, Monoclonal/metabolism , Antibody Specificity , Calcium/metabolism , Carps , Cattle , Chromatography, Gel , Collodion/chemistry , Cytosol/enzymology , DNA/metabolism , Deoxyribonuclease I/antagonists & inhibitors , Deoxyribonuclease I/isolation & purification , Electrophoresis, Polyacrylamide Gel , Hydrogen-Ion Concentration , Immunoblotting , Magnesium/metabolism , Molecular Weight , Parotid Gland/enzymology , Rats , Spectrophotometry, Ultraviolet
11.
Z Naturforsch C J Biosci ; 45(11-12): 1165-70, 1990.
Article in English | MEDLINE | ID: mdl-2095786

ABSTRACT

Monomeric G actin, total actin and the F:G actin ratio were determined in the rat liver cytosol and nucleoplasm by measuring the inhibition of standard crystalline DNase I. Actin was purified from rat liver nucleoplasm by Sephadex filtration. Accompanying the considerable amount of actin an endogenous DNase I like activity was found in rat liver cytosol and nucleoplasm. It was shown, that similarly to DNase I from bovine pancreas the liver DNase was inhibited by mammalian and avian skeletal muscle actin as well as by endogenous liver actin, as verified by electrophoresis of DNase containing extracts on polyacrylamide gels with incorporated DNA.


Subject(s)
Actins/metabolism , Deoxyribonuclease I/metabolism , Liver/enzymology , Animals , Cell Nucleus/enzymology , Chromatography, Gel , Chromatography, Ion Exchange , Cytosol/enzymology , Deoxyribonuclease I/isolation & purification , Isoenzymes/isolation & purification , Isoenzymes/metabolism , Rats , Rats, Inbred Strains
12.
J Inorg Biochem ; 32(1): 39-65, 1988 Jan.
Article in English | MEDLINE | ID: mdl-3346661

ABSTRACT

Studies of the solution properties of gold(III)tetrakis(4-N-methylpyridyl) porphine and its DNA binding characteristics have been conducted utilizing uv/vis absorption spectroscopy, circular dichroism (CD), Mossbauer spectroscopy, and temperature-jump relaxation techniques. These studies indicate that over the concentration range considered this water soluble gold(III) porphyrin does not aggregate, binds axial ligands only weakly with a preference for soft Lewis bases, and is capable of intercalation into nucleic acids of appropriate base pair content. The interaction of this and several other porphyrins with the synthetic polynucleotide poly(dA-dC).poly(dT-dG) has been studied. Spectroscopic signatures for intercalation were found for those derivatives not having axial ligands. Intercalation into chromatin in vitro can also occur with those porphyrins and metalloporphyrins which do not have axial ligands. Finally, studies utilizing microinjection techniques indicate that once within the cell, tetrakis(4-N-methylpyridyl)porphine tends to localize in the nucleus.


Subject(s)
DNA , Polydeoxyribonucleotides , Porphyrins , Animals , Chromatin/metabolism , Gold , Kinetics , Liver/metabolism , Male , Metalloporphyrins , Rats , Rats, Inbred Strains , Spectrophotometry , Spectrum Analysis
13.
Comp Biochem Physiol B ; 84(2): 207-9, 1986.
Article in English | MEDLINE | ID: mdl-3488171

ABSTRACT

Total actin content and F:G actin ratio were determined in the liver cytosol of fish, frogs and mouse by measurements of inhibition of exogenous crystalline bovine pancreatic DNase I. Endogenous DNase I-like activity, was found in all examined livers after electrophoresis in the presence of sodium dodecyl sulfate and subsequent enzyme renaturation. It is concluded that DNase I-like enzymes occur in the liver cytosol in a latent form, probably bound to cytoplasmic actin.


Subject(s)
Actins/analysis , Deoxyribonuclease I/metabolism , Liver/metabolism , Animals , Cyprinidae , Cytosol/analysis , Mice , Rana catesbeiana , Rana esculenta , Species Specificity
14.
FEBS Lett ; 153(1): 235-9, 1983 Mar 07.
Article in English | MEDLINE | ID: mdl-6219006

ABSTRACT

L1210 leukemia cell cytosol was analysed for the presence of DNase I activity. No free activity was determined in crude cytosol. DNase I enzyme was found to occur in a latent form bound to cytoplasmic actin. DNase-actin complex was partially isolated by Sephadex filtration and DNase I-like activity was demonstrated after SDS gel electrophoresis of the complex and enzyme renaturation. The results were compared with those for synthetic complex of pancreatic bovine DNase I and chicken muscle actin.


Subject(s)
Actins/metabolism , Endodeoxyribonucleases/metabolism , Leukemia L1210/enzymology , Animals , Chromatography, Gel , Cytosol/enzymology , Deoxyribonuclease I , Electrophoresis, Polyacrylamide Gel , Mice , Mice, Inbred DBA
16.
Acta Physiol Pol ; 32(3): 317-26, 1981.
Article in English | MEDLINE | ID: mdl-6975551

ABSTRACT

The aim of our work was to investigate the biological basis of the existence of multiple alkaline RNAses in the Rana esculenta liver with respect to formation of a RNAse-RNAse inhibitor complex (latent RNAse). Subcellular distribution indicated that most of the alkaline RNAse activity appeared in the high-speed supernatant and mainly in a latent form bound to an endogenous inhibitor. This RNAse-RNAse inhibitor complex was isolated, the bound RNAse activity was released and found to correspond to RNAse III and RNAse IV, the two most cathodic RNAses present in the supernatant fraction. The two RNAses differed markedly in the ability to interact with the inhibitor and this might be essential for the different biological roles the RNAses may play in the regulation of cytoplasmic RNA level. On the basis of molecular weight determinations we assume that the enzyme-inhibitor complex is shared by two different RNAses.


Subject(s)
Enzyme Inhibitors/metabolism , Microsomes, Liver/enzymology , Mitochondria, Liver/enzymology , Ribonucleases/metabolism , Animals , Binding Sites , Rana esculenta , Ribonucleases/antagonists & inhibitors
17.
Acta Biochim Pol ; 26(3): 275-83, 1979.
Article in English | MEDLINE | ID: mdl-40372

ABSTRACT

1. RNAases varying in pH optimum, activation with pCMB, sensitivity towards temperature and acid treatment, as well as electrophoretic mobility were found in Rana esculenta liver extract. 2. Of the three activity peaks of alkaline ribonuclease separated on CM-cellulose with 2000-fold purification, RNAase of peak C is thermo- and acid-stable and exhibits specificity for pyrimidine bases, preferring poly(U) over poly(C). 3. Differences in the specific "inhibitory effect" of frog liver supernatant on the frog liver alkaline RNAase were observed.


Subject(s)
Liver/enzymology , Ribonucleases/isolation & purification , Animals , Anura , Chromatography , Electrophoresis, Polyacrylamide Gel , Hydrogen-Ion Concentration , Rana esculenta/metabolism , Ribonucleases/antagonists & inhibitors , Substrate Specificity
18.
Acta Biochim Pol ; 25(4): 349-59, 1978.
Article in English | MEDLINE | ID: mdl-35912

ABSTRACT

1. Acid phosphatase (AcPase) from liver of the frog, Rana esculenta has been isolated and purified. The enzyme is heterogeneous, showing 4 activity zones on disc electrophoresis. The AcPase was separated into 3 peaks on DEAE-cellulose. Peak A corresponding to the electrophoretic AcPase IV represents an extensively purified enzyme form. 2. The separated enzyme forms are change isomers with a molecular weight of about 33,000. They differ markedly in substrate requirements and sensitivity towards activators and inhibitors. All of them are highly activated by dithiothreitol, show a rather restricted substrate specificity, and marked activity against ATP.


Subject(s)
Acid Phosphatase/isolation & purification , Liver/enzymology , Rana esculenta/metabolism , Acid Phosphatase/antagonists & inhibitors , Animals , Anura , Chromatography, DEAE-Cellulose , Chromatography, Gel , Enzyme Activation/drug effects , Hydrogen-Ion Concentration , Molecular Weight , Substrate Specificity , Temperature
19.
Acta Microbiol Pol A ; 8(1): 71-7, 1976.
Article in English | MEDLINE | ID: mdl-820164

ABSTRACT

Commercial nisin was fractionated using a Bio-Gel P-10 column and ion-exchange chromatography on CM-sephadex C-25. Pure nisin having a titre of 40 X 10(6) units per gram was obtained. In polyacrylamide-gel electrophoresis the pure nisin gave three bands. It is suggested that heterogeneity of nisin is due to the presence of several biological polypeptides. The pure nisin is digested by chymotrypsin but it is not affected by TPCK-trypsin and pepsin.


Subject(s)
Nisin/isolation & purification , Chromatography, Gel , Chromatography, Ion Exchange , Chymotrypsin/metabolism , Culture Media/analysis , Lactococcus lactis/metabolism , Molecular Weight , Nisin/analysis , Nisin/biosynthesis , Pepsin A/metabolism , Peptides/analysis , Trypsin/metabolism
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