RESUMO
BACKGROUND: Acute thrombotic microangiopathies (TMAs) are characterized by excessive microvascular thrombosis and are associated with markers of neutrophil extracellular traps (NETs) in plasma. NETs are composed of DNA fibers and promote thrombus formation through the activation of platelets and clotting factors. OBJECTIVE: The efficient removal of NETs may be required to prevent excessive thrombosis such as in TMAs. To test this hypothesis, we investigated whether TMAs are associated with a defect in the degradation of NETs. METHODS AND RESULTS: We show that NETs generated in vitro were efficiently degraded by plasma from healthy donors. However, NETs remained stable after exposure to plasma from TMA patients. The inability to degrade NETs was linked to a reduced DNase activity in TMA plasma. Plasma DNase1 was required for efficient NET degradation and TMA plasma showed decreased levels of this enzyme. Supplementation of TMA plasma with recombinant human DNase1 restored NET-degradation activity. CONCLUSIONS: Our data indicate that DNase1-mediated degradation of NETs is impaired in patients with TMAs. The role of plasma DNases in thrombosis is, as of yet, poorly understood. Reduced plasma DNase1 activity may cause the persistence of pro-thrombotic NETs and thus promote microvascular thrombosis in TMA patients.
Assuntos
Desoxirribonuclease I/metabolismo , Armadilhas Extracelulares/metabolismo , Neutrófilos/metabolismo , Microangiopatias Trombóticas/sangue , Humanos , HidróliseRESUMO
BACKGROUND: Injections with phosphatidylcholine- and deoxycholate-containing substances are used to treat localized fat accumulation and lipomas. It is believed that the injected substances induce fat cell destruction with subsequent acute panniculitis followed by a repair process of the treated fat tissue. OBJECTIVES: We investigated whether necrosis or apoptosis of fat cells was induced by the injected substances. METHODS: Samples of fat tissue of lipoma were collected at various times after injection and evaluated by light and electron microscopy, by immunostaining for active caspase-3 and antideoxyribonuclease I, in situ end-labelling (TUNEL staining), and biochemical caspase-3 assays. RESULTS: Light and electron microscopy showed fat cell necrosis in all areas of the treated lipomas. Low levels of active caspase-3 indicated the absence of apoptosis. CONCLUSIONS: Injection of the lipolytic substances phosphatidylcholine and deoxycholate leads to fat cell necrosis rather than apoptosis. However, additional studies evaluating different dosing and further time points after treatment are necessary.
Assuntos
Tecido Adiposo/efeitos dos fármacos , Ácido Desoxicólico/farmacologia , Necrose , Fosfatidilcolinas/farmacologia , Tecido Adiposo/citologia , Caspase 3/metabolismo , Desoxirribonuclease I/metabolismo , Humanos , Marcação In Situ das Extremidades Cortadas , Lipólise , Microscopia EletrônicaRESUMO
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.
Assuntos
Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Cofilina 1/metabolismo , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Proteínas do Citoesqueleto/metabolismo , Adesão Celular , Movimento Celular , Cofilina 1/análise , Proteínas do Citoesqueleto/análise , Humanos , Imuno-Histoquímica , Metástase Neoplásica , Células Tumorais CultivadasRESUMO
Relaying a signal across the plasma membrane requires functional connections between the partner molecules. Membrane microdomains or lipid rafts provide an environment in which such specific interactions can take place. The integrity of these sites is often taken for granted when signalling pathways are investigated in cell culture. However, it is well known that smooth muscle and endothelial cells undergo cytoskeletal rearrangements during monolayer culturing. Likewise affected--and with potentially important consequences for signalling events--is the organization of the plasma membrane. The expression levels of three raft markers were massively upregulated, and raft-associated 5'-nucleotidase activity increased in conventional monolayer cultures as compared with a spheroidal coculture model, shown to promote the differentiation of endothelial cells. Our data point to a shift of raft components in monolayer cultures and demonstrate potential advantages of the spheroid coculture system for investigation of raft-mediated signalling events in endothelial cells.
Assuntos
5'-Nucleotidase/metabolismo , Anexina A2/metabolismo , Anexina A6/metabolismo , Células Endoteliais/metabolismo , Microdomínios da Membrana/metabolismo , Miócitos de Músculo Liso/metabolismo , 5'-Nucleotidase/genética , Anexina A2/genética , Anexina A6/genética , Aorta/metabolismo , Biomarcadores , Humanos , Microdomínios da Membrana/enzimologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esferoides Celulares/metabolismo , Regulação para Cima/fisiologiaRESUMO
Deoxyribonuclease 1 (Dnase1)-deficient mice develop symptoms of Systemic lupus erythematosus (SLE). Here we analysed the renal histopathology of these animals in comparison to F1 hybrids of New Zealand black and white mice (NZB/W F1), an established model of SLE. Animals were divided into three groups according to the presence of anti-nuclear antibodies (ANA) and renal lesions. Groups 1a-1c were healthy, whereas group 2 and 3 were classified as lupus-prone and affected. Subendothelial and/or mesangial immune complex deposits, mesangial and endocapillary proliferation, haematoxylin bodies and platelet aggregation were detected in both mouse strains but were more severe in the NZB/W F1 mice. The lupus nephritis was classified as a proliferating (WHO type III or IV), which appeared to be preceded by a mesangial form (WHO type II). Subclassification of the ANA revealed a high prevalence of anti-nucleosome antibodies in Dnase1-deficient mice, whereas NZB/W F1 mice developed autoantibodies against a broad range of chromatin constituents. Mapping of the murine Dnase1 gene locus to chromosome 16A1-3 did not coincide with one of the reported susceptibility loci in the NZB/W F1 model, although a reduced Dnasel serum and urine activity has been described previously in these mice.
Assuntos
Desoxirribonuclease I/genética , Nefrite Lúpica/genética , Nefrite Lúpica/patologia , Animais , Complexo Antígeno-Anticorpo/análise , Modelos Animais de Doenças , Feminino , Glomérulos Renais/imunologia , Glomérulos Renais/patologia , Glomérulos Renais/ultraestrutura , Nefrite Lúpica/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NZB , Camundongos Knockout , MicrotomiaRESUMO
Actin ADP-ribosylated at Arg177 was previously shown not to polymerise after increasing the ionic strength, but to cap the barbed ends of filaments. Here we confirm that the polymerisation of ADP-ribosylated actin is inhibited, however, under specific conditions the modified actin copolymerises with native actin, indicating that its ability to take part in normal subunit interactions within filaments is not fully eliminated. We also show that ADP-ribosylated actin forms antiparallel but not parallel dimers: the former are not able to form filaments. ADP-ribosylated actin interacts with deoxyribonuclease I, vitamin D binding protein, thymosin beta(4), cofilin and gelsolin segment 1 like native actin. Interaction with myosin subfragment 1 revealed that the potential of the modified actin to aggregate into oligomers or short filaments is not fully eliminated.
Assuntos
Actinas/metabolismo , Adenosina Difosfato Ribose/metabolismo , Proteínas dos Microfilamentos/metabolismo , Fatores de Despolimerização de Actina , Actinas/química , Animais , Compostos de Dansil/metabolismo , Eletroforese em Gel de Poliacrilamida , Gelsolina/metabolismo , Humanos , Indicadores e Reagentes/metabolismo , Músculo Esquelético/química , Polímeros/química , Polímeros/metabolismo , CoelhosRESUMO
The intracellular distribution of gelsolin in NIH 3T3 cells was examined by immunostaining using affinity-purified polyclonal gelsolin antibodies before and after induction of apoptosis by serum withdrawal. Serum deprivation induced detachment of an increasing number of NIH 3T3 cells, but also apoptosis in attached cells as verified morphologically by chromatin condensation, nuclear fragmentation and labelling of their periphery by FITC-annexin V. Ongoing apoptosis was also demonstrated by activation of caspase-3 activity and chromatin cleavage into high-molecular-mass fragments, although no internucleosomal chromatin degradation (DNA-ladder formation) was detected. When cells were maintained in the presence of 10% foetal calf serum, gelsolin immunoreactivity was evenly distributed in the cytoplasm. No obvious co-localisation of gelsolin and the actin-containing stress fibres was detected under these conditions. At day one after serum withdrawal, a redistribution of gelsolin to actin filaments was detected within a few attached cells by double fluorescence staining. The number of cells exhibiting this redistribution increased at days two to four. In addition, the stress fibres increased in thickness and their length was continuously reduced. At day four, many cells contained shortened stress fibres, which had lost their longitudinal orientation. Additionally, the cytoplasm of a number of attached cells was highly condensed around their nuclei and a homogenous distribution of both gelsolin and actin was detected in the remaining cytoplasmic rim. Up to day two, these effects were reversible after re-addition of serum to attached cells. A similar redistribution of gelsolin immunore-activity was observed after induction of apoptosis by cycloheximide, but not after initiation of necrosis by hydrogen peroxide. In NIH 3T3 cells no alteration in the expression of gelsolin at the level of protein (Western blot) or specific mRNA (Northern blot) was observed after serum withdrawal. Using Western blotting, no proteolysis of gelsolin was detected up to day 4, although caspase-3 activity was found to have increased fivefold after serum withdrawal. These results suggested that in these cells F-actin severing might occur in the absence or advance of gelsolin cleavage by caspases. Intact gelsolin on its own may be sufficient for the dissolution of the microfilaments, since micro-injection of gelsolin into primary bovine lens cells led to a transient disappearance of the stress fibres and to a reduction of their attachment area to the substratum. In NIH 3T3 cells similar effects of micro-injected gelsolin were only observed at day one after serum withdrawal.
Assuntos
Actinas/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Proteínas Sanguíneas/farmacologia , Gelsolina/metabolismo , Células 3T3 , Actinas/análise , Actinas/genética , Animais , Northern Blotting , Western Blotting , Meios de Cultura Livres de Soro/farmacologia , Gelsolina/análise , Gelsolina/genética , Cápsula do Cristalino/citologia , Camundongos , RNA Mensageiro/análise , Ratos , Fibras de Estresse/química , Fibras de Estresse/metabolismoRESUMO
Recently, medium-dose UVA1 phototherapy (50 J/cm2) has been introduced as an effective treatment for severe atopic dermatitis (AD). In order to further elucidate the mechanisms by which medium-dose UVA1 irradiation leads to an improvement in skin status in patients with AD, biopsy specimens from ten patients before and after treatment were analysed immunohistochemically for features of apoptosis. We sought to determine the extent to which UVA1 irradiation was able to modulate the balance between p53 and bcl-2 expression in vivo using monoclonal antibodies labelling these proteins. As compared with lesional skin of patients with AD before UVA1 irradiation, the number of dermal cells, apparently lymphocytes, that were positive for p53 had significantly increased after treatment and, in addition, some basal keratinocytes showed slight positive staining for p53. An increased expression of the bcl-2 gene before treatment in predominately dermal lymphocytes was significantly downregulated by UVA1 therapy. The increase in p53+ cells and the decrease in bcl-2+ cells were closely linked to a significant reduction in dermal T cells (CD3+) and a substantial clinical improvement in skin condition. In summary, medium-dose UVA1 irradiation led to a marked modulation of the expression of p53 and bcl-2, and this plays a key role in regulating UVA1-induced apoptosis.
Assuntos
Dermatite Atópica/metabolismo , Dermatite Atópica/terapia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Terapia Ultravioleta , Idoso , Dermatite Atópica/patologia , Humanos , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , Contagem de Linfócitos , Linfócitos/metabolismo , Linfócitos/efeitos da radiação , Pessoa de Meia-Idade , Pele/efeitos dos fármacos , Pele/metabolismo , Pele/efeitos da radiação , Linfócitos T/metabolismo , Linfócitos T/patologia , Resultado do TratamentoRESUMO
Cutaneous T-cell lymphoma (CTCL) is a malignancy of mature T-cells, predominantly of the helper phenotype, that primarily invade the skin. Different photo- and chemotherapeutic treatments are known to be beneficial in early-stage CTCL. This observation has initiated prospective investigations into the efficacy of phototherapeutic regimens. The purpose of our study was to investigate the ability of medium-dose UVA1 phototherapy (60 J/cm2) to induce apoptosis (programmed cell death) in skin infiltrating T-cells of CTCL in vivo. We describe the results of three different staining methods for formalin-fixed, paraffin-embedded tissue sections. The in situ end-labeling (ISEL) procedure, nuclear staining using the DNA-binding fluorochrome Hoechst 33342, and immunohistochemistry using polyclonal antibodies against recombinant mouse deoxyribonuclease I (DNase I) demonstrated that UVA1 irradiation was able to induce marked apoptosis in CTCL. Thereby, ISEL and Hoechst staining clearly revealed DNA-condensation and nuclear fragmentation, accompanied by the formation of typical "apoptotic bodies". The accumulation of DNase I immunoreactivity in the cytoplasm of lymphocytes in UVA1 irradiated skin indicated that DNase I or DNase I-related endonucleases may have acted as apoptotic endonuclease(s) which were synthesized after UVA1 irradiation prior to their apoptotic elimination.
Assuntos
Apoptose/efeitos da radiação , Desoxirribonuclease I/metabolismo , Linfoma Cutâneo de Células T/metabolismo , Neoplasias Cutâneas/metabolismo , Pele/efeitos da radiação , Terapia Ultravioleta , Idoso , Benzimidazóis , Núcleo Celular/metabolismo , Desoxirribonuclease I/efeitos da radiação , Corantes Fluorescentes , Humanos , Imuno-Histoquímica , Queratinócitos/metabolismo , Linfoma Cutâneo de Células T/patologia , Linfoma Cutâneo de Células T/radioterapia , Masculino , Dosagem Radioterapêutica , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/radioterapiaRESUMO
The ability of two different Jurkat sublines, termed standard and JM, to form DNA ladders was investigated after various apoptotic stimuli. Exposure to a broad spectrum of drugs interfering with signal transduction or cellular metabolism revealed distinct differences between both Jurkat sublines with regard to the pattern of DNA degradation. In standard Jurkat cells, internucleosomal DNA cleavage occurred only after treatment with the protein kinase inhibitor staurosporine. In contrast, the JM subline responded with internucleosomal DNA fragmentation to exposure to gemcitabine, cycloheximide or staurosporine. All drugs induced the formation of DNA fragments of about 50 kb in both sublines, as revealed by pulse field electrophoresis, except H2O2, which caused unspecific DNA degradation. The staurosporine-induced DNA ladder formation was accompanied by an increase in caspase-3 activity in both lines which, however, was considerably lower in Jurkat JM cells after gemcitabine or cycloheximide exposure. When the analysis of internucleosomal DNA degradation was carried out after mycoplasma infection, both Jurkat lines responded with DNA ladder formation after exposure to all drugs used (here only shown for the standard subline). Employing the zymogram technique, nuclease activities of 47 kDa and 54 kDa were detected in culture supernatants, cell homogenates and nuclear extracts only when mycoplasma-infected, whereas the samples obtained from mycoplasma-free sublines were nuclease-negative using this technique, indicating that these endonucleases were of mycoplasmal origin. After drug exposure, the mycoplasmal nucleases must have gained access to the cytoplasm and nuclei of their host cells by an unknown mechanism.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Técnicas de Cultura de Células/métodos , Núcleo Celular/ultraestrutura , Fragmentação do DNA/efeitos dos fármacos , DNA/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Anticorpos/imunologia , Caspase 3 , Caspases/metabolismo , Núcleo Celular/enzimologia , DNA/ultraestrutura , Desoxirribonucleases , Humanos , Peróxido de Hidrogênio/farmacologia , Células Jurkat , Mycoplasma/enzimologia , Estaurosporina/farmacologia , Receptor fas/imunologiaRESUMO
Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease that affects over one million people in the United States. SLE is characterized by the presence of anti-nuclear antibodies (ANA) directed against naked DNA and entire nucleosomes. It is thought that the resulting immune complexes accumulate in vessel walls, glomeruli and joints and cause a hypersensitivity reaction type III, which manifests as glomerulonephritis, arthritis and general vasculitis. The aetiology of SLE is unknown, but several studies suggest that increased liberation or disturbed clearance of nuclear DNA-protein complexes after cell death may initiate and propagate the disease. Consequently, Dnase1, which is the major nuclease present in serum, urine and secreta, may be responsible for the removal of DNA from nuclear antigens at sites of high cell turnover and thus for the prevention of SLE (refs 7-11). To test this hypothesis, we have generated Dnase1-deficient mice by gene targeting. We report here that these animals show the classical symptoms of SLE, namely the presence of ANA, the deposition of immune complexes in glomeruli and full-blown glomerulonephritis in a Dnase1-dose-dependent manner. Moreover, in agreement with earlier reports, we found Dnase1 activities in serum to be lower in SLE patients than in normal subjects. Our findings suggest that lack or reduction of Dnase1 is a critical factor in the initiation of human SLE.
Assuntos
Desoxirribonuclease I/deficiência , Desoxirribonuclease I/metabolismo , Deleção de Genes , Lúpus Eritematoso Sistêmico/enzimologia , Ribonucleoproteínas Nucleares Pequenas , Células 3T3 , Animais , Anticorpos Antinucleares/imunologia , Autoantígenos/imunologia , DNA/imunologia , DNA/metabolismo , Desoxirribonuclease I/sangue , Desoxirribonuclease I/genética , Feminino , Genótipo , Histonas/imunologia , Humanos , Nefropatias/sangue , Nefropatias/enzimologia , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/patologia , Nefrite Lúpica/enzimologia , Nefrite Lúpica/genética , Nefrite Lúpica/imunologia , Nefrite Lúpica/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Nucleossomos/imunologia , Proteinúria/sangue , Proteinúria/enzimologia , RNA Mensageiro/análise , RNA Mensageiro/genética , Proteínas Centrais de snRNPRESUMO
Thymosin beta(4) possesses actin-sequestering activity and, like transglutaminases, is supposed to be involved in cellular events like angiogenesis, blood coagulation, apoptosis and wound healing. Thymosin beta(4) serves as a specific glutaminyl substrate for transglutaminase and can be fluorescently labeled with dansylcadaverine. Two (Gln-23 and Gln-36) of the three glutamine residues were mainly involved in the transglutaminase reaction, while the third glutaminyl residue (Gln-39) was derivatized with a low efficiency. Labeled derivatives were able to inhibit polymerization of G-actin and could be cross-linked to G-actin by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. Fluorescently labeled thymosin beta(4) may serve as a useful tool for further investigations in cell biology. Thymosin beta(4) could provide a specific glutaminyl substrate for transglutaminase in vivo, because of the fast reaction observed in vitro occurring at thymosin beta(4) concentrations which are found inside cells. Taking these data together, it is tempting to speculate that thymosin beta(4) may serve as a glutaminyl substrate for transglutaminases in vivo and play an important role in transglutaminase-related processes.
Assuntos
Actinas/metabolismo , Timosina/metabolismo , Transglutaminases/metabolismo , Sequência de Aminoácidos , Animais , Cadaverina/análogos & derivados , Cadaverina/farmacologia , Bovinos , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Corantes Fluorescentes/farmacologia , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Ligação Proteica/efeitos dos fármacos , Coelhos , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Timosina/fisiologia , Fatores de TempoRESUMO
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.
Assuntos
Actinas/isolamento & purificação , Desoxirribonuclease I/metabolismo , Fígado/metabolismo , Actinas/química , Actinas/metabolismo , Animais , Carpas , Cromatografia de Afinidade , Cromatografia em Gel , Cromatografia por Troca Iônica , Citosol/metabolismo , Desoxirribonuclease I/antagonistas & inibidores , Eletroforese em Gel Bidimensional , Immunoblotting , Polímeros , Espectrometria de FluorescênciaRESUMO
When cultured hepatocytes were incubated in cell culture medium at 4 degreesC for up to 30 h and then returned to 37 degreesC, blebbing of the plasma membrane, cell detachment, chromatin condensation and margination, enhanced nuclear stainability with Hoechst 33342, ruffling of the nuclear membrane, and DNA fragmentation occurred. Similar to hepatocytes, cultured liver endothelial cells exhibited blebbing, chromatin condensation and margination, marked nuclear condensation, and increased stainability with Hoechst 33342 when exposed to hypothermia/rewarming. In both cell types, the occurrence and extent of these alterations were dependent on the duration of the cold incubation period. This cold-induced apoptosis was inhibited by hypoxia, by an array of free radical scavengers/antioxidants, and by iron chelators. However, the extent of the protection by the different antioxidants was different in the two cell types: iron chelators provided complete protection in liver endothelial cells but only partial protection in hepatocytes, whereas lipophilic antioxidants such as alpha-tocopherol provided complete protection in both cell types. During cold incubation, and especially during rewarming, lipid peroxidation occurred. These results suggest that the formation of reactive oxygen species (ROS) is a key mediator of cold-induced apoptosis, with ROS formation being completely iron-mediated in liver endothelial cells and partially iron-mediated in hepatocytes.
Assuntos
Apoptose , Fígado/citologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Células Cultivadas , Temperatura Baixa , Endotélio/citologia , Masculino , Ratos , Ratos WistarRESUMO
A cell line (PaTu 8902LM) exhibiting an altered phenotypic appearance was selected from a highly dedifferentiated established human pancreatic tumour cell line (PaTu 8902) by repetitive exposure to laminin-1/nidogen substratum and subsequent selection for adherent cells. Polymerase chain reaction analysis for repetitive DNA indicated that both cell lines are genetically very closely related. The original PaTu 8902 line consisted of flat cells growing in monolayers. In contrast, the obtained PaTu 8902LM cells exhibited a spherical morphology and tended to form clusters. Immunofluorescence analysis using antibodies against apical and basolateral marker enzymes indicated that the PaTu 8902LM cells were polarized, arranging their apical surfaces around central lumenal structures when growing in clusters. In addition, the selected PaTu 8902LM cell line exhibited altered levels of a number of differentiation marker enzymes like 5'-nucleotidase, transglutaminase and plasminogen activators. The different morphological characteristics of both cell lines were maintained even after injection into nude mice. In xenografts, PaTu 8902LM cells were grouped around lumenal, duct-like structures, whereas the original PaTu 8902 cell line formed solid tumours composed of undifferentiated cells. Evidence is presented that the PaTu 8902LM cells are not merely selected from preexisting cells, but that the exposure of PaTu 8902 cells to laminin-1/nidogen had induced a stable transdifferentiation towards the phenotype of the epithelial cells lining the pancreatic secretory ducts. Thus the PaTu 8902LM cells resemble more closely those cells from which tumours of the pancreas originate in vivo and therefore might be a useful cell system in future analyses of the biology of pancreatic tumours which are of increasing incidence and clinical importance.
Assuntos
Adenocarcinoma , Laminina/farmacologia , Glicoproteínas de Membrana/farmacologia , Neoplasias Pancreáticas , Animais , Antineoplásicos Fitogênicos/farmacologia , Adesão Celular/efeitos dos fármacos , Membrana Celular/química , Membrana Celular/enzimologia , Polaridade Celular/fisiologia , Cromossomos/química , Colchicina/farmacologia , Citoesqueleto/química , Endopeptidases/análise , Endopeptidases/metabolismo , Matriz Extracelular/química , Matriz Extracelular/enzimologia , Imunofluorescência , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Nus , Microscopia Eletrônica , Microtúbulos/química , Microtúbulos/efeitos dos fármacos , Proteínas de Neoplasias/análise , Proteínas de Neoplasias/genética , Fenótipo , Coelhos , Transplante Heterólogo , Células Tumorais Cultivadas/química , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/ultraestrutura , Vacúolos/química , Vimblastina/farmacologiaRESUMO
Recombinant plant (birch) profilin was analyzed for its ability to promote actin polymerization from the actin:thymosin beta4 and beta9 complex. Depending on the nature of the divalent cation, recombinant plant (birch) profilin exhibited two different modes of interaction with actin, like mammalian profilin. In the presence of magnesium ions birch profilin promoted the polymerization of actin from A:Tbeta4. In contrast, in the presence of calcium but absence of magnesium ions birch profilin was unable to initiate the polymerization of actin from the complex with Tbeta4. However, under these conditions profilin formed a stable stoichiometric complex with skeletal muscle alpha-actin, as verified by its ability to increase the critical concentration of actin polymerization. Chemical cross-linking indicated that birch profilin competes with Tbeta4 for actin binding. Ternary complex formation of birch profilin with actin:DNase I complex was suggested by chemical cross-linking. However, the determination of the critical concentrations of actin polymerization in the simultaneous presence of birch profilin and DNase I indicated that profilin and DNase I did not form a ternary complex. These data indicated a negative co-operativity between the profilin and DNase I binding sites on actin.
Assuntos
Actinas/metabolismo , Proteínas Contráteis , Desoxirribonuclease I/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas de Plantas/metabolismo , Polímeros/metabolismo , Timosina/metabolismo , Animais , Sítios de Ligação , Reagentes de Ligações Cruzadas , Humanos , Profilinas , Coelhos , ÁrvoresRESUMO
It was previously shown (Paddenberg et al (1996) Eur J Cell Biol 69, 105 - 119) that cells of established lines like NIH3T3 fibroblasts and the human pancreatic adenocarcinoma PaTu 8902 line only degrade their chromatin at internucleosomal sites after an apoptotic stimulus when infected with Mycoplasma hyorhinis. In order to distinguish mycoplasma nucleases (Mr 47 - 54 kDa) from already described eukaryotic apoptotic enzymes, the mycoplasma nucleases were partially purified from serum-free culture supernatants and further characterized. Here we demonstrate directly that the enriched mycoplasma nucleases were able to fragment the DNA of nuclease-negative substrate nuclei at internucleosomal sites. The DNA degradation was accompanied by morphological changes typical of apoptosis like chromatin condensation and margination followed by shrinkage of the nuclei. The biochemical characterization revealed that the mycoplasma nucleases had a neutral to weakly basic pH-optimum. They required both calcium and magnesium in the mM range for maximal activation and were inhibited by zinc chloride, EGTA and EDTA. In two dimensional zymograms they migrated as three spots with isoelectic points between 8.1 and 9.5. They were not inhibited by monomeric actin. Our data also demonstrate that nuclear extracts prepared from nuclei isolated from Mycoplasma hyorhinis infected cells contained the mycoplasma nuclease activities leading to their internucleosomal DNA-degradation after incubation in the presence of calcium and magnesium.
Assuntos
Apoptose , Cromatina/metabolismo , Desoxirribonucleases/metabolismo , Mycoplasma/enzimologia , Cátions Bivalentes/farmacologia , Núcleo Celular/metabolismo , Fragmentação do DNA , Desoxirribonucleases/química , Desoxirribonucleases/isolamento & purificação , Humanos , Concentração de Íons de Hidrogênio , Ponto Isoelétrico , Células Tumorais CultivadasRESUMO
Rat vaginal epithelial cells (VEC) undergo division and differentiation under the influence of oestradiol in a programmed manner. The differentiation process of VEC leads to keratinization, cornification and subsequent desquamation of the dead cells. This process of programmed cell death, referred to as terminal differentiation may share some common pathways with cell death by apoptosis but differ substantially in many aspects. Terminal differentiation of VEC is accompanied by the loss of majority of the organelles including the nucleus. To understand the mechanisms that underlie this process we have analysed the regulation of DNase I (a key effector of apoptotic cell death) in rat VEC under the influence of oestradiol. The present study demonstrates that under physiological conditions, cell death in the VEC is mainly through terminal differentiation although a few cells may undergo apoptotic death involving DNA fragmentation. Unaltered levels of bcl-2 message upon oestradiol administration suggest an important role played by this molecule in preventing death of the VEC by apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Estradiol/farmacologia , Queratinócitos/efeitos dos fármacos , Vagina/efeitos dos fármacos , Animais , Divisão Celular/efeitos dos fármacos , Desoxirribonuclease I/metabolismo , Feminino , Genes bcl-2 , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Queratinócitos/metabolismo , Queratinócitos/ultraestrutura , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , RNA Mensageiro/biossíntese , Ratos , Ratos Wistar , Vagina/citologiaRESUMO
BACKGROUND/AIMS: Primary cultures of rat hepatocytes, rat (FAO) and human (HepG2) hepatoma cells were studied by immunocytochemistry for expression of transforming growth factor (TGF)-beta, for the release of TGF-beta into the medium, and generation of hepatocellular apoptosis by the respective cell-conditioned media. METHODS/RESULTS: Using the alkaline-phosphatase anti-alkaline-phosphatase technique, intense TGF-beta immunostaining was shown in all cell types. The cytokine is released almost entirely in the latent form into the culture medium; only the FAO-cells had a substantial fraction of bioactive TGF-beta in the native (unacidified) culture fluid. Exposure of hepatocytes with the respective cell-conditioned media in the activated, but not in the native form (except for FAO-cell media), induced severe detrimental effects as evidenced by: (i) gross morphological alterations, (ii) functional impairment (reduction of WST-1 test, detachment of cells, lactate dehydrogenase increase in the medium), and (iii) generation of apoptosis. The latter phenomenon was confirmed by an increase of internucleosomal DNA fragments, positive TUNEL reaction, and intense binding of the fluorochrome Hoechst 33342 to fragmented nuclei. All these effects, which were mimicked by addition of recombinant human TGF-beta 1, were almost entirely antagonized by pre-incubation of the conditioned media with latency associated peptide. In contrast to hepatocytes, both types of hepatoma cells were completely resistant to the multiple actions of TGF-beta and activated conditioned media. CONCLUSIONS: It is concluded that hepatocytes might have the ability to induce autocrine, TGF-beta-mediated apoptosis, whereas hepatoma cells, because of their TGF-beta resistance, might generate TGF-beta-mediated peritumorous apoptosis of hepatocytes in a paracrine way, which could facilitate their expansion in situ. Both mechanisms, however, are critically dependent on extracellular TGF-beta activation.
Assuntos
Apoptose/fisiologia , Carcinoma Hepatocelular/patologia , Fígado/citologia , Fator de Crescimento Transformador beta/fisiologia , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Resistência a Medicamentos , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/patologia , Masculino , Ratos , Ratos Sprague-Dawley , Proteínas RecombinantesRESUMO
After androgen ablation by castration, the epithelial cells of the rat ventral prostate are eliminated by apoptosis. The number of cells showing apoptotic chromatin degradation increases with time up to day 3 after castration as verified by in situ end labeling of fragmented DNA. Apoptotic chromatin degradation is catalyzed by a Ca2+, Mg2+-dependent endonuclease. Recently, evidence has been presented that suggests deoxyribonuclease I (DNase I) is identical or very closely related to the apoptotic endonuclease (Peitsch, M.C., B. Polzar, H. Stephan, T. Crompton, H.R. MacDonald, H.G. Mannherz, and J. Tschopp. 1993. EMBO [Eur. Mol. Biol. Organ.] J. 12:371-377). Therefore, the expression of DNase I in the ventral prostate of the rat was analyzed before and after androgen ablation at the level of protein, enzymatic activity, and gene transcripts using immunohistochemical and biochemical techniques. DNase I immunoreactivity was detected only in a few single epithelial cells before androgen ablation. After castration, a time-dependent increase in DNase I immunoreactivity was observed within the epithelial cells. It first appeared after about 12 h in the apical region of a large number of epithelial cells. Up to day 3 after castration, the intracellular DNase I antigenicity continuously increased, and the cell nuclei gradually became DNase I positive. At day 5, almost all nuclei of the epithelium were stained by anti-DNase I. DNase I immunoreactivity was particularly concentrated in cells showing morphological signs of apoptosis, like nuclear fragmentation, and in many cases was found to persist in apoptotic bodies. DNase I gene transcripts were detected in control animals using dot and Northern blotting as well as RNase protection assay. After androgen ablation, the amount of DNase I gene transcripts in total extractable RNA was found unchanged or only slightly decreased up to day 5. Their exclusive localization within the epithelial cells was verified by in situ hybridization. Before castration, the DNase I gene transcripts were homogeneously distributed in all epithelial cells. At day 3, DNase I-specific mRNA was found to be highly concentrated in cells of apoptotic morphology. Using the zymogram technique, a single endonucleolytic activity of about 32 kD was detected in tissue homogenates before castration. After androgen ablation, the endonucleolytic activity increased about four- to sevenfold up to day 3. At day 5, however, it had dropped to its original level. At day 1, three new endonucleolytic variants of higher molecular mass were expressed. At day 3, the predominant endonucleolytic activity exhibited an apparent molecular mass of 32 kD. Enzymatic analysis of the endonucleases present in prostate homogenates before and after castration demonstrated properties identical to DNase I. They were inhibited by chelators of divalent cations, Zn2+ ions and monomeric actin. Immunodepletion was achieved by immobilized antibodies specific for rat parotid DNase I. A polyclonal antibody raised against denatured DNase I was shown by Western blotting to stain a 32-kD band after enrichment of the endonuclease from day 0 and 3 homogenates by preparative gel electrophoresis. The data thus indicate that androgen ablation leads to translational upregulation of an endonucleolytic activity with properties identical to DNase I in rat ventral prostate, followed by its intracellular retention and final nuclear translocation in those epithelial cells that are destined to apoptotic elimination.