RESUMO
Transgenic mice studies have suggested that transforming growth factor-alpha (TGF-alpha) influences the postnatal differentiation of astrocytes. To understand the role of TGF-alpha during astrocytic differentiation, it is important to determine how this factor affects astrocytes in the absence of other influences. We have thus examined in vitro under serum-free medium conditions the effect of TGF-alpha on the properties of astrocytes derived from the cerebral cortex of newborn rats. When TGF-alpha is added to serum-free medium, most astrocytes lose their polygonal shape and extend two long processes running in opposite directions. This bipolar morphology strikingly resembles that of radial glial cells. Intriguingly, serum inhibits this morphological transformation. TGF-alpha also triggers an increase in glial fibrillary acidic protein (GFAP) expression and a decrease in nestin expression. Another major effect of TGF-alpha is to practically abolish the motility of astrocytes. TGF-alpha, however, does not appear to influence the proliferation and apoptosis of astrocytes. These results suggest that polygonal astrocytes are derived primarily from radial glial cells, and that in vivo TGF-alpha may be instrumental in determining the shape and migratory potential of radial glial cells.
Assuntos
Astrócitos/metabolismo , Padronização Corporal/fisiologia , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Células Cultivadas/metabolismo , Córtex Cerebral/crescimento & desenvolvimento , Proteínas do Tecido Nervoso , Fator de Crescimento Transformador alfa/metabolismo , Envelhecimento/fisiologia , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Padronização Corporal/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Movimento Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Tamanho Celular/fisiologia , Células Cultivadas/citologia , Células Cultivadas/efeitos dos fármacos , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Meios de Cultura Livres de Soro/farmacologia , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/fisiologia , Imunofluorescência , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Nestina , Fenótipo , Ratos , Fator de Crescimento Transformador alfa/efeitos dos fármacosRESUMO
Synemin, a developmentally regulated protein first described in muscle cells, has recently been recognized as an intermediate filament (IF) protein. Because IF proteins are invaluable markers of cell origin within the nervous system, we were interested in determining the expression pattern of synemin in the brain. Our results show that, during development of the rat cortex, synemin is expressed only in a subpopulation of astrocytic cells expressing GFAP as well as vimentin and nestin. Unlike GFAP, however, synemin is not expressed in mature astrocytes and, unlike vimentin and nestin, synemin is not present in astrocytic precursors before GFAP expression. Taken together with morphological evidence, the time course of synemin expression, as determined by Western blotting, suggests that synemin is expressed in radial glial cells undergoing morphological transformation into astrocytes. Studies of synemin expression in vitro demonstrate that, early in primary culture, the majority of polygonal astrocytes are derived from synemin(+) radial glial cells. With time in culture, however, polygonal astrocytes either stop expressing synemin or are overgrown by cells not expressing synemin. The unique pattern of synemin expression, both in vivo and in vitro, suggests that the use of synemin as a marker will add a new dimension to studies of astrocytic differentiation.
Assuntos
Astrócitos/metabolismo , Córtex Cerebral/embriologia , Proteínas Musculares/biossíntese , Proteínas do Tecido Nervoso , Animais , Astrócitos/química , Astrócitos/citologia , Células Cultivadas , Córtex Cerebral/citologia , Feminino , Imunofluorescência , Proteína Glial Fibrilar Ácida/análise , Proteína Glial Fibrilar Ácida/imunologia , Proteínas de Filamentos Intermediários/análise , Proteínas de Filamentos Intermediários/imunologia , Nestina , Gravidez , Ratos , Ratos Sprague-Dawley , Vimentina/análise , Vimentina/imunologiaRESUMO
To begin understanding the regulation and biological significance of changes in the expression of intermediate filament proteins in astrocytic tumors, we have recently shown that TGF-alpha alters the protein level of glial fibrillary acidic protein (GFAP), nestin, and vimentin in U-373 MG glioblastoma cells. Here, we have determined the molecular mechanisms regulating these changes. In addition, to evaluate the significance of these changes we have examined whether TGF-alpha affects various cellular properties related to differentiation. Our results show that, in U-373 MG cells treated with TGF-alpha, GFAP gene transcription, mRNA level, and specific protein synthesis decrease by approximately 50%. This suggests that, in U-373 MG cells, TGF-alpha down-regulates the expression of this marker of astrocytic differentiation at the transcriptional level, resulting in decreased GFAP mRNA level and specific protein synthesis. In contrast, TGF-alpha does not change vimentin gene transcription, but increases by about 50% the transcription of the gene for nestin, a marker for undifferentiated astrocytic precursors. This differential regulation of GFAP, nestin, and vimentin gene expression indicates that TGF-alpha induces further dedifferentiation of U-373 MG cells. This notion is also supported by our findings that TGF-alpha increases the motility of U-373 MG cells and induces a less stellate morphology.
Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/genética , Proteínas de Filamentos Intermediários/genética , Proteínas do Tecido Nervoso , Fator de Crescimento Transformador alfa/farmacologia , Vimentina/genética , Diferenciação Celular , Divisão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/fisiologia , Tamanho Celular/efeitos dos fármacos , Glioblastoma , Humanos , Nestina , Biossíntese de Proteínas/efeitos dos fármacos , RNA Mensageiro/genética , Transcrição Gênica/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
Transforming growth factor-alpha (TGF-alpha) and its receptor are frequently co-expressed in high-grade astrocytomas, suggesting a role for TGF-alpha autocrine/paracrine loops in the malignant progression of astrocytomas. To identify genes that may be critical in mediating TGF-alpha impact on the malignant progression of astrocytomas, we have used cDNA arrays to investigate TGF-alpha effects on the gene expression profile of U-373 MG glioblastoma cells. We found that in these cells approximately 50% of the TGF-alpha regulated genes code for cell motility/invasion-related proteins. TGF-alpha action on the expression of four of these proteins, alpha-catenin, IQGAP1, RhoA, and cadherin-11, was further investigated by immunoblotting in four astrocytoma cell lines and in normal astrocytes. The results demonstrate that the effects of TGF-alpha on IQGAP1, alpha-catenin, and RhoA expression are cell-line dependent. On the other hand, under TGF-alpha treatment, cadherin-11 expression is consistently decreased in all astrocytoma cell lines tested but is increased in normal astrocytes. In addition, we found that cadherin-11 is consistently down-regulated in astrocytomas versus normal brain tissues. Altogether, these results suggest that the down-regulation of cadherin-11 is a frequent molecular event in the neoplastic transformation of astrocytes and that this down-regulation may be initiated and/or amplified by TGF-alpha autocrine/paracrine loops during tumor progression.
Assuntos
Astrocitoma/genética , Astrocitoma/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Caderinas/biossíntese , Caderinas/genética , Fator de Crescimento Transformador alfa/farmacologia , Animais , Transformação Celular Neoplásica , Regulação para Baixo/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Proteínas Recombinantes/farmacologiaRESUMO
Various growth factors and basement membrane proteins have been implicated in the pathobiology of astrocytomas. The goal of this study was to determine the relative contribution of these two factors in modulating the phenotype of U-373 MG glioblastoma cells as determined by the expression of the intermediate filament proteins glial fibrillary acidic protein, vimentin, and nestin. For these determinations, cells plated in serum-free medium were treated either with growth factors binding to tyrosine kinase receptors including transforming growth factor-alpha, epidermal growth factor, platelet-derived growth factor-AA, basic fibroblast growth factor, and insulin-like growth factor-1 or with basement membrane proteins including collagen IV, laminin, and fibronectin. The changes in the expression levels of intermediate filament proteins in response to these treatments were analyzed by quantitation of immunoblots. The results demonstrate that collagen IV and growth factors binding to tyrosine kinase receptors decrease the glial fibrillary acidic protein content of U-373 MG cells. Growth factors binding to tyrosine kinase receptors also decrease the vimentin content of these cells but do not affect their nestin content. On the other hand, basement membrane proteins decrease the nestin content of U-373 MG cells but do not affect their vimentin content. The significance of these results with respect to the role played by different factors in modulating the phenotype of neoplastic astrocytes during tumor progression is discussed.
Assuntos
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Substâncias de Crescimento/farmacologia , Proteínas de Filamentos Intermediários/metabolismo , Proteínas de Membrana/farmacologia , Membrana Basal/química , Neoplasias Encefálicas/patologia , Transformação Celular Neoplásica/efeitos dos fármacos , Técnica Indireta de Fluorescência para Anticorpo , Glioblastoma/patologia , Humanos , Immunoblotting , Fenótipo , Receptores Proteína Tirosina Quinases/metabolismo , Células Tumorais CultivadasRESUMO
Several studies have shown that immunoenzymatic staining of formalin-fixed, paraffin-embedded astrocytomas with keratin antibodies frequently yields positive labelling, but no biochemical evidence of keratin expression in astrocytomas has been reported. We have investigated the presence of keratin in astrocytoma and normal brain tissues both by immunofluorescence on frozen sections and by 1D and 2D immunoblotting using seven monoclonal antibodies that, collectively, recognize most keratin polypeptides. Four of these antibodies did not stain neural tissues by immunofluorescence and were also negative by immunoblotting. The remaining three keratin antibodies stained normal brain and/or a high proportion of astrocytomas. Two of these three antibodies only stained glial fibrillary acidic protein (GFAP)-positive cells, while the third only stained GFAP-negative cells. 1D and 2D immunoblotting analysis showed that positive immunofluorescence staining of normal brain and/or astrocytomas seen with these three keratin antibodies was due to cross-reactivity with non-keratin proteins, such as GFAP. These results demonstrate that, contrary to earlier suggestions, keratin polypeptides are not frequently expressed in astrocytomas. Our studies also emphasize that keratin antibodies should be used cautiously for the differential diagnosis of undifferentiated gliomas from tumours of non-glial origin.
Assuntos
Astrocitoma/metabolismo , Neoplasias Encefálicas/metabolismo , Encéfalo/metabolismo , Queratinas/metabolismo , Anticorpos Monoclonais/imunologia , Astrocitoma/patologia , Neoplasias Encefálicas/patologia , Reações Cruzadas/imunologia , Reações Falso-Positivas , Técnica Direta de Fluorescência para Anticorpo , Proteína Glial Fibrilar Ácida/imunologia , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Immunoblotting , Queratinas/imunologiaRESUMO
Recent research highlights the roles of cytoskeletal intermediate filaments (IFs) and their interactions with both the cell surface and other cytoskeletal systems in maintaining cellular integrity and the mechanical properties of cytoplasm. This has been demonstrated by analyses of mutations in IF-associated proteins (IFAPs) that are involved in connecting IFs to cell surface junctions. New data also point to the role of IFAPs as molecular 'nuts and bolts' in the construction of an integrated cytoplasmic architecture. This is highlighted by the initial descriptions of a family of multifunctional molecules that are capable of bridging IFs to other cytoskeletal elements. These findings, together with the development of specific peptide inhibitors capable of disassembling IF networks in vivo, are paving the way to the identification of new cellular functions for IFs and IFAPs.
Assuntos
Citoplasma/química , Filamentos Intermediários/fisiologia , Citoplasma/fisiologiaRESUMO
Under normal culture conditions, epithelial cells of the FG line, derived from a pancreatic tumor, characteristically grow in mounds and fail to flatten efficiently onto their substrate. In such cells, keratin intermediate filaments (IFs) are concentrated in the perinuclear region. Furthermore, the IF associated protein, IFAP300, primarily localizes along these keratin bundles. Additionally, alpha 6 beta 4 integrin heterodimers localize in streaks or spots towards the edges of cells while alpha 3 beta 1 integrin is predominantly at cell-cell surfaces. Neither show any obvious interaction with IF. Remarkably, upon plating FG cells into medium containing soluble rat laminin-5, FG cells rapidly adhere and spread onto their substrate. Moreover, FG cells "capture" rat laminin-5 and place it basally in circles or arcs at areas of cell-substrate interaction. Double label immunofluorescence microscopy reveals colocalization of IFAP300 as well as alpha 6 beta 4 and alpha 3 beta 1 integrin with the polarized laminin-5. Concomitantly, alpha 6 integrin undergoes dephosphorylation on serine residue 1041. Laminin-5-induced rapid adhesion can be blocked by antibodies against the alpha 3 integrin subunit. In contrast, while alpha 6 integrin antibodies do not block laminin-5-induced rapid adhesion, they prevent FG cells from assuming an epithelial-like morphology. Keratin IF bundles associate with IFAP300-alpha 6 beta 4/alpha 3 beta 1 integrin complexes along the cell-substratum-attached surface of FG cells coincubated in laminin-5-containing medium. Coprecipitation results suggest that in these complexes, IFAP300 may associate with the alpha 6 beta 4 integrin heterodimer. Based on our results and published evidence that IFAP300 binds keratin in vitro [Skalli et al., 1994; J. Cell Biol. 125:159-170], we propose that laminin-5/FG cell interaction results in a novel integrin dephosphorylation event, which subsequently induces IFAP300 association with alpha 6 beta 4 integrin. IFAP300 then mediates the interaction of IFs with the cell surface via the alpha 6 beta 4 integrin heterodimer.
Assuntos
Antígenos CD/metabolismo , Moléculas de Adesão Celular/fisiologia , Citoesqueleto/ultraestrutura , Integrinas/metabolismo , Queratinas/ultraestrutura , Neoplasias Pancreáticas/patologia , Sequência de Aminoácidos , Animais , Meios de Cultura , Integrina alfa3beta1 , Integrina alfa6 , Integrinas/análise , Proteínas de Filamentos Intermediários/análise , Dados de Sequência Molecular , Fosforilação , Testes de Precipitina , Ratos , Receptores de Laminina/análise , Células Tumorais Cultivadas , CalininaRESUMO
The distribution of IFAP 300, a protein previously characterized as cross-linking vimentin intermediate filaments (IF), has been investigated in epithelial cells. In frozen sections of bovine tongue epithelium the staining obtained with IFAP 300 antibodies is concentrated in the peripheral cytoplasm of keratinocytes, including the entire peripheral region of basal cells. Further immunofluorescence studies reveal that in primary cultures of mouse keratinocytes the distribution of IFAP 300 is similar to that of the desmosomal protein desmoplakin. In rat bladder carcinoma 804G cells the staining pattern of IFAP 300 antibodies coincides with that obtained with antibodies against the hemidesmosomal protein BP 230. By immunogold electron microscopy IFAP 300 is mainly located at sites where IF appear to attach to desmosomes and hemidesmosomes. Morphometric analyses of the distribution of the gold particles show that IFAP 300 overlaps with desmoplakin and BP 230, but also that it extends deeper into the cytoplasm than these latter two proteins. The staining reaction seen in epithelial cells by immunofluorescence and immunogold is specific for IFAP 300 as shown by immunoblotting. Immunoblotting also reveals that IFAP 300 is present in both cell-free preparations of desmosomes and hemidesmosomes. These morphological and biochemical results are intriguing since, in recent years, the proteins appearing in these two types of junctions have been found to be different. One possible exception is plectin, a protein that has been suggested to be very similar to IFAP 300. However, we show here that IFAP 300 differs from plectin in several respects, including differences at the primary sequence level. We also show that purified IFAP 300 pellets with in vitro polymerized IF prepared from desmosome-associated keratins under conditions in which IFAP 300 alone is not sedimentable. This indicates that IFAP 300 can associate with keratin IF. These data, taken together with the immunogold results, suggest that IFAP 300 functions in epithelial cells as a linker protein connecting IF to desmosomes as well as to hemidesmosomes, possibly through structurally related proteins such as desmoplakin and BP 230, respectively.
Assuntos
Desmossomos/ultraestrutura , Proteínas de Filamentos Intermediários/metabolismo , Filamentos Intermediários/ultraestrutura , Sequência de Aminoácidos , Animais , Bovinos , Cricetinae , Imunofluorescência , Imuno-Histoquímica , Proteínas de Filamentos Intermediários/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Plectina , Ratos , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
During mitosis in BHK-21 baby hamster kidney cells the hyperphosphorylation of the type III intermediate filament (IF) protein vimentin is accompanied by the disruption of the IF network into punctate, protofilamentous structures. In this study, the morphological and biochemical changes of IFAP 300, a 300-kDa IF-crossbridging protein, are examined during mitosis. Double-label immunofluorescence shows that the distribution of IFAP 300 coincides with the typical filamentous pattern displayed by vimentin in interphase cells, whereas in mitotic cells it is reorganized into a punctate, nonfilamentous pattern. Accompanying these latter morphological changes, IFAP 300 is phosphorylated at a unique, mitosis-specific site. Comparison of the sites phosphorylated in cultured cells with those phosphorylated in vitro by various kinases suggests that IFAP 300 is phosphorylated by the same two kinases that phosphorylate vimentin during mitosis. One of these is p34cdc2 protein kinase, which appears to be responsible for the phosphorylation of the mitosis-specific site. The other kinase phosphorylates IFAP 300 in vitro at a site that is also found in the protein immunoprecipitated from either mitotic or interphase cells. In contrast to vimentin, the phosphorylation levels of IFAP 300 are not obviously altered between interphase and mitosis. Our results show that IFAP 300 is a physiological substrate for p34cdc2 and that this kinase may be involved in the mitotic reorganization of IFAP 300 by phosphorylating a mitosis-specific site. Taken together with our previous results, this study suggests that the activation of p34cdc2 coordinates the mitotic reorganization of the vimentin IF network both by severing IF-IF connections mediated by IFAP 300 and by disassembling individual IFs into protofilaments.
Assuntos
Proteína Quinase CDC2/metabolismo , Ciclo Celular , Proteínas de Filamentos Intermediários/metabolismo , Filamentos Intermediários/metabolismo , Vimentina/metabolismo , Animais , Células Cultivadas , Cricetinae , Imunofluorescência , Técnicas In Vitro , Filamentos Intermediários/ultraestrutura , Mitose , Mapeamento de Peptídeos , Fosforilação , Fosfotirosina , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
The dynamic properties of cellular protein polymers such as microtubules and microfilaments depend to a large extent on the cell's capacity to modify rapidly the exchange rate between polymerized and unpolymerized pools of subunits. Until quite recently the dynamic nature of intermediate filaments was underestimated because of their biochemical stability in vitro and a paucity of studies on their characteristics in vivo. However, the recent studies described in this review show that the karyoskeletal and cytoskeletal structures that assemble from many intermediate filament proteins possess the properties expected of dynamic protein polymer networks.
Assuntos
Proteínas de Filamentos Intermediários/metabolismo , Filamentos Intermediários/metabolismo , Animais , Proteínas do Citoesqueleto/metabolismo , Proteínas do Citoesqueleto/fisiologia , Citoesqueleto/metabolismo , Citoesqueleto/fisiologia , Humanos , Proteínas de Filamentos Intermediários/fisiologia , Filamentos Intermediários/fisiologiaRESUMO
BALB/c mice neonatally injected with 10(8) semiallogeneic (C57BL/6 x BALB/c)F1 spleen cells become tolerant to the H-2b alloantigens, but also develop a wide range of autoimmune manifestations characteristic of systemic lupus erythematosus (SLE). Indeed, in these mice, the presence of a hypergammaglobulinaemia, autoantibodies--including anti-ssDNA, anti-platelet, thymocytotoxic and rheumatoid factor antibodies--circulating immune complexes, cryoglobulins as well as renal glomerular deposition of immunoglobulins have been observed. In this study, we have shown that the allogenic effect and B cell chimaerism which characterize these F1 cell-injected mice is associated with the expression of a large spectrum of autoantibodies, including anti-ssDNA and anti-cytoskeleton antibodies, and that these autoantibodies are not multispecific. We took advantage of the fact that, in this model, autoantibodies are exclusively produced by F1 donor B cells to inject newborn BALB/c mice with F1 Xid spleen cells lacking the CD5+ B cell subset. Injection of 2 x 10(8) F1 Xid spleen cells triggers the production of anti-ssDNA as well as anti-BrMRBC antibodies, and these mice developed tissue lesions. Finally, analysis of the VH gene family expressed by monoclonal autoantibodies derived from F1 cell-injected mice showed that they used the 2 largest families J558 and 7183. These results suggest that the allogenic effect and B cell chimerism which characterize the neonatal induction of tolerance to MHC alloantigens is associated with the selective triggering of autoreactive B cells producing monospecific IgG autoantibodies. They also imply that upon stimulation by persisting alloreactive CD4+ T cells, either CD5- B cells are able to produce autoantibodies or autoantibody-producing CD5+ B cells can differentiate from Xid spleen cells.
Assuntos
Autoanticorpos/biossíntese , Doenças Autoimunes/etiologia , Animais , Animais Recém-Nascidos , Especificidade de Anticorpos , Antígenos CD , Autoanticorpos/genética , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Subpopulações de Linfócitos B/imunologia , Antígenos CD5 , Genes de Imunoglobulinas , Antígenos H-2 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , SíndromeRESUMO
We have studied the expression of alpha-smooth muscle actin, smooth muscle myosin, and desmin in granulation tissue during the healing of an open wound in the rat by means of electron microscopy and immunohistochemistry, at the light and electron microscopic levels, using specific antibodies directed against these proteins. Important amounts of the three antigens were always expressed in pericytic and/or smooth muscle cells of neoformed small vessels. In fibroblastic cells, microfilaments were absent 4 days after wounding but accumulated gradually, starting from the 6th up to the 15th day; at this time, they were evident in about 70% of fibroblastic cells (myofibroblasts). They then regressed progressively, and on the 30th day, microfilaments were no longer present in scar fibroblasts. alpha-Smooth muscle actin, but not smooth muscle myosin and desmin, was always present in microfilament bundles of myofibroblasts. The staining intensity was progressive from the 6th to the 15th day and decreased thereafter; no staining was seen at the 30th day. Between the 20th and the 25th day, many apoptotic figures were seen in fibroblastic and endothelial cells, suggesting that apoptosis is the mechanism of their disappearance. We conclude that myofibroblasts develop gradually from granulation tissue fibroblasts and temporarily express a marker of smooth muscle differentiation. These results may be relevant for the understanding of the mechanisms of normal and pathologic wound healing.
Assuntos
Actinas/biossíntese , Pele/patologia , Cicatrização/fisiologia , Actinas/análise , Animais , Desmina/análise , Fibroblastos/patologia , Fibroblastos/ultraestrutura , Imunofluorescência , Granuloma/patologia , Microscopia Eletrônica , Músculo Liso/patologia , Músculo Liso/ultraestrutura , Ratos , Ratos Endogâmicos , Pele/ultraestrutura , Fatores de TempoRESUMO
Intermediate filament proteins and actin isoforms of a series of 12 malignant hemangiopericytomas and five glomus tumors were examined by light microscopy, transmission electron microscopy, two-dimensional gel electrophoresis (2D-GE), and by immunohistochemistry, the latter using monoclonal or affinity-purified polyclonal antibodies to desmin, vimentin, cytokeratins, alpha-smooth muscle, and alpha-sarcomeric actins. By light microscopy, all hemangiopericytomas disclosed a predominant vascular pattern with scant storiform, myxoid and spindle cell areas, and with variable degrees of perivascular fibrosis. By ultrastructure, smooth muscle differentiation was observed in each hemangiopericytoma. Immunohistochemically, neoplastic cells of hemangiopericytomas expressed vimentin as the sole intermediate filament protein and lacked alpha-smooth muscle or alpha-sarcomeric actins. 2D-GE revealed only beta and gamma actins, in proportions typical for fibroblastic tissues. Glomus tumors revealed vimentin and alpha-smooth muscle actin within glomus cells by immunohistochemical techniques and disclosed ultrastructurally distinct smooth muscle differentiation. Therefore hemangiopericytomas represent a distinct soft-tissue neoplasm with uniform morphologic, immunohistochemical, and biochemical features most likely related to glomus tumors, the former representing an aggressive and potentially malignant neoplasm of vascular smooth muscle cells and the latter a well-differentiated neoplasm of vascular smooth muscle cells. Because malignant hemangiopericytomas disclose smooth muscle differentiation by ultrastructure, but do not express alpha-smooth muscle actin, as normal pericytes and glomus cells, it is suggested that these neoplasms represent highly vascularized smooth muscle neoplasms, ie, poorly differentiated leiomyosarcomas derived from vascular smooth muscle cells or their equivalent, the pericytes, which have lost alpha-smooth muscle actin as a differentiation marker that is similar to many conventional poorly differentiated leiomyosarcomas.
Assuntos
Actinas/fisiologia , Tumor Glômico/patologia , Hemangiopericitoma/patologia , Proteínas de Filamentos Intermediários/fisiologia , Anticorpos Monoclonais , Capilares/citologia , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Filamentos Intermediários/ultraestrutura , Microscopia EletrônicaRESUMO
Using light and electron-microscopic immunolocalization techniques, and gel electrophoresis combined with immunoblotting, we have examined the expression of cytoskeletal proteins in normal human fetal, child and adult lenses, in human anterior capsular cataract and in bovine lens cells in vivo and in vitro. In this report, we focus our observations on the pattern of actin-isoform expression during normal and pathological situations in vivo and culture conditions. We have noted that cells of developing and mature human lenses as well as bovine lens cells in situ contain only beta- and gamma-actins. In contrast, alpha-smooth muscle (alpha-sm) actin, an isoform typical of smooth muscle differentiation, was demonstrated in bovine lens cells at different times of culture. Moreover, the multilayered cells observed in the subcapsular zone of human anterior capsular cataract were characterized by the presence of alpha-sm actin. Thus, extensive changes in actin-isoform expression take place in lens cells growing in culture and may also occur during cataractogenesis. The biological meaning of the appearance of a marker of myoid differentiation in the ectodermally derived lens-forming cells is discussed.
Assuntos
Actinas/metabolismo , Catarata/patologia , Cristalino/citologia , Músculo Liso/análise , Actinas/análise , Adulto , Idoso , Animais , Catarata/metabolismo , Bovinos , Diferenciação Celular , Células Cultivadas , Criança , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Humanos , Imuno-Histoquímica , Cristalino/embriologia , Cristalino/metabolismo , Cristalino/ultraestrutura , Microscopia Eletrônica , Pessoa de Meia-Idade , FenótipoRESUMO
Arterial smooth muscle cells express alpha- and gamma-smooth muscle, as well as beta- and gamma-cytoplasmic actins. Two actin antibodies, one recognizing smooth muscle and cytoplasmic actin isoforms, the other recognizing specifically alpha-smooth muscle actin, were microinjected into cultured aortic smooth muscle cells. The effect of these antibodies on stress fiber organization was examined by staining with rhodamine-labeled phalloidin and by immunofluorescence with the same antibodies. Microinjection of the general actin antibody abolished most of the stress fiber staining with all reagents, but did not significantly affect the shape of the injected cells. This suggests that stress fiber integrity is not absolutely necessary for the maintenance of cell shape within the time of observation. Microinjection of the specific alpha-smooth muscle antibody abolished to various extents the staining of stress fibers with this antibody, but left practically intact their staining with rhodamine-labeled phalloidin and with the general actin antibody. This suggests that the incorporation of alpha-smooth muscle actin is not absolutely necessary for the maintenance of stress fiber integrity in cultured smooth muscle cells.
Assuntos
Actinas/fisiologia , Anticorpos/administração & dosagem , Músculo Liso Vascular/fisiologia , Actinas/imunologia , Animais , Aorta Torácica/citologia , Aorta Torácica/fisiologia , Células Cultivadas , Imunofluorescência , Imunoglobulina G/administração & dosagem , Microinjeções , Músculo Liso Vascular/citologia , Ratos , Ratos Endogâmicos , Estresse MecânicoRESUMO
To investigate a possible correlation between cytodifferentiation, proliferation, and actin expression, smooth muscle cells from the 9-week-old rabbit aortic media were enzyme-dispersed into single cells and were plated in primary culture at different initial seeding densities. The volume fraction of myofilaments (Vv myo) in cells seeded moderately densely fell from 39.5% +/- 1.2% in the intact aortic media to 11.5% +/- 1.6% on Day 5, one day before the onset of logarithmic growth. The Vv myo remained low over the next 3 days, then began to rise as the density of cells increased, returning almost to the original levels after confluency and 1.84 cumulative population doublings (CPD). The expression of alpha-smooth muscle actin mRNA followed a similar time course of change, falling from 84.7% +/- 1.2% of total actin mRNA in freshly isolated cells to 54.0% +/- 6.5% on Day 5, returning to 87.5% +/- 0.5% after confluency. In these cultures, the alpha-smooth muscle actin protein content was 93.7% +/- 2.9% of total actin in freshly isolated cells, 68.7% +/- 3.1% on Day 5, and 73.3% +/- 2.5% 3 days after confluency. In densely seeded cultures, the Vv myo and expression of alpha-smooth muscle actin mRNA fell only slightly on Day 5 and rose to original levels upon confluency after 0.33 CPD. However, at the protein level, alpha-smooth muscle actin decreased on Day 5 and remained low on Day 12. The Vv myo, alpha-smooth muscle actin mRNA, and actin protein of sparsely seeded cells fell on Day 5 and then remained low throughout the culture period, including 5 days after confluency (Day 24), when the cells had undergone 5.37 CPD. Cells that were maintained subconfluent but quiescent on Day 7 in culture had the same low Vv myo, low alpha-actin mRNA expression, and low alpha-actin protein content as actively proliferating cells. The results show that Vv myo and alpha-smooth muscle actin mRNA undergo parallel changes during primary culture according to seeding density, but not to replication, and that alpha-smooth muscle actin protein decreases in culture then remains low irrespective of culture conditions.
Assuntos
Actinas/genética , Proteínas Musculares/metabolismo , Músculo Liso Vascular/metabolismo , RNA Mensageiro/metabolismo , Animais , Aorta/citologia , Aorta/metabolismo , Contagem de Células , Diferenciação Celular , Divisão Celular , Células Cultivadas , Técnicas Citológicas , Músculo Liso Vascular/citologiaRESUMO
The diagnosis of orbital rhabdomyosarcoma (RMS) in childhood gives rise to several clinical and anatomo-pathological problems. Antibodies recognizing structural proteins and cytoskeletal components have been shown to increase the diagnostic accuracy of different neoplastic lesions. In this study we examined anatomo-clinically and, where possible, by means of immunohistochemistry and electron microscopy, a series of 14 cases of orbital RMS in childhood. In the 12 cases studied by immunohistochemistry, desmin was always present, although showing variable patterns, and alpha-sarcomeric actin was found in 10 cases. alpha-Smooth muscle actin was always absent. The other markers tested (myoglobin, polyclonal actin, vimentin and enolase) proved unreliable for several reasons. We conclude that antibodies against desmin and alpha-sarcomeric actin are useful for the diagnostic definition of RMS. In addition, immunohistochemical analysis supplies data regarding the degree of tumor differentiation and may be applied to monitor radio- and chemotherapy.