Cytoskeletal organization in acute leukemias.
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Actins↗
Biomedical subjects
G Gabbiani
Publications and source records attributed to G Gabbiani.
Analysis of alpha-smooth-muscle actin mRNA expression in rat aortic smooth-muscle cells using a specific cDNA probe.
We constructed two cDNA probes, the first of which hybridizes with all rat actin mRNAs while the second is specific for alpha-smooth muscle (SM) actin mRNA. Northern hybridization using these probes showed that, in normal rat aortic media, the proportion of alpha-SM actin mRNA expression increases during development, reaching about 90% of the total actin mRNA level in adult animals. As compared to the situation in normal aortic media, the proportion of alpha-SM actin mRNA was found to decrease significantly in intimal thickening 15 days after endothelial injury, i.e. when SM cells (SMCs) are actively replicating. At 60 days after injury, the SMCs were observed to have stopped dividing and to have recovered a normal content of alpha-SM actin mRNA. The content of alpha-SM actin mRNA was also selectively decreased (as compared to controls) in the hypotensive abdominal aortic media located below an aortic ligature, while it was not modified in the thoracic hypertensive segment above the same ligature. Primary cultures of rat aortic SMCs synthesize and contain low amounts of alpha-SM actin, but their alpha-SM actin mRNA content is similar to that of SMCs in vivo. As compared to primary cultures, the proportion of alpha-SM actin mRNA was found to be significantly decreased in SMCs at the fifth passage, at which stage it became comparable to the level of synthesized alpha-SM actin. Thus, the synthesis and expression of alpha-SM actin in SMCs appear to be regulated predominantly at the level of gene transcription in certain situations (e.g. aortic ligature in vivo and culture at the fifth passage), and predominantly at a post-transcriptional level in other situations (e.g. primary culture).
Actins↗
Actin-isoform pattern as a marker of normal or pathological smooth-muscle and fibroblastic tissues.
The relative proportions of actin isoforms present in smooth-muscle (SM) and fibroblastic human and non-human tissue extracts were examined by densitometric evaluation of Coomassie-Blue-stained spots in two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) as well as by quantification of radiolabeled actin NH2-terminal peptide spots separated by two-dimensional paper electrophoresis. SM tissues contained alpha- and gamma-SM as well as beta- and gamma-cytoplasmic (CY) actins in different proportions in different organs. Species differences with respect to the ratios of the isoactins were also observed. Moreover, during pregnancy, both human and rat myometrium exhibited a changed actin-isoform pattern, there being an increased proportion of gamma-actin. Analysis of the NH2-terminal peptides showed that, in human myometrium, this was essentially due to an increase in the amount of the gamma-SM isoform. Fibroblastic tissues were found to contain only the beta- and gamma-CY isoforms, the ratio being approximately 2.6:1. Thus, the presence or absence of alpha-actin provides a reliable biochemical criterion for distinguishing between fibroblastic and SM cell populations and/or tissues. This distinction and the evaluation of changes in isoactin ratios may be useful in the study of differentiation as well as physiological and pathological phenomena, and for determining the origin of certain soft-tissue tumours.
Actins↗
Post-irradiation leiomyosarcoma. Case report with immunohistochemical studies.
We report here one case of post-irradiation leiomyosarcoma. The diagnosis of this was confirmed using anti-intermediate filament antibodies: tumor cells were positive for anti-vimentin and anti-desmin but negative for anti-prekeratin and anti-epithelial membrane antigen. The positive staining with anti-desmin clearly indicates a muscular origin although the tumor cells were not stained by two anti-actin antibodies, one directed against alpha-smooth muscle actin and the other against alpha-striated muscle actin. Irradiation was motivated by a wrong diagnosis of breast carcinoma when the patient was 13 year-old. The laps of time separating irradiation and the occurrence of the leiomyosarcoma was 13 years. The total dose was 6,000 rads. This is the ninth case of post-irradiation leiomyosarcomas reported in the literature.
Actin Cytoskeleton↗
Intermediate filament proteins and actin isoforms as markers for soft tissue tumor differentiation and origin. I. Smooth muscle tumors.
A series of 3 benign and 10 malignant smooth muscle (SM) neoplasms and of 2 malignant fibrous histiocytomas was examined by light microscopy, transmission electron microscopy, two-dimensional gel electrophoresis (2D-GE) and indirect immunofluorescence, using polyclonal monospecific or monoclonal antibodies to desmin, vimentin, cytokeratin, alpha-SM and alpha-sarcomeric (alpha-SR) actins. Benign neoplasms displayed typical light-microscopic features of SM, whereas leiomyosarcomas demonstrated variations in their histologic pattern. In 6 sarcomas, light microscopy suggested a SM differentiation, whereas in the other 4, a predominant nondistinctive spindle-cell pattern was observed. By transmission electron microscopy, all 13 neoplasms showed the minimal essential features of SM differentiation. Immunofluorescence disclosed heterogeneity of cytoskeletal protein expression: 5 neoplasms (3 benign and 2 malignant well-differentiated) expressed desmin, vimentin, and alpha-SM-actin; 2 malignant neoplasms expressed desmin and vimentin; 1 malignant neoplasm expressed desmin, vimentin and alpha-SR actin; 1 malignant neoplasm expressed vimentin and alpha-SR actin; and 4 malignant neoplasms expressed vimentin alone. By 2D-GE, 3 benign and 4 malignant SM neoplasms expressed alpha, beta, and gamma actins, and the remaining expressed only beta and gamma actins. The presence of alpha-SM actin in all benign neoplasms and in 2 well-differentiated leiomyosarcomas suggests that this actin isoform reflects a high degree of cellular differentiation. In 2 leiomyosarcomas, alpha-SR actin was detected by immunofluorescence, which suggested a skeletal muscle differentiation of these neoplasms. This study supports the assumption that leiomyosarcomas represent a heterogeneous group of neoplasms and furnishes new criteria for their characterization.
Actins↗
The cytoskeleton of rat aortic smooth muscle cells: normal conditions, experimental intimal thickening and tissue culture.
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Actins↗
Cytoskeletal organization of peripheral blood normal and leukemic lymphocytes and lymphoblasts.
Content of actin and vimentin and organization of actin were studied in peripheral blood lymphocytes from healthy donors, circulating lymphocytes from patients with chronic lymphocytic leukemia (CLL) and lymphoblasts from children with common acute lymphoblastic leukemia (c-ALL). Actin and vimentin were quantified as percentage of total proteins by densitometric scanning of SDS-polyacrylamide gels loaded with Triton-X-100 cell lysates. In addition, the amounts of F- and G-actin were evaluated by ultracentrifugation of total lysates prior to electrophoresis. Compared to peripheral blood lymphocytes from healthy donors, CLL lymphocytes, but not blasts expressing c-ALL antigen, show a decrease in total actin and vimentin contents. Analysis of SDS gels after ultracentrifugation shows that F-actin is diminished of about 50% in both CLL and ALL cells compared to peripheral lymphocytes from healthy donors. The changes in actin and vimentin content and of actin organization may account for some of the functional abnormalities observed in leukemic cell populations.
Actin Cytoskeleton↗
Characterization of stromal cells with myoid features in lymph nodes and spleen in normal and pathologic conditions.
Stromal cells with myoid features were identified in rat or human lymph nodes and spleen in normal and pathologic conditions, using antibodies to desmin, alpha-smooth muscle actin, and smooth muscle myosin. In normal lymph nodes, myoid cells (MCs) were present in the superficial and deep paracortex as well as in the medulla, and absent in lymphoid follicles. In the spleen, they were numerous in the red pulp, less abundant in periarteriolar lymphocyte sheaths of the white pulp, and absent in lymphoid follicles. On double immunostaining, alpha-smooth muscle actin and smooth muscle myosin were coexpressed with desmin only in the deep paracortex and parafollicular areas of the lymph nodes, as well as in the MCs of the periarteriolar lymphocyte sheaths and marginal zone of the spleen; the remaining MCs expressed only desmin. When examined by means of electron microscopy, MCs showed a dendritic shape and cytoplasmic bundles of microfilaments with dense bodies scattered between them. When compared with normal conditions, MCs showed changes of distribution and number in several pathologic situations. Additional findings were 1) staining of pericytes surrounding high endothelium venules of lymph nodes with alpha-smooth muscle actin antibodies in man and rat and with desmin antibodies in rats; 2) staining of endothelial cells in these venules with desmin antibodies in rats. It is concluded that a subset of reticular cells in lymph nodes and spleen, as well as pericytes and endothelial cells in high endothelium venules display cytoskeletal features suggesting a myoid differentiation and function.
Actins↗
Cytoskeletal features of normal and atheromatous human arterial smooth muscle cells.
The distribution of actin, vimentin, desmin, and tropomyosin was studied in the media of the human aorta and femoral and coronary arteries, as well as in atheromatous plaques from the same arteries, by means of immunofluorescence, densitometric analysis of sodium dodecylsulfate-polyacrylamide gel electrophoresis, and bidimensional gel electrophoresis. The proportions of desmin-containing cells varied in the media of different arteries; 4 per cent of the cells in the aorta, 11 per cent in the coronary artery, and 37 per cent in the femoral artery contained desmin. In fibrous atheromatous plaques, independently of the artery, desmin-containing cells were almost absent, but they reappeared in complicated lesions. The content of vimentin per smooth muscle cell increased in fibrous atheromatous plaques, whereas the content of actin and tropomyosin was less than in normal media. Moreover, the alpha-actin predominance observed in the media was transformed to beta-actin predominance in the atheromatous plaques. These cytoskeletal changes provide new, possibly useful, biochemical markers for the characterization of smooth muscle cells during early and advanced phases of atheroma formation.
Adult↗
A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation.
A monoclonal antibody (anti-alpha sm-1) recognizing exclusively alpha-smooth muscle actin was selected and characterized after immunization of BALB/c mice with the NH2-terminal synthetic decapeptide of alpha-smooth muscle actin coupled to keyhole limpet hemocyanin. Anti-alpha sm-1 helped in distinguishing smooth muscle cells from fibroblasts in mixed cultures such as rat dermal fibroblasts and chicken embryo fibroblasts. In the aortic media, it recognized a hitherto unknown population of cells negative for alpha-smooth muscle actin and for desmin. In 5-d-old rats, this population is about half of the medial cells and becomes only 8 +/- 5% in 6-wk-old animals. In cultures of rat aortic media SMCs, there is a progressive increase of this cell population together with a progressive decrease in the number of alpha-smooth muscle actin-containing stress fibers per cell. Double immunofluorescent studies carried out with anti-alpha sm-1 and anti-desmin antibodies in several organs revealed a heterogeneity of stromal cells. Desmin-negative, alpha-smooth muscle actin-positive cells were found in the rat intestinal muscularis mucosae and in the dermis around hair follicles. Moreover, desmin-positive, alpha-smooth muscle actin-negative cells were identified in the intestinal submucosa, rat testis interstitium, and uterine stroma. alpha-Smooth muscle actin was also found in myoepithelial cells of mammary and salivary glands, which are known to express cytokeratins. Finally, alpha-smooth muscle actin is present in stromal cells of mammary carcinomas, previously considered fibroblastic in nature. Thus, anti-alpha sm-1 antibody appears to be a powerful probe in the study of smooth muscle differentiation in normal and pathological conditions.
Actins↗
Expression of actin mRNAs in rat aortic smooth muscle cells during development, experimental intimal thickening, and culture.
The expression of actin-isoform mRNAs in the smooth muscle cells (SMC) of the aortic media in rats has been studied by Northern-blot hybridization, using a general actin-cRNA probe, and two cRNA probes specific for beta- and gamma-cytoplasmic actins, during: (1) development, (2) intimal thickening after endothelial injury induced by balloon catheterization, and (3) growth in culture. In 5-day-old rats, the ratio between alpha-smooth-muscle-actin mRNA and beta- and gamma-cytoplasmic-actin mRNAs was close to 1. It increased to about 4 in 6-week-old rats. Replicating SMC from regions of intimal thickening 15 days after endothelial injury, and SMC growing in culture contained a predominance of cytoplasmic actin mRNAs. Intimal SMC 60 days after endothelial injury (at which time the endothelium had fully regenerated) demonstrated a pattern of actin mRNAs similar to that of normal media. Functional mRNA measured by translation in a reticulocyte lysate showed increases in the level of alpha-actin and decreases in beta-actin in rats from 5 days to 6 weeks of age. These results suggest that during development, under pathological conditions, and in cell culture, the expression of actin isoforms in arterial SMC depends on many factors, including the amount and translation efficiency of mRNAs, and the relative stabilities of the proteins involved.
Actins↗
Distribution of intermediate filament proteins in normal and diseased human glomeruli.
The distribution of intermediate filament proteins (vimentin, desmin, and cytokeratin) was studied by means of immunofluorescence in the normal human and rat glomerulus and in pathologic human glomeruli. Antifibronectin antibodies were used as mesangial markers. In normal human glomeruli, vimentin antibodies stained endothelial cells, podocytes, and mesangial cells; desmin antibodies, surprisingly, stained podocytes. In normal rat glomeruli, the pattern of vimentin staining was the same as in humans, but desmin antibodies stained both mesangial cells and podocytes. In human and rat glomeruli cytokeratin staining was confined to segments of Bowman's capsule. In human pathologic glomeruli, vimentin and desmin antibodies stained the structures that were positive in normal glomeruli, giving a characteristic pattern for each pathologic condition examined. These results are compatible with the mesenchymal origin of podocytes and mesangial cells and suggest that both cells have smooth muscle-like phenotypic features. Mesangial cells may have slightly different differentiation paths in humans and rats, leading to a distinct expression of intermediate filament proteins.
Animals↗
The cytoskeleton of rat aortic smooth muscle cells. Normal conditions, experimental intimal thickening, and tissue culture.
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Actins↗
Cytoskeletal remodeling of rat aortic smooth muscle cells in vitro: relationships to culture conditions and analogies to in vivo situations.
Cytoskeletal features of arterial smooth muscle cells (SMC) vary characteristically during development and during atheromatous plaque formation (Gabbiani et al., 1984; Kocher et al., 1985). We have analyzed the cytoskeletal features of rat aortic SMC placed in culture in the presence of 10% foetal calf serum (thus containing growth factors probably playing a role in SMC development and atheroma formation), as compared to SMC freshly isolated from the rat aortic media. Under these conditions, SMC show a typical cytoskeletal remodeling characterized by: 1) increased content of vimentin per cell, increased number of cells containing only vimentin, and decreased number of vimentin plus desmin containing cells; 2) decreased contents of actin, tropomyosin and myosin; 3) a switch in the pattern of actin isoforms with the appearance of a beta-type predominance. Some of these changes (e.g. increase of vimentin and decrease of alpha-type actin) are seen already in cells entering for the first time in S-phase after plating. Pulse-chase experiments with 3H-thymidine (3H-TdR) indicate that vimentin containing SMC possess a higher replicative activity than vimentin plus desmin containing SMC, thus explaining the selection of vimentin containing cells during culture. Our results indicate that during culture SMC develop features similar to those observed in normal foetal SMC or in SMC present in atheromatous plaques; this model may be useful for the understanding of mechanisms leading to SMC differentiation and to atheroma formation.
Animals↗
Actin isoform synthesis and mRNA levels in quiescent and proliferating rat aortic smooth muscle cells in vivo and in vitro.
The relative levels of actin isoform synthesis in rat aortic smooth muscle cells (SMC) have been studied in vivo and in culture by means of [35S]methionine incorporation. They have been compared to the functional levels of actin isoform mRNAs, assayed by translation of total cell RNA in a reticulocyte lysate or, in some cases, to the actin isoform RNA content, assayed by Northern-blot hybridization to a total actin cRNA probe. In normal media and in freshly isolated SMC, the relative levels of actin isoform synthesis and the actin mRNA translation products show a remarkable similarity, but differ from the proportions of actin isoforms present in a total cell extract (Gabbiani G, Kocher O, Bloom WS, Vandekerckhove J, Weber K: Actin expression in smooth muscle cells of rat aortic intimal thickening, human atheromatous plaque, and cultured rat aortic media. J Clin Invest 73:148, 1984); (Skalli O, Bloom WS, Ropraz P, Azzarone B, Gabbiani G: Cytoskeletal remodeling of rat aortic smooth muscle cells in vitro: relationship to culture conditions and analogies to in vivo situations. J Submicrosc Cytol, in press 1986). This suggests that different actin isoforms have different stabilities. Fifteen days after balloon induced endothelial denudation in vivo, and after being placed in culture, SMC show a decrease in the proportions of alpha-actin synthesis and alpha-actin mRNA levels with a corresponding increase in these parameters for beta- and lambda-actins. The proportions of actin isoform synthesis and actin mRNA translation products in intimal SMC revert to normal values 60 days after balloon induced endothelial denudation, when the aorta is reendothelialized; however, in culture decreased alpha-actin synthesis and mRNA level persist up to the fifth passage (P5). These changes may be helpful for the understanding of SMC adaptation mechanisms during arterial development and atheromatous plaque formation.
Actins↗
The cytoskeleton of rat aortic smooth muscle cells. Normal conditions, experimental intimal thickening and tissue culture.
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Actins↗
On the stress of working with Dr Selye.
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History, 20th Century↗
Human plasma actin-depolymerizing factor. Purification, biological activity and localization in leukocytes and platelets.
The plasma and serum of humans and various animal species exert an actin-depolymerizing activity. Human actin-depolymerizing factor (ADF) has been purified by ammonium sulfate fractionation, DEAE-cellulose and blue-Sepharose chromatography. It is a single polypeptide of approximately 90 kDa, with a pI between 6.0 and 6.5. ADF is heat and trypsin-sensitive, inactivated by EGTA, not stained by HIO4/Schiff on sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE), and not retained on a concanavalin-A-Sepharose column. Incubation of ethanol-fixed cultured cells or unfixed cryostat tissue sections with ADF abolishes immunofluorescent actin staining, by a mechanism which involves extraction of actin from the preparations. ADF promotes fragmentation and depolymerization of actin filaments as shown by electron microscopy, differential ultracentrifugation and DNAse I inhibition assay. This depolymerized actin retains its mobility on SDS/PAGE and is able to repolymerize in the presence of EGTA. Human white blood cells and platelets (but neither human fibroblasts nor white blood cells and platelets from pig, rat and rabbit) contain a 90-kDa protein reacting with an antibody raised in rabbit against human ADF as judged by immunofluorescence and immunoblotting techniques. Immunoblots of human granulocyte subcellular fractions suggest that the protein reacting with ADF antibody is present in the soluble cytoplasmic fraction. ADF may play a role in solubilization of plasma actin and in the intracellular organization of actin, and should be useful for the evaluation of the relative stability of cytoplasmic actin filaments in various physiological and pathological processes.
Actin Depolymerizing Factors↗