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1.
Immunofluorescence analysis of reexpression and activation: the origin of phenotypic diversity of rat hepatoma-mouse fibroblast hybrid colonies 总被引:1,自引:0,他引:1
M Mével-Ninio 《Differentiation; research in biological diversity》1984,26(1):68-76
The capacity of young hybrid colonies between 2s rat hepatoma cells and mouse L-fibroblasts to reexpress rat albumin and become activated for mouse albumin production, was examined at the level of individual cells using immunofluorescent staining of intracellular albumin. Most of the colonies that happened to reexpress albumin were observed to present a stable, homogeneous, hepatoma-like morphology, visible long before the first signs of albumin reexpression. These colonies switch from an extinct to an albumin-producing state between one and four weeks after fusion. Colonies that do not express albumin, present an epithelial or fibroblastic morphology. Karyologic analysis of hybrid clones representative of the various morphologic types revealed that their phenotypic diversity is correlated with their rat over mouse chromosome ratio. The results suggest that the potential of hybrid colonies to express albumin is determined at the time of nuclear fusion in the heterokaryons, possibly by the number of parental genomes participating in the formation of the mother hybrid cell. Double immunofluorescent staining of rat and mouse albumin in the same cell has been used to determine whether reexpression and activation are correlated phenomena or appear independently in any single cell. The analysis demonstrates that activation of mouse albumin never takes place without reexpression of the previously expressed rat albumin gene, while the converse is frequent. 相似文献
2.
Immunofluorescence analysis of the time-course of extinction, reexpression, and activation of albumin production in rat hepatoma- mouse fibroblast heterokaryons and hybrids 总被引:27,自引:8,他引:19
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《The Journal of cell biology》1981,90(2):339-350
We have used a combination of a sensitive immunocytochemical stain for intracellular albumin, and Hoechst 33258 dye for identification of parental nuclei to investigate the time-course of extinction, reexpression, and activation of albumin production in fusion products of 1s (hyperdiploid) or 2s (hypertetradiploid) rat hepatoma cells with mouse fibroblasts (L cells or embryonic cells). In all combinations, the initial event is extinction of albumin production. Extinction occurs immediately after fusion when the mouse fibroblast is a normal embryonic (senescent?) cell. In the case of an L cell, rat albumin is synthesized and secreted during the first 12 h after fusion; no production of mouse albumin occurs. Thereafter, albumin production ceases. 8-12 d after fusion, young hybrid colonies are found to resume the synthesis of rat albumin (reexpression), and several days later the production of mouse albumin begins (activation). The patterns of reexpression and activation indicate (a) that chromosome loss is not necessary for either event to occur and (b) that the cells active in the synthesis of mouse albumin are a subpopulation of those cells already engaged in the production of rat albumin. We conclude that (a) extinction is mediated by diffusible factor(s) from the L-cell parent that act in the hepatoma nucleus to prevent the formation of new albumin messenger RNA; (b) reexpression and activation are gene dosage- dependent but extinction is not; and (c) previously active genes are more rapidly expressed than previously silent ones. 相似文献
3.
Phenotypic exclusion in mouse melanoma-rat hepatoma hybrid cells: pigment and albumin production are not reexpressed simultaneously. 总被引:11,自引:0,他引:11
Hybridization of cells of defined and different histotypes has been carried out to investigate whether the expression (or reexpression) of parental functions is mutually exclusive, as is expected if the generally assumed rule of discreteness of differentiation applies to hybrid cells. A cross of pigmented mouse melanoma cells and albumin-producing rat hepatoma cells gave rise to hybrids containing essentially one set of chromosomes from each parent and producing neither melanin nor albumin. Cells of one hybrid clone are shown to retain the potential to reexpress both parental differentiations. Successive subclonings of this hybrid have shown that cells which reexpress one function may retain the potential to reexpress the other, and that freshly isolated, morphologically homogeneous subclones may produce pigment or albumin, but not both; there successive and exclusive shifts of phenotype are documented, and in these cases, chromosome loss is very slight. The use of immunoadsorbed antisera has revealed that most (if not all) of the albumin produced by the hybrid cells is of the mouse type. We conclude that both parental determinations are retained by the hybrid cells, and that the parental differentiations are reexpressed only in a mutually exclusive fashion. 相似文献
4.
Doris Cassio 《Differentiation; research in biological diversity》1984,26(1-3):77-82
Abstract. Neonatal hepatic functions are selectively extinguished in hybrids between mouse hepatoma cells, that express only fetal hepatic functions, and rat hepatoma cells expressing neonatal as well as fetal functions. A search for hybrid cells reexpressing these neonatal functions was undertaken to determine; (1) whether the selective extinction of neonatal functions is reversible and at what frequency, and (2) whether the reexpression of neonatal functions would be accompanied by modifications in the expression of fetal functions. The criterion used to obtain hybrids showing reexpression was glucose-free medium (G-) where growth requires the presence of the extinguished gluconeogenic enzymes. Even though the parental cells are of the same histotype it proved difficult to obtain re-expression. Survivors in G- were obtained only from hybrids containing a greater than Is complement of rat chromosomes; they reexpress not only gluconeogenic enzymes but also basal tyrosine aminotransferase activity, and the fetal hepatic function a-fetoprotein continues to be expressed in most of the clones. All survivors in G- display a significant loss of chromosomes and this loss concerns essentially mouse chromosomes. 相似文献
5.
Conditions required for activation of the mouse albumin or α-fetoprotein gene in hybrids between mouse lymphoblastoma and rat hepatoma cells 总被引:1,自引:0,他引:1
Carole H. Sellem Mary C. Weiss Doris Cassio 《Differentiation; research in biological diversity》1988,39(1):66-77
Activation of two previously silent mouse hepatic genes has been investigated in hybrid cells between pseudodiploid mouse lymphoblastoma cells and hyperdiploid or hypertetraploid rat hepatoma cells. In this material, activation of the mouse albumin gene is a frequent event, whereas activation of mouse alpha-fetoprotein (AFP) occurs only in those cells that produce large amounts of albumin. Quantitative tests of hybrid populations for the activated proteins and their mRNAs revealed the expected sizes and structures: moreover, as in hepatoma cells, the amount of both rat and mouse albumin produced was directly proportional to the intracellular concentration of the corresponding mRNA. The cellular environment required for activation of the liver-specific genes was investigated by cell-by-cell analysis of each hybrid clone. Immunostaining for the presence of rat and mouse albumin and mouse AFP revealed unexpected heterogeneity in the phenotypes of the hybrid populations, which were found to contain cells that: (a) failed to express either of the proteins; (b) produced all three; (c) produced both rat and mouse albumin; or (d) produced rat albumin only. Karyotypic analysis indicated that the hybrid-cell phenotype depended on parental chromosome ratios rather than absolute numbers of chromosomes. It was found for albumin and mouse AFP that the fraction of immunostained cells was equal to the fraction of metaphases that contained a minimal rat-to-mouse chromosome ratio of 2.5 and 9, respectively. It is concluded that in those hybrids, expression of liver-specific genes is regulated by extinguishers, but in a dose-dependent fashion, suggesting the intervention of antagonistic activators from the rat hepatoma chromosomes. 相似文献
6.
J Schwaber 《Somatic cell genetics》1977,3(3):295-302
Fusion of unfractionated human lymphocytes with mouse myeloma cells resulted in proliferating hybrid colonies, almost all producting human Ig. We examined whether this high frequency of Ig production was the result of selective formation of human B lymphocyte-mouse myeloma hybrids, rather than induction of Ig genes in T lymphocytes. Unfractionated peripheral lymphocytes and B lymphocytes from patients with the common variable form of agammaglobulinemia formed proliferating somatic cell hybrid colonies. In contrast, peripheral lymphocytes from a patient with agammaglobulinema who lacked B lymphocytes, as well as albumin gradient fractions of peripheral blood which do not contain B lymphocytes, failed to produce somatic cell hybrids with three different myeloma parent cell lines. B, T, and precursor lymphocytes all had Sendai virus receptors, as witnessed by viral agglutination. We conclude that fusion of human lymphocytes with mouse myeloma cells results in selective hybrid formation, rather than activation of Ig genes in disparate cell types. Only B lymphocyte-mouse myeloma heterokaryons form hybrid cells. 相似文献
7.
Activation of a silent gene is accompanied by its demethylation 总被引:3,自引:0,他引:3
The phenomenon of gene activation by cell fusion makes it possible to study a gene when it passes from a silent to an active state. The relationship between methylation and activation of the mouse albumin gene has been investigated in two types of hybrid clones: mouse lymphoblastoma--rat hepatoma hybrids where activation is very frequent, and mouse L-cell--rat hepatoma hybrids where activation is a rare event. Analysis of the methylation pattern of seven MspI/HpaII sites that occur along the first 8000 bases of the mouse albumin gene has been performed. The entire 5' region is unmethylated only in albumin-producing cells (adult liver and hepatoma); in non-hepatic cells this region is heavily methylated. In hybrids between rat hepatoma cells and mouse cells of mesenchymal origin, the only regular change is the demethylation of the most 5' site (M1), which is systematically observed in clones where expression of the mouse albumin gene has been activated. Demethylation of this site, like activation of the mouse albumin gene, is gene dosage-dependent; it is systematic in the lymphoblastoma--hepatoma hybrids and rare in L-cell--hepatoma hybrids. We conclude that demethylation of this site is tightly coupled with activation of the gene and may well be a necessary prerequisite for activation. 相似文献
8.
By an in vitro colony assay and cytochemical staining, we investigated the capacity of mouse bone marrow fibroblasts to differentiate into adipocytes and to express alkaline phosphatase (ALP) activity. Glucocorticoids enhanced colony formation of the fibroblasts, and stimulated their adipose conversion (55-65% of the colonies became adipocyte-positive), but they did not affect ALP activity. The fibroblasts became heterogeneous in size and morphology after growing in vitro then differentiated into adipocytes. All the cell types had ALP activity, and more than 95% of the colonies contained ALP-positive cells. ALP staining was strongest in cells in the early stage of adipose conversion, gradually decreasing with maturation. Our results indicate that the majority of the mouse bone marrow fibroblasts that formed colonies under our culture conditions are preadipocytes. We conclude that these fibroblasts originate from adventitial reticular cells present in bone marrow stroma because reticular cells have been reported to possess high ALP activity and have been suggested to differentiate into adipocytes. 相似文献
9.
The ability of skeletal muscle myoblasts to differentiate in the absence of spontaneous fusion was studied in cultures derived from chicken embryo leg muscle, rat myoblast lines L6 and L8, and the mouse myoblast line G8. Following 48–96 hr of culture in a low-Ca2+ (25 μm), Mg2+-depleted medium, chicken myoblasts exhibited only 3–5% fusion whereas up to 64% of the cells fused in control cultures. Depletion of Mg2+ led to preferential elimination of fibroblasts, with the result that 97% of the mononucleated cells remaining at 120 hr exhibited a bipolar morphology and stained with antibodies directed against M-creatine kinase, skeletal muscle myosin, and desmin. Mononucleated myoblasts rarely showed visible cross-striations or M-line staining with anti-myomesin unless the medium was supplemented with 0.81 mM Mg2+, suggesting that Mg2+ plays a role in sarcomere assembly. Conditions of Ca2+ and Mg2+ depletion inhibited myoblast fusion in the rodent cell lines as well, but mononucleated myoblasts failed to differentiate under these conditions. Differentiated individual myoblasts from rat cell lines and from chicken cell cultures were obtained when fusion was inhibited by growth in cytochalasin B (CB). CB-treated rat myoblast cultures accumulated MM-CK to nearly twice the specific activity found in extensively fused control cultures of comparable age. Spherical cells which accumulated during CB treatment were isolated and shown to contain nearly eight times the CK specific activity present in nonspherical cells from the same cultures. Approximately 90% of these cells exhibited immunofluorescent staining with antibodies to skeletal muscle myosin, failed to incorporate [3H]thymidine or to form colonies in clonal subculture, and thus represent terminally differentiated rat myoblasts. Quantitative microfluorometric DNA measurements on individual nuclei demonstrated that the terminally differentiated myoblasts obtained in these experiments from both chicken and rat contain 2cDNA levels, suggesting arrest in the G0 stage of the cell cycle. 相似文献
10.
Jeanne Bentley Lawrence Stephen F. Konieczny Maria Shaffer Annette W. Coleman John R. Coleman 《Experimental cell research》1982,142(2)
Several authors have described the extinction of myogenic competence in hybrids produced by fusion of myogenic and non-myogenic cells. Interpretations of such experiments rest upon the assumption that extinction does not occur with any appreciable frequency as a non-specific consequence of the cell hybridization process itself. In order to test this assumption we have analyzed the myogenic competence of over 140 independent homotypic hybrid clones produced by PEG-mediated fusion of rat L6 myoblasts. Based upon an evaluation of myotube formation in hybrid colonies, we demonstrate that 99% of primary hybrid clones are myogenic. The fact that 97% of secondary hybrid colonies also differentiate indicates that myogenic competence is a stable characteristic of the hybrids. Four hybrid clones were isolated and expanded for analyses of chromosome numbers, myotube formation, creatine kinase activities, and microfluorimetric DNA determinations of myotube nuclei. Our results demonstrate that polyploid homotypic hybrid cells produced by fusion of non-neoplastic, developmentally determined rat myoblasts retain and express their program of differentiation. This work provides a foundation for future studies which will investigate the expression of myogenic properties in hybrids between myogenic and non-myogenic cells. 相似文献
11.
T Gallagher Y Chardonnet J Viac J Patet A Lefèvre 《Virchows Archiv. B, Cell pathology including molecular pathology》1989,57(6):339-347
Somatic cell hybrids were obtained with electric pulse by fusion of human epithelial HeLa cells derived from a carcinoma of the uterine cervix and mouse fibroblasts 3T3.4E, deficient in thymidine kinase. Hybrids were selected and propagated in HAT media; some experiments were carried out in medium with delipidized serum. The hybrid cells were characterized by indirect immunofluorescence with a biotin-streptavidin system using a panel of nine monoclonal antibodies specific for membrane and cytoplasmic antigens of parental cells: intermediate filaments (keratins and vimentin), HLA class 1 (beta 2-microglobulin), cell activation (EGF and transferrin receptors) and cellular adhesion (fibronectin and laminin). All of these antigens were expressed in HeLa cells cultured in conventional medium or with delipidized serum. Conversely mouse fibroblasts contained only vimentin, fibronectin and laminin. All the parental antigens were present in first passage hybrid cells cultured in conventional medium. Vimentin, fibronectin and laminin were maintained in fourth passage hybrids whereas keratins, beta 2-microglobulin, EGF and transferrin receptors were no longer detected. When propagated in medium with delipidized serum, hybrid cells re-expressed these antigens after 5 days of culture. These findings suggest that the reexpression of HeLa cell antigens in hybrid cells was related to deficiency in vitamin A. 相似文献
12.
Hybrids formed by the fusion of mouse hepatoma (BWTG3) and rat fibroblast (JF1) cells exhibit the extinction of mouse albumin and alpha-fetoprotein synthesis. Karyotype analyses suggest that all parental chromosomes are present in the hybrids. The extinction, therefore, of mouse hepatocyte genes is attributed to the inhibitory action of the rat genome. In these studies, we show that these hybrids possess and express the mouse beta-glucuronidase gene (which is encoded on the same chromosome as the mouse albumin and alpha-fetoprotein gene), and we present data of Southern blot analysis which demonstrate that such hybrids have indeed retained both mouse and rat albumin DNA sequences. In addition, using mouse albumin cDNA, we have shown by cDNA-RNA reassociation kinetics that albumin mRNA is virtually absent in these hybrids. We conclude from these studies that the extinction of albumin synthesis involves a mechanism which results in the loss of cytoplasmic albumin mRNA. 相似文献
13.
Mouse splenic macrophages were fused with cells of the mouse myeloma line P3-X63-Ag8 in the presence of inactivated Sendai virus. Two continuously growing hybrid cell lines were established from fusion mixtures. These hybrid cell lines exhibited macrophage-like morphology and continued to express macrophage derived Fc receptor activity even after prolonged culture in vitro. 相似文献
14.
A rat hepatoma cell line (H4AZC2) was characterized with respect to seven liver-specific phenotypes. Ten clones from the fusion of H4AZC2 and mouse L cell were analyzed for the expression of these phenotypes. The only hepatic function retained by the hybrid clones was rat albumin synthesis which continued at reduced levels relative to the hepatoma parent. Rat albumin cDNA analysis of RNA from parental and hybrid cells indicated that the reduction in albumin production observed in the hybrids was reflected in coordinate reduction of cytoplasmic rat albumin mRNA. 相似文献
15.
Independent hybrid clones resulted from the whole cell and microcell-mediated transfer of hamster or mouse fibroblast chromosomes into mouse hepatoma XXIIa cells. The fusion was promoted with PEG, ethidium bromide alone, or in combination with HAT and ouabain, was used for selecting the hybrids. Using indirect immunoautoradiography, three clones (one intra- and one interspecies microcellular; one interspecies, whole cell fusion) have been found to express their hepatic function to synthesize transferrin. The liver specific protein--albumin--was extinguished in all the hybrid combinations. Possible mechanisms of gene expression are discussed. The hybrids selected could be used for mapping chromosomes, coding proteins, as well as for studying regulation in the tandem of albumin and alpha-fetoprotein genes in the mouse genome. The microcell mediated chromosome transfer into differentiated cells has been used to construct original genetical combinations of regulatory and structural elements of the mouse genome. 相似文献
16.
Hybrid cells derived from rat L6 myoblasts and mouse primary fibroblasts (M x F hybrids), as well as those derived from rat L6 myoblasts and mouse primary myoblasts (M x M hybrids), were examined for their ability to engage in myogenesis as judged by muscle fiber formation plus the expression of skeletal muscle myosin and creatine kinase (CK). Of 172 primary hybrid colonies scored, 59% were myogenic in the M x F fusion and 97% exhibited muscle fiber formation in the M x M fusion. Individual hybrid clones from each cross were isolated, expanded and analyzed for myogenic capabilities as well. All three M x M and all ten M x F isolated clones exhibited preferential elimination of mouse chromosomes. Nonetheless, all were capable of fusing spontaneously and of elaborating skeletal muscle myosin and CK. The three M x M hybrids expressed only MM-CK whereas nine out of ten M x F hybrids produced all three CK isoenzymes (MM, MB, BB). These results suggest that M X M hybrids express CK patterns reminiscent of the rat L6 parental cells while M X F hybrids apparently mimic mouse muscle fiber CK patterns. Various models are discussed which address these phenomena. 相似文献
17.
R Bertolotti 《Somatic cell genetics》1977,3(6):579-602
Selective glucose-free media have been used to study the reexpression of liver-specific gluconeogenic enzymes in rat hepatoma X mouse lymphoblastoma somatic hybrids. The utilization for gluconeogenesis of dihydroxyacetone or oxaloacetate requires two enzymes: fructose diphosphatase as well as either triokinase for the former or phosphoenolpyruvate carboxykinase for the latter. By sequential selection with these substrates, the reexpression of the three gluconeogenic enzymes has been dissociated. The reexpression of these enzymes is correlated with the loss of mouse chromosomes. In addition, the characterization of the parental forms of aldolase B, another liver-specific enzyme, shows that reexpression corresponds to the simultaneous production of the rat and mouse enzymes. These results demonstrate the chromosomal origin of extinction and suggest that activation of mouse silent genes which accompanies reexpression can occur without loss of the parental determinations. The hypothesis that determination involves regulatory rather than structural genes is discussed. 相似文献
18.
Syncytin-A mediates the formation of syncytiotrophoblast involved in mouse placental development. 总被引:1,自引:0,他引:1
Rui Gong Liqin Huang Jian Shi Kan Luo Gang Qiu Huixing Feng Po Tien Gengfu Xiao 《Cellular physiology and biochemistry》2007,20(5):517-526
Syncytin-A, a new mouse endogenous retroviral envelope protein expressed in placenta, can mediate cell fusion in vitro. But its physiological function was still unknown. We proposed a role for syncytin-A in syncytiotrophoblast (SynT) formation derived from the differentiation of trophoblast stem (TS) cells during placental development. To evaluate this hypothesis, we analyzed the involvement of syncytin-A in the differentiation of mouse TS cells. After withdrawing fibroblast growth factor 4 (FGF4), TS cells can fuse to form SynT cells. We found syncytin-A mRNA and protein expression are colinear with fusion index increase during TS cell differentiation. Expression of syncytin-A is localized in SynT cells through in situ immunofluorescent staining. By using specific antibody and antisense oligonucleotides, we demonstrated that inhibition of syncytin-A lead to obvious decrease of SynT cell formation. These results present evidence in support of the direct role for syncytin-A in mouse TS cell fusion and differentiation involved in placental development. 相似文献
19.
20.
Tsygankova OM Prendergast GV Puttaswamy K Wang Y Feldman MD Wang H Brose MS Meinkoth JL 《Molecular and cellular biology》2007,27(19):6647-6658
Although abundant in well-differentiated rat thyroid cells, Rap1GAP expression was extinguished in a subset of human thyroid tumor-derived cell lines. Intriguingly, Rap1GAP was downregulated selectively in tumor cell lines that had acquired a mesenchymal morphology. Restoring Rap1GAP expression to these cells inhibited cell migration and invasion, effects that were correlated with the inhibition of Rap1 and Rac1 activity. The reexpression of Rap1GAP also inhibited DNA synthesis and anchorage-independent proliferation. Conversely, eliminating Rap1GAP expression in rat thyroid cells induced a transient increase in cell number. Strikingly, Rap1GAP expression was abolished by Ras transformation. The downregulation of Rap1GAP by Ras required the activation of the Raf/MEK/extracellular signal-regulated kinase cascade and was correlated with the induction of mesenchymal morphology and migratory behavior. Remarkably, the acute expression of oncogenic Ras was sufficient to downregulate Rap1GAP expression in rat thyroid cells, identifying Rap1GAP as a novel target of oncogenic Ras. Collectively, these data implicate Rap1GAP as a putative tumor/invasion suppressor in the thyroid. In support of that notion, Rap1GAP was highly expressed in normal human thyroid cells and downregulated in primary thyroid tumors. 相似文献