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At least 343 records · Page 19Linked to original sources
PubMed1998

An autocrine function for transforming growth factor (TGF)-beta3 in the transformation of atrioventricular canal endocardium into mesenchyme during chick heart development.

Transformation of atrioventricular canal endocardium into invasive mesenchyme is a critical antecedent of cardiac septation and valvulogenesis. Previous studies by Potts et al. (Proc. Natl. Acad. Sci. USA 88, 1510-1520, 1991) showed that treatment of atrioventricular canal endocardial and myocardial cocultures with TGFbeta3 antisense oligodeoxynucleotides blocked mesenchyme formation. Based on this observation, we sought to: (i) identify the target tissue of TGFbeta3 antisense oligos in this transformation bioassay, and (ii) more clearly define the mechanism of TGFbeta3 function in atrioventricular canal mesenchyme formation. In situ hybridization and immunohistochemistry showed little or no TGFbeta3 mRNA or protein in the atrioventricular canal myocardium or endocardium prior to mesenchyme formation (stage 14; paraformaldehyde fixation). However, by stage 18 transforming atrioventricular canal endocardial cells and mesenchyme as well as myocardium were positive for both TGFbeta3 mRNA and protein. In culture bioassays, atrioventricular canal endocardial monolayers pretreated with antisense phosphorothioate oligodeoxynucleotides to TGFbeta3 did not transform into invasive mesenchyme in response to cardiocyte conditioned medium: the subsequent addition of exogenous TGFbeta3 protein relieved this inhibition. Control cultures without pretreatment or those receiving missense oligos generated similar numbers of invasive mesenchyme in response to cardiocyte conditioned medium. Direct addition of TGFbeta3 protein to atrioventricular canal endocardial monolayers in the absence of cardiocyte conditioned medium resulted in loss of cell:cell associations and stimulated cellular hypertrophy, but did not engender invasive mesenchyme formation or alter endocardial proliferation after 24 h of culture. Similar results were obtained with TGFbeta2 protein, either alone or in combination with TGFbeta3. The results of this study indicate that: (i) atrioventricular canal endocardium expresses TGFbeta3 in response to a myocardially derived signal other than TGFbeta3, (ii) atrioventricular canal endocardial TGFbeta3 functions in an autocrine fashion to elicit selected characteristics necessary for cushion tissue formation, and (iii) TGFbeta3 alone or in combination with TGFbeta2 is insufficient to transform atrioventricular canal endocardium into invasive mesenchyme in culture.

Animals↗
PubMed1986

Positive screening and transformation of ura5 mutants in the fungus Podospora anserina: characterization of the transformants.

To develop a transformation system in the filamentous fungus Podospora anserina we have selected ura5 mutants deficient in orotidylic acid pyrophosphorylase using a positive screening. These mutants could be transformed to prototrophy by an hybrid vector carrying the ura5 gene of this organism. The properties of the transformants have been analysed. In most cases integration of the transforming vector occurred outside the ura5 locus and frequently repeated tandem copies of the vector were found. Reversion of the transformants could also be selected and we found that it can occur by exact or only partial excision of the integrated vector.

Ascomycota↗
PubMed1986

Heterologous insertion of transforming DNA and generation of new deletions associated with transformation in Aspergillus nidulans.

The analysis of four transformants for the proline catabolism (prn) gene cluster of Aspergillus nidulans is reported. Using a combination of traditional genetic methodology and Southern hybridisation we have shown that in two cases multiple copies of the transforming plasmid have been integrated into linkage groups other than VII, which contains the prn cluster. In the other two cases integration of the plasmid has probably occurred homologously. The phenotype of these transformants is broadly consistent with increased copy number resulting in increased expression. Genetic manipulation of these transformants using the sexual or parasexual cycles has shown that recombination events during and possibly also subsequent to integration of the transforming DNA can generate new mutational lesions, in particular, deletions.

Aspergillus nidulans↗
PubMed1982

Reversibility of the transformed and neoplastic phenotype. III. Long-term treatment with electrophoretically pure mouse interferon leads to the progressive reversion of the phenotype of X-ray transformed C3H/10T1/2 cells.

Electrophoretically pure mouse interferon induced the progressive reversion of the transformed phenotype of a clone of X-ray transformed C3H/10T1/2 cells. Cells of this clone did not harbor C-type particles and reverse transcriptase activity was not detected. Interferon-treated transformed cells were aligned without cellular overlapping and attained low cell densities. Morphologic changes were associated with the appearance of a thick layer of submembranous microfilaments. The tumorigenicity of interferon-treated cells was markedly reduced. Back reversion to the transformed phenotype occurred progressively when these cells were passaged in the absence of interferon. These results suggest that interferon may induce the reversion of the transformed phenotype by a mechanism other than by its antiviral activity.

Animals↗
PubMed2001

Enhancement of 3-methylcholanthrene-induced neoplastic transformation by 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone in the two-stage transformation assay in C3H 10T1/2 cells.

3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)furanone (MX) is a mutagenic by-product found in chlorinated drinking water. It is a multi-site carcinogen in Wistar rats although the mechanisms of action of the carcinogenesis remain unresolved. We evaluated the ability of MX to promote development of transformation foci in a two-stage cell transformation assay in vitro. C3H 10T1/2 mouse embryonic fibroblasts were exposed to 3-methylcholanthrene (MC, 5 microg/ml) in the initiation phase and to MX (0.5, 1 or 2 microg/ml) or the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA, the positive control, 0.3 microg/ml) during the promotion phase of the assay on dishes. In other experiments, the cells were exposed to MX (0.5, 5 or 10 microg/ml) only in the initiation phase. At the end of the assay (6 weeks from the start of the assay), the transformation foci were counted and scored after fixation and staining of the cells. MC increased the total number of transformation foci per dish and the number of malignant type III foci, and TPA further promoted this phenomenon. When MX was added during the promotion phase in the MC-initiated cells, it promoted the development of the transformation foci in a dose-dependent manner. MX alone (added as an initiator) also slightly increased the development of the foci, including the malignant forms (type II and III), but the effect was not dose-dependent. In contrast to MC-induced foci, TPA did not promote the development of MX-initiated foci, it even decreased their number. The results suggest that MX may also have potential to promote tumor development.

Animals↗
PubMed2003

Effects of chlorohydroxyfuranones on 3-methylcholanthrene-induced neoplastic transformation in the two-stage transformation assay in C3H 10T1/2 cells.

3-Chloro-4-(chloromethyl)-5-hydroxy-2(5 H)-furanone (CMCF), 3-chloro-4-methyl-5-hydroxy-2(5 H)-furanone (MCF) and 3,4-dichloro-5-hydroxy-2(5 H)-furanone (MCA) are chlorination byproducts in disinfected drinking water. These compounds are positive in genotoxicity tests in vitro. We have previously shown that 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5 H)-furanone (MX) can induce malignant transformed foci in the two-stage cell transformation assay in C3H 10T1/2 cells in vitro in both the initiation and promotion phases. In the present study we compared the effects of CMCF, MCF and MCA in the same assay. C3H 10T1/2 mouse embryonic fibroblasts were exposed to these chlorohydroxyfuranones (CHFs) at three different concentrations in the initiation phase or the promotion phase of the assay. In the latter experiments 3-methylcholanthrene (MC, 5 micro g/ml) was used as the initiating chemical. The phorbol ester 12- O-tetradecanoylphorbol-13-acetate (TPA, 0.3 micro g/ml) was used as a positive control promoter. At the end of the assay (6 weeks from the start), the transformation foci were counted and scored after fixation and staining of the cells. When added at the initiation phase of the assay on their own, CMCF and MCF, but not MCA, increased the transformation foci formation. TPA added in the promotion phase did not modify the responses of CMCF and MCF but TPA increased the number of foci in MCA-treated cells. When CHFs were added during the promotion phase to the MC-initiated cells, MCF and MCA enhanced the development of the transformation foci. The effect of CMCF was equivocal since at higher concentrations CMCF actually decreased the number of the MC-induced foci. Including the previous data for MX in this assay and considering the lowest active concentrations, the initiation activity of the foci formation decreased in the order MX >CMCF >MCF, i.e. with the decreasing number of chlorine atoms of the methyl group in the 4-position of the CHF molecule (two, one, and zero, respectively). In contrast, the activity in the promotion phase did not follow the same pattern. MX, MCF and MCA were all active over the same concentration range. Hence, in addition to MX, MCF and MCA may also possess some potential to promote tumor development.

Animals↗
PubMed2003

Stable transformants of the azaphilone pigment-producing Monascus purpureus obtained by protoplast transformation and Agrobacterium-mediated DNA transfer.

The high-level pigment-producing Monascus strain IBCC1 was characterized by random amplification of polymorphic DNA as M. purpureus. This technique allowed us to distinguish between M. purpureus and M. ruber strains. Transformation of Monascus species has not been previously reported. Protoplast formation and regeneration from M. purpureus IBCC1 was optimized by modification of growth media, lytic enzyme mixture, osmotic stabilizer and regeneration media. Of the Monascus transformants, 60% were found to be mitotically stable and retained the plasmid inserted in the chromosome after repeated sporulation cycles. Additionally, an Agrobacterium-mediated DNA transfer system was developed. The transformants obtained by Agrobacterium-mediated DNA transfer remained fully stable (98%) after four sporulation rounds and showed bands of hybridization corresponding to integration of the plasmid in different sites of the genome. The green fluorescent protein marker was well expressed in the M. purpureus transformants. The development of transformation systems is a basic tool for advanced genetic manipulation of the natural pigment producers, M. purpureus and M. ruber.

DNA, Bacterial↗
PubMed2006

Transgenic Medicago truncatula plants obtained from Agrobacterium tumefaciens -transformed roots and Agrobacterium rhizogenes-transformed hairy roots.

Medicago truncatula, barrel medic, is a forage crop that has been developed into a model legume. The development of new transformation methods is important for functional genomic studies in this species. Based on Agrobacterium tumefaciens-mediated transformation of root explants, we developed an effective system for producing M. truncatula (genotype R108) transgenic plants. Among the four A. tumefaciens strains (AGL1, C58C1, EHA105 and LBA4404) tested, EHA105 and AGL1 were most effective in regenerating transgenics. Callus induction frequency from root explants was 69.8%, and plantlet/shoot regeneration frequency was 41.3% when EHA105 was used. Transgenic nature of the regenerated plants was confirmed by PCR and Southern hybridization analyses. Progeny analysis revealed stable Mendelian meiotic transmission of transgenes. Because M. truncatula is particularly useful for the study of root endosymbiotic associations, we further developed a plant regeneration system from A. rhizogenes-transformed hairy roots of M. truncatula. Fertile true transgenic plants were regenerated from the hairy roots, thus allowing the assessment of gene functions at the whole plant level. Segregation analysis revealed that the hairy root genes could be segregated out in the progenies. By coupling A. rhizogenes-mediated hairy root transformation and the regeneration system reported here, once potential genes of interest are identified, the transformed hairy roots carrying such genes could be directly regenerated into plants for more detailed characterization of the genes.

Acetyltransferases↗
PubMed1976

Interactions of concanavalin A with chick embryo fibroblasts transformed by Rous sarcoma virus. Study with an RSV mutant thermosensitive for transformation.

The interactions between concanavalin A and chick embryo fibroblasts, normal and infected with Rous sarcoma virus (RSV-BH) or its thermosensitive mutant RSV-BH-Ta, have been studied. Normal chick embryo cells and RSV-BH transformed cells showed at 4 and 25 degrees C a similar number of concanavalin A receptors per cell. Analysis of the binding data by the Scatchard relation showed that apparent changes in binding as a function of temperature are due to the thermodynamic properties of the process and not to endocytosis. The lectin receptors on the cell surface of normal and RSV-BH infected cells showed homogeneity in their binding properties. Chick cells infected with RSV-BH-Ta showed a lectin binding behavior that was dependent on the temperature at which the cells were grown. At the permissive temperature for transformation (37 degrees C), the binding process was similar to that observed for normal and RSV-BH infected cells. At the nonpermissive temperature (41 degrees C), the cells showed at least two sets of concanavalin A receptors. The new set of receptors on the cell surface had a lower lectin affinity than those observed in the same cells at 37 degrees C. Chick cells infected with RSV-BH showed an enhanced agglutinability by concanavalin A, as compared with normal cells. Cells infected with RSV-BH-Ta showed a reversal of the correlation between increased concanavalin A agglutinability and the transformed state. At the permissive temperature for transformation, the cells were not agglutinable, whereas at the nonpermissive temperature they presented agglutinability indexes as high as those observed with RSV-BH infected cells. This enhanced agglutinability observed with cells maintained at the nonpermissive temperature for transformation may be related to the new set of low affinity receptors present at 41 degrees C.

Agglutination Tests↗
PubMedDate not supplied

Involvement of deoxyribonuclease activity in the differential sedimentation rates of nucleoids from non-transformed and transformed mouse embryo fibroblasts.

Lysis of non-transformed confluent C3H10T1/2C18 mouse embryo fibroblasts in the presence of detergents, high concentrations of salt and EDTA on top of neutral sucrose gradients revealed a reduced sedimentation rate of the resulting nucleoids from these cells compared to those from exponentially growing non-transformed cells or from transformed cells. Exposure of confluent cells to 1000 rads of X-ray had no effect on this rate of nucleoid sedimentation; and ethidium bromide titration and alkaline sucrose analysis suggested the presence of discontinuities in the DNA. An endonucleolytic activity could be extracted from nuclei of these cells with 0.5 M NaCl, indicating a very tight association with the chromatin. Such an enzyme in non-transformed confluent cells may account for the differences in nucleoid structure and may be related to changes in cell function with normal arrest of cell growth. There was no growth-phase effect on the properties of nucleoids from transformed cells.

Animals↗
PubMed1975

Fibrin overlay methods for the detection of single transformed cells and colonies of transformed cells.

Fibrin overlay methods are described which can detect the plasminogen activator produced by single transformed cells or small colonies of transformed cells. These methods were applied to malignant cells derived from humans, mice, hamsters, rats, and chicks. The lysis observed was plasminogen dependent. Transformation of chicken cells by Rous sarcoma virus was detected 4 days after infection. The number of lysis zones produced was proportional to the virus inoculum and was identical to the number of morphologically determined foci. These methods may also have application in model systems for scoring transformation by chemicals. Transformed mouse and chicken cells were detected at the single cell level and the number of lysis zones produced was dependent on the number of cells present, the time of incubation, and the concentration of plasminogen.

Animals↗
PubMed1982

Detection of a transforming gene product in cells transformed by Moloney murine sarcoma virus.

We identified, in cells transformed by Moloney murine sarcoma virus (M-MuSV clone 124), a protein encoded by the M-MuSV transforming gene, v-mos. An antiserum against a synthetic peptide corresponding to the C terminus of a protein predicted from the v-mos nucleotide sequence specifically recognizes a protein doublet of approximately 37,000 daltons from 35S-methionine-labeled M-MuSV 124-transformed producer cells. By peptide mapping, this protein is almost identical to the 37 kd in vitro translation product from the M-MuSV v-mos gene. Immunoprecipitates from 32P-labeled cells contain a single v-mos-specific phosphoprotein, which has at least six sites of phosphorylation containing phosphoserine. Pulse-chase experiments show that the lower band in the 35S-methionine-labeled doublet is the primary translation product, which is modified, probably by phosphorylation, to yield the upper band. A similar mos protein is immunoprecipitated from HT1-MuSV-transformed cells, but not from uninfected NIH/3T3 cells. These mos proteins are present at very low levels in transformed cell lines. Cells acutely infected with M-MuSV 124, however, transiently contain much higher levels of the mos protein. These high levels coincide with extensive cell mortality.

Animals↗
PubMed1987

Revertants of v-fos-transformed fibroblasts have mutations in cellular genes essential for transformation by other oncogenes.

Morphologic revertants of FBJ murine sarcoma virus (v-fos)-transformed rat-1 fibroblasts were isolated using a novel selection procedure based on prolonged retention of rhodamine 123 within mitochondria of v-fos-transformed versus normal fibroblasts. Two classes of revertants were isolated: class I revertants have sustained mutations in cellular genes, and a class II revertant has a nonfunctional v-fos provirus. Somatic-cell hybridization studies suggested that the revertant phenotype was recessive to the transformed phenotype. Class I revertants were also resistant to retransformation by v-gag-fos-fox, v-Ha-ras, v-abl, and v-mos, but could be retransformed by the trk oncogene and polyoma virus middle T antigen. These results suggest that the class I revertants sustained mutations in one or more cellular genes essential for transformation by some, but not all, oncogenes. Our data suggest the existence of common biochemical pathways for transformation.

Animals↗
PubMed1995

Overexpression of the hyaluronan receptor RHAMM is transforming and is also required for H-ras transformation.

Overexpression of the RHAMM gene by transfection into fibroblasts is transforming and causes spontaneous metastases in the lung. H-ras-transformed fibrosarcomas transfected with a dominant suppressor mutant of RHAMM exhibit a so-called revertant phenotype and are completely nontumorigenic and nonmetastatic. Conversely, fibroblasts stably expressing low levels of RHAMM as a result of antisense transfection are resistant to ras transformation. Collectively, these results indicate that RHAMM acts downstream of ras. The loss of functional RHAMM ablates signaling within focal adhesions, in particular changes in focal adhesion kinase phosphorylation, and as a result these focal adhesions are unable to turn over in response to hyaluronan. These results provide evidence of the oncogenic potential of a novel extracellular matrix receptor and establish a functional link between transformation by ras and signaling within focal adhesions that are required for transformation by this oncogene.

Amino Acid Sequence↗
PubMed1979

Change of isozyme pattern during activation of a transforming gene product: its relation to other biochemical markers of cellular transformation and differentiation.

Isozyme patterns of leucine aminotransferase were studied in connection with glucose transport and DNA synthesis during the activation and deactivation of the transforming gene product in rat kidney cells transformed by one Rous sarcoma virus mutant (which has a temperature-sensitive lesion in its transforming gene. On temperature shift-down of confluent transformed cells grown at 40 degrees C in the presence of fresh serum, isozyme III of leucine aminotransferase appeared in 12--20 h, with increasing amounts from 24 to 48 h. Upon temperature shift-up, isozyme I became the predominant form in these cells within 4 days, the major change occurring within the first 24 h. The rate of protein turnover was similar to the rate of loss of isozymes I and III during temperature shift-down and shift-up, respectively. A stimulation of incorporation of [3H]thymidine into DNA was observed within 8--12 h after temperature shift-down of the transformed cells. For the maintenance of stimulated DNA synthesis for at least 16 h, continued exposure to the permissive temperature is not necessary. Stimulation of glucose transport occurred prior to the stimulation of [3H]thymidine incorporation. The isozymes of leucine aminotransferase also changed during the in vitro differentiation of Yaffee L6A cells in such a way that isozyme I represented the major part of this enzyme in the fused myotube, and isozyme III was more predominant in the less differentiated state (mononucleated cells).

Animals↗
PubMed1993

Multiple tandem integrations of transforming DNA sequences in yeast chromosomes suggest a mechanism for integrative transformation by homologous recombination.

In yeast, the fate of linear DNA molecules upon transformation is determined by the existence of sequence homology between chromosomes and the ends of the transforming molecule. To understand the mechanism of integration of transforming DNA, we have studied the influence of DNA concentration on the frequency and type of transformants obtained, using either non-replicative or replicative plasmids. In both cases, increasing DNA concentration results in multiple tandem repeats integrated into the chromosome containing the homologous target sequence. When a diploid strain is transformed, multiple tandem repeats occur in only one of the two homologous chromosomes at a time. The frequency distribution of the different types of integrants observed indicates non-independent integration events likely to result from plasmid-plasmid interaction prior to chromosome integration. In addition, our results define the proper conditions for optimized gene targetting or gene rescue experiments.

Base Sequence↗
PubMed2004

The malignant transformation event as a clonal transformation of apoptosis to anti-apoptosis pathways and of cell cycle dynamics.

Anti-apoptosis, as a more vital aspect of a whole series of progressive steps in carcinogenesis, would appear primarily a phenomenon of variable nonresponsiveness. Programmed cell death, in addition, would appear to affect the individual cell as attributes also of an integral field phenomenon constituted by clones of tumor cells that grow, proliferate and spread. Various modes of development of anti-apoptosis subsequently involving the development of genetic abnormalities would possibly account for transformation as a neoplastic phenomenon. It is in terms of such pathways as telomeric length preservation, of anti-apoptosis as failed activation of apoptosis, and of failed DNA binding events that clonally selected tumor cells would tend to inherently progress to higher grade and stage and to spread systemically. Moreover, clonal and field events would possibly influence in their turn individual cell cyclical activity and programmed cell pathways in neoplastic transformation. Anti-apoptosis would constitute, in fact, a predicted system of pathways that switch from apoptosis as an essential pathway in malignant transformation. Angiogenesis, as a full series of cascade events of a paracrine/autocrine nature, would also participate with anti-apoptosis in a tumor cell cycle transformation that is self-amplifying. Given different modes of how apoptosis would convert to anti-apoptosis in tumorigenesis, perhaps abnormal cell cycle dynamics might also contribute to the evolving nature of such neoplastic transformation.

Apoptosis↗
PubMed2002

Diverse effects of RacV12 on cell transformation by Raf: partial inhibition of morphological transformation versus deregulation of cell cycle control.

Activated Raf kinases and Rac GTPases were shown to cooperate in the oncogenic transformation of fibroblasts, which is characterised by the disassembly of the cellular actin cytoskeleton, a nearly complete loss of focal adhesion complexes and deregulated cell proliferation. This is surprising since the Rac GTPase induces actin structures and the adhesion of suspended cells to extracellular matrix proteins. NIH 3T3 cells expressing a hydroxytamoxifen-inducible oncogenic c-Raf-1-oestrogen receptor fusion protein (c-Raf-1-BxB-ER, N-BxB-ER cells) undergo morphological transformation upon stimulation of the Raf kinase. We show that treatment with the Rac, Rho and Cdc42 activating Escherichia coli toxin CNF1 or coexpression of an activated RacV12 mutant partially inhibits and reverses the disassembly of cellular actin structures and focal adhesion complexes by oncogenic Raf. Activation of the Rac GTPase restores actin structures and focal adhesion complexes at the cellular boundary, leading to spreading of the otherwise spindle-shaped Raf-transformed cells. Actin stress fibres, however, which are regulated by the function of the Rho GTPase, are disassembled by oncogenic Raf even in the presence of activated Rac and Rho. With respect to the RacV12-mediated spreading of Raf-transformed cells, we postulate an anti-oncogenic function of the activated Rac. Another feature of cell transformation is the deregulation of cell cycle control. NIH 3T3 cells expressing high levels of the c-Raf-1-BxB-ER protein undergo a cell cycle arrest upon stimulation of the oncogenic Raf kinase. Our results show that in N-BxB-ER-RacV12 cells the expression of the activated RacV12 mediates cell proliferation in the presence of high-intensity Raf signals and high levels of the Cdk inhibitor p21(Cip1). These results indicate a pro-oncogenic function of the Rac GTPase with respect to the deregulation of cell cycle control.

3T3 Cells↗