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A Eva

Publications and source records attributed to A Eva.

At least 37 records · Page 2Linked to original sources
PubMed1991

Dominant dysplasia of the lens in transgenic mice expressing the dbl oncogene.

To study the transforming activity of the dbl oncogene and its effect on normal development in vivo, we linked dbl cDNA to promoters with different cell type specificities and used the constructs to generate transgenic mice. The promoters included the mouse alpha A-crystallin promoter, the rat insulin II promoter, and a mouse metallothionein promoter. We also generated transgenic mice carrying a recombinant cosmid clone that contains the entire dbl gene. Mice with the crystallin promoter construct developed cataracts and expressed the dbl protein in their lenses. The architecture of the lenses suggested a block to the normal pattern of differentiation and elongation of the secondary fiber cells. Mice with the insulin II promoter expressed dbl protein in the pancreas but showed no evidence of diabetes and no apparent pancreatic beta-cell defects. Similarly, mice with the metallothionein promoter expressed dbl protein in heart and testes, but showed no pathologic abnormalities in these tissues even after treatment with heavy metals. However, one family of mice carrying the metallothionein promoter construct showed cataracts and a dramatic fibroblastic dysplasia of the lens. One family with the cosmid-dbl gene showed a nearly identical lenticular dysplasia, but with a slower developmental time course. Thus, although the dbl oncogene did not induce neoplasia in any of the mice studied, it is apparently capable of interfering with the ability of the lens epithelial cells to differentiate into lens fiber cells, and of inducing metaplasia of the epithelial cells into fibroblastic cells.

Animals↗
PubMed1991

A region of proto-dbl essential for its transforming activity shows sequence similarity to a yeast cell cycle gene, CDC24, and the human breakpoint cluster gene, bcr.

Proto-dbl is a human proto-oncogene, whose oncogenic activation was initially detected by DNA transfection. We report significant sequence similarity between the predicted proto-dbl product and the products of CDC24, a Saccharomyces cerevisiae cell division cycle gene required for correct budding and establishment of cell polarity, and bcr, a gene implicated in the pathogenesis of chronic myelogenous leukemia (CML). Of 925 residues of the predicted proto-dbl protein, a stretch of 238 residues showed 29% and 22% identity over a region of similar length of the CDC24 and bcr proteins, respectively. When evolutionarily conservative substitutions were taken into account, the similarities were 68.8% and 71.6% for proto-dbl/CDC24 and proto-dbl/bcr gene products, respectively. Moreover, all three sequences were predicted to be markedly hydrophilic over this region. Very small deletions within the conserved region completely abolished transforming activity of dbl, while extensive deletion outside of this region had no effect. Even substitutions over a small stretch of close similarity with the other proteins substantially impaired transforming activity. Cells transformed by the dbl oncogene, like cdc24 mutants arrested at the nonpermissive temperature, form multinucleate cells. Thus, our findings indicate that the conserved region is an essential domain that may reflect important functional similarities among these otherwise highly divergent molecules.

Amino Acid Sequence↗
PubMed1989

Chromosomal localization of DBL oncogene sequences.

The DBL oncogene was generated by rearrangements involving three discontinuous regions of the human genome. Analyses of panels of human X rodent somatic cell hybrids demonstrated that the DBL proto-oncogene located on the X chromosome (just proximal or distal to bands q26-27.2) underwent recombination at its 5' and 3' ends with sequences derived from chromosomes 3 (p13q-ter) and 16 (p13-q22), respectively. DBL was localized to chromosome Xq27-q28 by in situ hybridization. Another oncogene, MCF2, was previously shown to contain sequences derived from Xq27 as well. Comparison of the restriction maps and nucleotide sequences of genomic and cDNA clones representing the chromosome X-specific sequences of the DBL oncogene and MCF2, taken together with their chromosomal localization, indicates that these oncogenes were derived from the same genetic locus.

Animals↗
PubMed1989

NIH3T3 fibroblasts transformed by the dbl oncogene show altered expression of bradykinin receptors: effect on inositol lipid turnover.

We have examined polyphosphoinositide turnover in mouse fibroblasts (NIH3T3) transformed by the dbl oncogene as compared to normal cells. The dbl-transformed fibroblasts did not show alterations of the basal level of inositol polyphosphates, polyphosphoinositides, diacylglycerol or phosphatidic acid. This indicates that the activity of C-type phospholipases, inositol lipid kinases and diacylglycerol kinase is not altered in dbl-induced transformation. However, dbl-transformed NIH3T3 cells exhibited increased inositol lipid turnover in response to bradykinin. Further analysis revealed significantly higher number of bradykinin receptors in dbl transfectants as compared to control NIH3T3. When several clonally-derived dbl NIH3T3 transfectants were analyzed, we observed a large variation of their bradykinin receptor number. Cell lines exhibiting increased bradykinin binding, however, failed to show augmented mitogenic response to the peptide agonist. Among other oncogenes, only ras showed a similar effect. We conclude that increased bradykinin receptor number is a phenomenon observed with several cell lines transformed by different oncogenes, and it does not correlate with either enhanced mitogenic responsiveness of transformed cells to the peptide, or with the presence of a specific oncogene in the transformant.

Animals↗
PubMed1989

The human dbl-proto-oncogene product is a cytoplasmic phosphoprotein which is associated with the cytoskeletal matrix.

The translational product of the dbl oncogene is a 66 kDa (p66) protein with no apparent sequence similarity to any of the known oncogene products, whereas the human dbl proto-oncogene encodes a translational product of 115 kDa (p115). We compared proto-dbl p115 and dbl p66 with respect to their subcellular localization, biogenesis and post-translational modifications. Like p66, p115 was found to be a cytoplasmic phosphoprotein present in both cytosol and crude membrane preparations. Membrane fractionation studies revealed that p115 as well as p66 were primarily associated with fractions enriched in plasma membranes, suggesting that this subcellular compartment is a likely site of action of dbl proteins. The membrane-associated forms of p115 and p66 were fairly resistant to solubilization by nonionic detergents, suggesting that dbl proteins associate with the cytoskeletal matrix. p115 was also found to be phosphorylated primarily on serine residues. However, p115 was phosphorylated to a lesser extent as compared to the phosphorylated form of p66. The half-life of proto-dbl p115 was significantly shorter (1 hour) than that of dbl p66 (5-6 h). The higher stability of p66 is likely due to the acquisition of unrelated human sequences and/or to the deletion of the N-terminal region of proto-dbl.

Cell Line, Transformed↗
PubMed1989

Expression of the dbl proto-oncogene in Ewing's sarcomas.

We have investigated the expression of the dbl proto-oncogene in childhood tumors of known or suspected neuroectodermal origin. We found that while the dbl gene is consistently found expressed in Ewing's sarcoma as a single mRNA species, of approximately 5.0 kb, it is generally absent in two seemingly related categories of tumors, neuroblastoma and neuroepithelioma. The specificity of expression of the dbl proto-oncogene in Ewing's sarcoma supports the concept that Ewing's sarcoma may be differentiated from two closely related tumors, neuroblastoma and neuroepithelioma, on the basis of the presence of specific molecular markers.

Guanine Nucleotide Exchange Factors↗
PubMed1989

The N-terminal region of proto-dbl down regulates its transforming activity.

The dbl proto-oncogene can transform NIH3T3 cells when overexpressed, but its transforming activity is about 50 to 70 fold lower than that of the dbl oncogene. The dbl oncogene encodes a protein of 478 amino-acids while proto-dbl encodes a protein of 925 amino-acids. The genesis of dbl involved the loss of the first 497 amino-acids of proto-dbl and the acquisition of a new N-terminus from another human locus. The last 428 amino-acids of proto-dbl and dbl product are identical with the exception of a single conservative amino-acid change. Any of these alterations could be responsible for the greater transforming activity of dbl. In order to define the role of these alterations more precisely, we constructed two deletion mutants, one derived from proto-dbl and the second from dbl in which only their last 428 amino-acids were retained. Under the control elements of the same promoter, the transforming activity of each of these mutants was similar to that of the dbl oncogene, i.e. 60-80 fold greater than that of proto-dbl. This finding suggests that the loss of the first 497 amino-acid of proto-dbl, rather than the acquisition of a new N-terminus, is crucial to the enhanced transforming activity of the dbl oncogene. Both mutant proteins were equally distributed between the membrane and cytosolic fractions, a pattern similar to that of their corresponding parental proteins. These results suggest that the subcellular distribution of proto-dbl is determined by its C-terminal 428 amino-acids. Unlike their parental proteins, neither mutant was phosphorylated, indicating that phosphorylation is not required for dbl transforming activity. In addition to the lack of phosphorylation, each mutant protein had a half-life of 5-6 h while the half-life of proto-dbl was about 1 h. Thus, our data suggest that the N-terminal half of proto-dbl can down regulate its transforming activity and that sequences within this region are responsible for rapid turnover of the protein.

Animals↗
PubMed1988

Molecular cloning and characterization of the human dbl proto-oncogene: evidence that its overexpression is sufficient to transform NIH/3T3 cells.

We isolated cDNA clones representing the human dbl proto-oncogene transcript. Nucleotide sequence analysis revealed an open reading frame encoding a predicted protein of 925 amino acids. Using peptide antisera directed against specific proto-dbl peptides, a 115-kd protein was detected in COS cells transfected with an expression vector containing the entire coding region of proto-dbl. This mol. wt is consistent with that predicted from the open reading frame. We have previously shown that the dbl oncogene was generated by substitution of the 5' portion of proto-dbl with an unrelated human sequence. In this study we show that this rearrangement resulted in the loss of the 497 amino-terminal codons of the dbl proto-oncogene. Under the influence of a strong promoter proto-dbl could readily transform NIH/3T3 cells but its transforming activity was less than that of the dbl oncogene driven by the same promoter. Proto-dbl overexpression is, therefore, sufficient to transform NIH/3T3 cells, but specific structural alterations of its coding region significantly enhance its transforming activity. No apparent similarity was detected between the predicted proto-dbl product and other known proto-oncogenes. However, a stretch of 300 amino acids within the N-terminal half of proto-dbl showed structural similarity to the intermediate filament vimentin. This region in proto-dbl contains a heptad repeat motif characteristic of an alpha-helical coiled-coil structure. Taken together, these findings indicate that the human proto-dbl represents a new class of cellular oncogenes that may be related to cytoskeletal elements of the cell.

Amino Acid Sequence↗
PubMed1988

The predicted DBL oncogene product defines a distinct class of transforming proteins.

The DBL transforming gene was originally identified by transfection of NIH 3T3 cells with DNA from a human B-cell lymphoma. This gene was found to have arisen as a result of recombination of the 3' portion of the DBL protooncogene coding sequences with an unrelated segment of human DNA. It encodes a cytoplasmic protein that is equally distributed between cytosol and crude membrane fractions. To further characterize this transforming gene, a biologically active cDNA clone of the DBL transforming gene mRNA was isolated. Analysis of the sequence of the DBL oncogene cDNA revealed a long open reading frame that encodes a hybrid protein whose first 50 amino acids (at least) derive from a complete exon of a different locus. No significant homology with known oncogenes or any known protein sequences was demonstrated. The computer analysis of the predicted DBL protein indicated it is highly hydrophilic with no hydrophobic domains characteristic of a membrane-spanning region or signal peptide. Thus, the DBL oncoprotein is distinct among known transforming gene products.

Amino Acid Sequence↗
PubMed1988

Detection of activated proto-oncogenes in N-nitrosodiethylamine-induced liver tumors: a comparison between B6C3F1 mice and Fischer 344 rats.

DNA from B6C3F1 mouse and Fischer 344 rat liver tumors induced by N-nitrosodiethylamine (DEN) were examined for the ability to induce morphological transformation of NIH3T3 cells. DNAs from 14 of 33 of the mouse liver tumors induced by a single injection of DEN at 12 or 15 days of age were positive in this assay while DNA from only one of 28 DEN-induced rat liver tumors was active. Southern blot analysis of the NIH3T3 transformants derived from the mouse liver tumors revealed amplified and/or rearranged restriction fragments homologous to the H-ras proto-oncogene. DNA from two independent foci induced by the rat tumor DNA did not hybridize to probes for members of the ras gene family or c-raf. Activating mutations in the H-ras genes from the DEN-induced mouse liver tumors were characterized by selective oligonucleotide hybridization and the detection of a new XbaI restriction site by Southern blot analysis. In activated H-ras genes from the DEN-induced mouse liver tumor DNA, seven of 14 had a CG----AT transversion at the first base of the 61st codon, three of 14 had an AT----GC transition and four of 14 had the AT----TA transversion at the second base of codon 61. This spectrum of mutations is very similar to that recently observed in activated H-ras genes found in spontaneously occurring B6C3F1 mouse liver tumors. Taken together, the data suggest that the DEN-induced rat and mouse liver carcinogenesis may involve genetic targets other than or in addition to the H-ras gene.

Animals↗
PubMed1987

Independently activated dbl oncogenes exhibit similar yet distinct structural alterations.

The dbl oncogene was initially isolated following transfection of NIH3T3 cells with DNA of a human diffuse B cell lymphoma. Its transcribed sequences were shown to be distributed over a 30-kb span within a molecularly cloned 45-kb segment of human DNA which contained the transforming gene. By restriction mapping, its transcribed region corresponded to that of its normal allele, except at the 5' end where a rearrangement involved transcribed dbl oncogene sequences from another locus. An independent isolate of a dbl-related transforming gene was obtained following transfection of NIH3T3 cells with DNA of a human nodular poorly differentiated lymphoma (NPDL). Physical mapping indicated that this transforming gene, designated NPDL-dbl, shared considerable homology with the dbl oncogene, but differed at both 5' and 3' termini. Its point of divergence from the normal allele at the 5' end was at least 10 kb upstream from that of the dbl oncogene. The oncogenes each expressed truncated transcripts compared to the 5.3-kb normal transcript. The dbl and NPDL-dbl oncogene translational products of 66 and 76 kDa, respectively, were consistent with their corresponding major 2.8- and 3.5-kb transcripts. It was not possible to detect evidence of the 5' structural rearrangements associated with these oncogenes in either of the original tumors. Thus, if these rearrangements were critical to their activation, they occurred in the process of gene transfer or in vivo in only a minority of tumor cells.

Animals↗
PubMed1986

Identification of the protein encoded by the human diffuse B-cell lymphoma (dbl) oncogene.

The dbl oncogene was initially isolated from a human diffuse B-cell lymphoma. Antisera from mice bearing tumors induced by this oncogene specifically detected a protein of about 66 kDa (p66) in dbl transformants. dbl cDNA-selected poly(A)+ RNA isolated from a transfectant clone expressing p66 directed the in vitro synthesis of this protein, establishing that it is encoded by dbl. Subcellular localization studies revealed that p66 is a cytoplasmic protein distributed between cytosol and crude membrane fractions. Moreover, p66 was shown to be a phosphoprotein, with phosphorylation specific to serine residues. Our characterization of the dbl-encoded protein appears to distinguish this transforming gene product from those of other known oncogenes.

B-Lymphocytes↗
PubMed1986

High frequency of c-K-ras activation in 3-methylcholanthrene-induced mouse thymomas.

Thymic lymphomas were induced in RJF mice by percutaneous application of 3-methylcholanthrene (MCA). DNAs from 83% of the tumors analyzed contained a transforming c-K-ras gene. The high frequency of c-K-ras oncogene activation in response to MCA seems to favor the concept that the activation of c-K-ras is related to the specificity of the mutagenic effect of MCA.

Animals↗
PubMed1983

Frequent activation of c-kis as a transforming gene in fibrosarcomas induced by methylcholanthrene.

The DNA's from two of four methylcholanthrene-induced mouse fibrosarcomas contained transforming genes that were identical in their pattern of restriction endonuclease resistance to inactivation of biologic activity. This transforming gene was identified as the activated homolog of the Kirsten murine sarcoma virus onc gene, v-kis. The finding that a defined carcinogen reproducibly leads to activation of kis as a transforming gene should be of value in elucidating the role of oncogenes in the neoplastic process.

Animals↗
PubMed1983

Transforming genes of human hematopoietic tumors: frequent detection of ras-related oncogenes whose activation appears to be independent of tumor phenotype.

We surveyed 22 human hematopoietic tumors and tumor cell lines for sequences capable of transforming NIH 3T3 cells by DNA transfection. A primary human acute myelogenous leukemia, a chronic myelogenous leukemia cell line, and cell lines derived from three independent acute lymphocytic leukemias demonstrated oncogenes capable of conferring the transformed phenotype to NIH 3T3 cells through serial cycles of transfection. One of three transforming genes associated with acute lymphocytic leukemia cells (classified as thymocyte developmental stage II) was identified as the activated cellular homologue of the Kirsten murine sarcoma virus onc gene, kis, a member of the ras family of onc genes. A transforming gene, which was demonstrated to be common to several human myeloid and lymphoid tumor cells, was shown to be a distantly related member of the ras gene family. Thus, the NIH 3T3 transfection assay commonly detects related oncogenes in human hematopoietic tumor cells. Moreover, the activation of these oncogenes appears to be independent of the specific stage of cell differentiation or tumor phenotype.

Alleles↗