PubMed Health⌕ Search

Biomedical subjects

T Graf

Publications and source records attributed to T Graf.

At least 109 records · Page 6Linked to original sources
PubMed1987

v-myb dominance over v-myc in doubly transformed chick myelomonocytic cells.

Chick myelomonocytic cells transformed by the v-myc oncogene resemble mature macrophages; those transformed by v-myb or v-myb,ets exhibit an immature phenotype. We have analyzed whether these oncogenes are capable of altering the differentiation phenotype of transformed cells by introducing both v-myc plus either v-myb or v-myb,ets into the same cells. Surprisingly, the doubly transformed cells were found to be essentially indistinguishable from cells transformed by v-myb or v-myb,ets alone even when they expressed a high level of v-myc protein. These results demonstrate that v-myb is dominant over v-myc and that, while v-myc induces cell proliferation without affecting differentiation, v-myb induces in the same target cells both proliferation and a block or reversal of differentiation.

Animals↗
PubMed1987

A single point mutation in erbA restores the erythroid transforming potential of a mutant avian erythroblastosis virus (AEV) defective in both erbA and erbB oncogenes.

We have characterized the v-erbA and v-erbB oncogenes of td359, a transformation-defective mutant of avian erythroblastosis virus (AEV) unable to transform erythroblasts, and the revertant r12, obtained after in vivo passage of the mutant. Molecular cloning, sequencing, construction of chimeric viruses and testing of their oncogenic capacities revealed that both oncogenes of td359 are mutated and biologically defective. The r12 virus, although still containing a mutant v-erbB gene, recovered its erythroid transforming potential by acquiring a highly active gag-erbA gene. These results demonstrate that two co-operating oncogenes, an active v-erbA and a defective v-erbB, can transform a cell type not transformed by either oncogene alone. Furthermore, a single amino acid substitution inactivated the td359 v-erbA protein and we show that its reversion led to the reactivation of the protein. This lesion is located in the same region as several previously described inactivating mutations of glucocorticoid receptors, suggesting that the structure/function relationship of the virally transduced form of the c-erbA/thyroid hormone receptor is closely similar to that of steroid hormone receptors.

Alpharetrovirus↗
PubMed1987

A temperature-sensitive mutant of Abelson murine leukemia virus confers inducibility of IgM expression to transformed lymphoid cells.

Lymphoid cell lines were isolated that were inducible for the expression of surface immunoglobulin by shift from 35.5 to 39.5 degrees C after infection of mouse bone marrow cells with a mutagen-treated Abelson murine leukemia virus. Virus produced by one of the cell lines (ts49) transmitted the temperature-sensitive phenotype to new lymphoid transformants as well as to NIH/3T3 cells. In addition, the tyrosine autophosphorylating activity of the p120gag-abl protein synthesized in ts49-transformed cells was found to be temperature-sensitive. Shift experiments using ts49-transformed lymphoid cells showed that at 39.5 degrees C they synthesize increased amounts of mu and kappa chain RNA and protein, and that they can be further induced to secrete IgM when treated with lipopolysaccharide.

Abelson murine leukemia virus↗
PubMed1987

Reversibility of differentiation and proliferative capacity in avian myelomonocytic cells transformed by tsE26 leukemia virus.

Chicken hematopoietic cells infected with E26 leukemia virus can be transformed into growth factor-dependent, rapidly proliferating cells that exhibit properties of immature myelomonocytic cells. Cells infected with a mutant of E26 that carries a temperature-sensitive lesion, presumably residing in the myb oncogene, differentiate into resting, macrophage-like cells when shifted from 37 degrees to 42 degrees C (Beug et al. 1984). Here we show that differentiated tsE26 cells gradually reacquire an immature phenotype and proliferative capacity when shifted back to 37 degrees C, provided that they are kept at 42 degrees C no longer than 4-8 days. We also show that DNA synthesis inhibitors do not prevent terminal differentiation at 42 degrees C but inhibit the complete reexpression of the immature phenotype in downshift experiments. Our results suggest that the reactivation of the E26 protein function can both induce a "retro-differentiation" and cell proliferation in myelomonocytic target cells.

Animals↗
PubMed1987

Defective v-erbB genes can be complemented by v-erbA in erythroblast and fibroblast transformation.

We have introduced 3'-terminal deletions of increasing size in the v-erbB oncogene and analysed the effects of these mutations on the transformation of fibroblasts and erythroblasts. The results show that the transforming activity of the mutants is gradually diminished, and completely abolished in those mutants that do not produce stable v-erbB proteins. The capacity to transform erythroblasts is lost before fibroblast transformation is severely affected, suggesting that a larger part of the C-terminal domain is required for mitogenic signalling in erythroid cells than in fibroblasts. In addition, the v-erbA oncogene was found to cooperate with v-erbB not only in erythroblast but also in fibroblast transformation, inducing a fully transformed phenotype in fibroblasts partially transformed by a mutant erbB oncogene.

Alpharetrovirus↗
PubMed1987

Common site of mutation in the erbB gene of avian erythroblastosis virus mutants that are temperature sensitive for transformation.

The genome of the avian erythroblastosis virus temperature sensitive mutant ts34 was cloned from a cell line that was shown to contain a single integrated copy of the virus. The mutation was localized to the v-erbB gene by making chimeric viruses between the mutant genome and that of wildtype. Sequencing of the mutant v-erbB gene revealed a single amino acid change of a histidine to an aspartate residue at a position equivalent to amino-acid 826 of the human epidermal growth factor receptor. Interestingly this is the identical mutation to that recently reported for another temperature sensitive mutant ts167, indicating that this may be a hot spot for mutations in the v-erbB gene that give rise to ts transformation mutants. In addition, the different biological phenotypes of ts34 and ts167 are suggested to be due to an additional mutation in v-erbA in ts167.

Alpharetrovirus↗
PubMed1987

The transforming activity of the chicken c-myc gene can be potentiated by mutations.

It was previously demonstrated that four different avian v-myc oncogenes harbor several point mutations. At least one of these leads to an amino acid substitution located in the proximity of position 61 in the second exon, whereas additional substitutions are found in exon 3. We have investigated whether these mutations affect the transforming activity of myc. By constructing avian retroviral genomes expressing hybrid gag-myc oncogenes, in which all or parts of the v-myc domains were replaced by corresponding parts of c-myc, we show here that a substitution of threonine 61 of c-myc for a methionine (as in v-mycmc29) significantly enhances the fibroblast transforming capacity of the recombinant oncogene. However, such a hybrid v/c-myc gene is still several fold less active than the v-mycmc29 oncogene. We have also expressed c-myc from subgenomic retroviral mRNAs: in these constructions the AUG of gag in the RNA leader sequence is in the same reading frame as that of c-myc, apparently leading to the production of a myc protein with 11 N-terminal amino acids encoded by gag and non-coding c-myc sequences. These myc proteins also transform chicken embryo fibroblasts, albeit with a lower efficiency than v-myc, again suggesting that mutations can increase the transforming capacity of myc.

Animals↗
PubMed1986

v-erbA cooperates with sarcoma oncogenes in leukemic cell transformation.

The v-erbB, v-src, v-fps, v-sea, and v-Ha-ras oncogenes induce avian erythroid progenitor cells to self-renew in an erythropoietin-independent manner. These transformed erythroblasts retain both their capacity to differentiate into erythrocytes and their requirement for complex growth media. However, previous studies showed that erythroblasts transformed by v-erbB plus v-erbA (which by itself is not oncogenic) are blocked in differentiation and grow in standard media. Here we show that the introduction of v-erbA into erythroblasts transformed with v-src, v-fps, v-sea, or v-Ha-ras likewise induces a fully transformed phenotype. It also reduces the capacity of ts sea- and ts erbB-transformed erythroblasts to differentiate terminally in an erythropoietin-dependent manner after a temperature shift. Cooperativity involving v-erbA also occurs in vivo since chicks infected with a retroviral construct encoding v-erbA and v-src develop both acute erythroblastosis and sarcomas.

Alpharetrovirus↗
PubMed1986

v-mil induces autocrine growth and enhanced tumorigenicity in v-myc-transformed avian macrophages.

MH2, an avian retrovirus containing the v-myc and v-mil oncogenes, rapidly transforms chick hematopoietic cells in vitro. The transformed cells belong to the macrophage lineage and proliferate in the absence of exogenous growth factors. Here we analyze a series of MH2 deletion mutants and show that these two oncogenes together establish an autocrine growth system in which v-myc stimulates cell proliferation, while v-mil induces the production of chicken myelomonocytic growth factor (cMGF). We also demonstrate that these two oncogenes cooperate in vivo. MH2 efficiently induces monocytic leukemias and liver tumors, while deletion mutants lacking either a functional v-mil or v-myc do not.

Animals↗
PubMed1986

Temperature-sensitive mutants of MH2 avian leukemia virus that map in the v-mil and the v-myc oncogene respectively.

MH2 is an avian retrovirus that contains the v-mil and v-myc oncogenes. In vitro it transforms chick macrophages that are capable of proliferation in the absence of growth factor. Earlier work showed that v-myc induces macrophage transformation and that v-mil induces the production of chicken myelomonocytic growth factor (cMGF), thus generating an autocrine system. We describe the isolation of temperature-sensitive (ts) mutants of MH2 virus. As suggested by marker rescue experiments, one mutant bears a ts lesion in v-mil, whereas the other carries a mutation in v-myc. Ts v-mil MH2-transformed macrophages become factor-dependent at the non-permissive temperature (42 degrees C), while ts-v-myc MH2-transformed macrophages cease growing and acquire a more normal macrophage phenotype at 42 degrees C irrespective of the presence of cMGF. Both phenotypes can be reversed by backshift to the permissive temperature. These results suggest that the gene products of v-mil and v-myc function independently of each other and that v-mil is necessary for the maintenance of autocrine growth, whereas v-myc is required to maintain the transformed phenotype.

Animals↗
PubMed1986

A single amino acid substitution in v-erbB confers a thermolabile phenotype to ts167 avian erythroblastosis virus-transformed erythroid cells.

A library of recombinant bacteriophage was prepared from ts167 avian erythroblastosis virus-transformed erythroid precursor cells (HD6), and integrated proviruses from three distinct genomic loci were isolated. A subclone of one of these proviruses (pAEV1) was shown to confer temperature-sensitive release from transformation of erythroid precursor cells in vitro. The predicted amino acid sequence of the v-erbB polypeptide from the mutant had a single amino acid change when compared with the wild-type parental virus. When the wild-type amino acid was introduced into the temperature-sensitive avian erythroblastosis virus provirus in pAEV1, all erythroid clones produced in vitro were phenotypically wild type. The mutation is a change from a histidine to an aspartic acid in the temperature-sensitive v-erbB polypeptide. It is located in the center of the tyrosine-specific protein kinase domain and corresponds to amino acid position 826 of the human epidermal growth factor receptor sequence.

Alpharetrovirus↗
PubMed1986

Two new retroviral onc genes, sea and jun.

Avian leukemia virus S13 induces erythroblastosis, granulocytic leukemia, fibrosarcoma, anemia, and endothelial neoplasia. It transforms chick embryo fibroblasts and primitive erythroid cells in culture and is defective in replication. Its onc gene, sea, is expressed as transformation specific env-sea fusion glycoprotein of 155 kDa. Gp155 is proteolytically processed into gp85env and gp70env-sea. The latter shows tyrosine specific protein kinase activity. Avian sarcoma virus 17 induces fibrosarcoma and transforms chick embryo fibroblasts in culture. Its cell derived onc gene, jun, is not related to known onc genes and appears to be expressed as a gag-jun fusion protein of 55 kDa. The amino acid sequence of jun shows homology in its C-terminal region to the C-terminal DNA binding region of the yeast regulatory protein GCN4, suggesting that the jun protein may bind to DNA.

Amino Acid Sequence↗
PubMed1985

How do retroviral oncogenes induce transformation in avian erythroid cells?

The v-erb B oncogene, as well as other oncogenes of the src-gene family transform immature erythroid cells from chick bone marrow in vivo and in vitro. The erb B-transformed erythroid cells differ from normal late erythroid precursors (CFU-E) in that they have acquired the capacity to undergo self-renewal as well as to differentiate terminally. They also do not require the normal erythroid differentiation hormone, erythropoietin, for either process. Cooperation of v-erb B with a second oncogene, v-erb A, results in a differentiation arrest of the transformed cells, which now only use the self-renewal pathway. Studies with conditional and non-conditional mutants in both v-erb B and v-erb A will be presented to elucidate further how the transforming proteins encoded by these oncogenes, gp74erb B and gp75gag-erb A, affect the differentiation programme of the infected erythroid precursor with the outcome of hormone-independent leukaemic cells arrested at an early stage of erythroid differentiation.

Animals↗
PubMed1985

Protein synthesis in differentiating normal and leukemic erythroid cells.

Erythroleukemic cells transformed by the AEV or S13 strains of avian erythroblastosis virus differentiate in vitro either spontaneously (S13) or following a temperature induction (temperature-sensitive mutants of AEV). To study differentiation in these cells at the molecular level, homogeneous fractions of maturing cells at discrete stages of differentiation were prepared by Percoll density-gradient centrifugation. This method was also used for the fractionation of differentiating normal erythroid cells separated from total bone marrow by an immunological "panning" technique. Total protein synthesis in these cells was then analyzed by two-dimensional gel electrophoresis. The expression of several proteins was altered in differentiating leukemic cells but not in their normal counterparts. However, in general, the normal and leukemic cells from comparable stages of maturity showed closely related protein synthetic patterns. Similar early and late changes in the synthesis of a number of polypeptides were detected during maturation from early erythroid precursors to terminally differentiated erythrocytes. Further, the leukemic as well as the normal cells appeared to undergo a major switch in total protein synthetic pattern during late differentiation. These results demonstrate that normal and erythroleukemic cells differentiate along similar pathways.

Alpharetrovirus↗