PubMed Health⌕ Search

Biomedical subjects

T Graf

Publications and source records attributed to T Graf.

At least 91 records · Page 5Linked to original sources
PubMed1991

v-myb and v-ets transform chicken erythroid cells and cooperate both in trans and in cis to induce distinct differentiation phenotypes.

E26 is an acute avian leukemia virus that encodes the transcriptional activator oncogenes v-myb and v-ets in a single fusion protein. This virus is also unique in that it is able to transform hematopoietic cells of both the myeloid and the erythroid lineage. To determine the contributions of v-myb and v-ets to the transforming potential of the virus, derivatives expressing separate Myb and Ets proteins, either alone or in combination, were constructed. We found that in the myeloid lineage v-myb, but not v-ets, induces cell transformation. In the erythroid lineage both v-myb and v-ets weakly transform erythroblast-like cells. These cells exhibit a mature phenotype and a low self-renewal capacity. The transforming efficiency of the two oncogenes is enhanced if they are coexpressed as separate proteins or as a fusion protein, the transformed cells displaying an increased self-renewal capacity. Interestingly, however, cells transformed by the Myb-Ets fusion protein have a distinct phenotype in that they are very immature. These results demonstrate that v-myb and v-ets can cooperate in the transformation of erythroid cells both in trans and in cis and that the mode of cooperation is reflected by the differentiation phenotypes of the transformed cells.

Animals↗
PubMed1991

Protein truncation is required for the activation of the c-myb proto-oncogene.

The protein product of the v-myb oncogene of avian myeloblastosis virus, v-Myb, differs from its normal cellular counterpart, c-Myb, by (i) expression under the control of a strong viral long terminal repeat, (ii) truncation of both its amino and carboxyl termini, (iii) replacement of these termini by virally encoded residues, and (iv) substitution of 11 amino acid residues. We had previously shown that neither the virally encoded termini nor the amino acid substitutions are required for transformation by v-Myb. We have now constructed avian retroviruses that express full-length or singly truncated forms of c-Myb and have tested them for the transformation of chicken bone marrow cells. We conclude that truncation of either the amino or carboxyl terminus of c-Myb is sufficient for transformation. In contrast, the overexpression of full-length c-Myb does not result in transformation. We have also shown that the amino acid substitutions of v-Myb by themselves are not sufficient for the activation of c-Myb. Rather, the presence of either the normal amino or carboxyl terminus of c-Myb can suppress transformation when fused to v-Myb. Cells transformed by c-Myb proteins truncated at either their amino or carboxyl terminus appear to be granulated promyelocytes that express the Mim-1 protein. Cells transformed by a doubly truncated c-Myb protein are not granulated but do express the Mim-1 protein, in contrast to monoblasts transformed by v-Myb that neither contain granules nor express Mim-1. These results suggest that various alterations of c-Myb itself may determine the lineage of differentiating hematopoietic cells.

Animals↗
PubMed1991

Cavitation potential of mechanical heart valve prostheses.

Just like technical check valves, the function of mechanical heart valve prostheses may presumably also lead to cavitation effects during valve closure. Due to the waterhammer effect, cavitation may primarily occur in the mitral position leading to high mechanical loading of the valve itself and of corpuscular blood elements. Ten different types of commercial mechanical heart valves were investigated in the mitral position of a pulsatile mock loop, to detect cavitation thresholds under physiologically similar conditions by cinematographic techniques. Almost all these valve prostheses show cavitation up to a ventricular pressure gradient of 5000 mmHg/s. The threshold depends on valve type and size and is sometimes within the physiological range below 2000 mmHg/s. Visible cavitation bubbles with a diameter of up to 1.8 mm and a collapse time of less than 0.1 ms suggest that vapour cavitation may play an important role for material and blood damage in mechanical heart valve prostheses.

Coronary Circulation↗
PubMed1990

Mutations in v-myb alter the differentiation of myelomonocytic cells transformed by the oncogene.

Chick myelomonocytic cells transformed by the v-myb oncogene-containing viruses E26 and AMV differ in that the former resemble myeloblasts and express the v-myb-regulated granulocyte-specific mim-1 gene, while the latter resemble monoblasts and are mim-1 negative. We constructed a series of AMV-E26 chimeras and localized the critical differences between these viruses to three point mutations within the second repeat of the v-myb DNA binding domain. These three positions are altered in the v-myb protein of AMV relative to the proteins encoded by c-myb or E26 v-myb. Back mutating AMV v-myb at any of these three sites restored the oncogene's ability to activate the mim-1 gene. Surprisingly, two of these changes led to the transformation, in vitro and in vivo, of cells having a promyelocyte-like phenotype. These results indicate that different forms of v-myb impose alternate phenotypes of differentiation on transformed myeloid cells, probably by regulating unique sets of differentiation-specific genes.

Animals↗
PubMed1990

Transformation by v-myb correlates with trans-activation of gene expression.

The v-myb oncogene of avian myeloblastosis virus causes acute myelomonocytic leukemia in chickens and transforms avian myeloid cells in vitro. Its protein product p48v-myb is a nuclear, sequence-specific, DNA-binding protein which activates gene expression in transient DNA transfection studies. To investigate the relationship between transformation and trans-activation by v-myb, we constructed 15 in-frame linker insertion mutants. The 12 mutants which transformed myeloid cells also trans-activated gene expression, whereas the 3 mutants which did not transform also did not trans-activate. This implies that trans-activation is required for transformation by v-myb. One of the transformation-defective mutants localized to the cell nucleus but failed to bind DNA. The other two transformation-defective mutants localized to the cell nucleus and bound DNA but nevertheless failed to trans-activate. These latter mutants define two distinct domains of p48v-myb which control trans-activation by DNA-bound protein, one within the amino-terminal DNA-binding domain itself and one in a carboxyl-terminal domain which is not required for DNA binding.

Amino Acid Sequence↗
PubMed1989

The v-myb oncogene product binds to and activates the promyelocyte-specific mim-1 gene.

The v-myb oncogene induces myeloid leukemias in chickens, transforms myeloid cells in vitro, and encodes a sequence-specific DNA binding protein. We used differential hybridization to screen for v-myb-regulated genes in cells transformed by a temperature-sensitive mutant of the oncogene and identified a new gene, mim-1, which encodes a specifically expressed, secretable protein contained in the granules of both normal and v-myb-transformed promyelocytes. The promoter of the mim-1 gene contains three closely spaced binding sites for v-myb protein and is strongly activated by v-myb in a cotransfection assay. Synthetic copies of the binding sites are both necessary and sufficient to confer v-myb protein-dependent activation to a heterologous promoter. We conclude that mim-1 is a cellular gene that is directly regulated by the product of the v-myb oncogene.

Amino Acid Sequence↗
PubMed1989

Early reversible induction of leukotriene synthesis in chicken myelomonocytic cells transformed by a temperature-sensitive mutant of avian leukemia virus E26.

We used chicken myelomonocytic cells transformed by a temperature-sensitive mutant of the myb/ets oncogene-containing avian leukemia virus E26 to study the regulation of leukotriene (LT) synthesis during macrophage differentiation. Cells exposed to arachidonic acid and the Ca2+ ionophore 23187 produced up to 180 times more LTs at the nonpermissive temperature (42 degrees C) than at the permissive temperature (37 degrees C). Induction of LT synthesis was detectable within 2 hr after temperature shift, whereas conventional macrophage markers became evident after 2-3 days. N-Formylmethionylleucylphenylalanine, opsonized zymosan, and complement factor C5a induced LT synthesis in temperature-sensitive mutant-transformed cells only when the cells were maintained at 42 degrees C, and this effect was blocked by pertussis toxin. When cells were kept at 42 degrees C for 48 hr and then shifted back to 37 degrees C to induce retrodifferentiation, LT synthesis rates declined within 8 hr and reached near control values within 36 hr. Retrodifferentiation also led to decreased LT synthesis in response to N-formylmethionylleucylphenylalanine, opsonized zymosan, and C5a. These results indicate that activation of the 5-lipoxygenase pathway is a very early event in the macrophage differentiation pathway that is directly or indirectly controlled by the temperature-sensitive v-myb protein.

Animals↗
PubMed1989

A single amino-acid substitution in the DNA-binding domain of the myb oncogene confers a thermolabile phenotype to E26-transformed myeloid cells.

A biologically active provirus of the ts 143 E26 mutant that is temperature-sensitive (ts) for myeloblast transformation was molecularly cloned. The predicted amino-acid sequence of the v-myb-encoded domain of the mutant P135gag-myb-ets protein displayed two single amino-acid changes, one of which was non-conservative when compared to the wild-type E26 v-myb sequence. This mutation, which substitutes a threonine residue (wild-type) for an arginine residue (mutant), is located within the amino-terminal part of v-myb in the DNA-binding domain at a position which is conserved between the c-myb genes of chicken, humans, mice and Drosophila. Introduction of this mutation into the genome of a wild-type E26 virus was sufficient to induce a ts phenotype similar to that obtained with the original ts 143 E26 virus.

Base Sequence↗
PubMed1988

A single point mutation in the v-ets oncogene affects both erythroid and myelomonocytic cell differentiation.

The v-myb, ets-containing avian leukemia virus E26 is unique in its capacity to transform both erythroblasts and myeloblasts. Previous studies showing that v-myb is sufficient for the transformation of myeloid cells failed to definitively establish the role of the v-ets gene. We have now isolated a mutant of E26, ts1.1, that is temperature-sensitive for erythroid cell transformation and that we found to contain a single mutation in the v-ets gene. Surprisingly, myeloid cells transformed by this mutant showed an altered phenotype relative to wild-type-transformed cells, in that they resemble promyelocytes. In addition, infection of mature macrophages with ts1.1 led to their transformation and conversion into promyelocyte-like cells. We conclude that the v-ets domain of the p135gag-myb-ets protein of E26 has an effect on both erythroid and myeloid cell differentiation, suggesting a possible role for the c-ets/c-myb genes in the commitment of hematopoietic cells towards specific lineages.

Animals↗
PubMed1988

Hematopoietic growth factor glycosylation. Multiple forms of chicken myelomonocytic growth factor.

The production of chicken myelomonocytic growth factor (cMGF) can be rapidly induced by bacterial lipopolysaccharide from the macrophage cell line HD11. Immunoprecipitation analysis of lipopolysaccharide-induced HD11 cells labeled with various radioactive precursors showed the secretion of a variety of cMGF forms. The precursor-product relationships of the different cMGF forms were studied by pulse-chase experiments, by long-term metabolic labeling in the presence or absence of glycosylation- and oligosaccharide-processing inhibitors, as well as by glycosidase treatment of immunoprecipitates. Our results show that the half-time for intracellular processing/secretion is less than 10 min, making cMGF one of the most rapidly processed proteins. The different forms of the factor are generated from a 24-kDa polypeptide precursor by co- and post-translational acquisition of one or two N-linked oligosaccharides and by O-linked glycosylation. In addition, a fraction of cMGF is modified by long chain, chondroitinase-sensitive, sulfated glycans. This modification is tunicamycin-sensitive, suggesting that the sulfated glycans are attached to N-linked rather than to O-linked oligosaccharides.

Animals↗
PubMed1988

Replacement of lys 622 in the ATP binding domain of P100gag-mil abolishes the in vitro autophosphorylation of the protein and the biological properties of the v-mil oncogene of MH2 virus.

Lysine 622 in the ATP-binding domain of P100gag-mil, the translation product of the v-mil oncogene of MH2, has been replaced with methionine using oligonucleotide site-directed mutagenesis. This substitution results in the inactivation of the serine/threonine-specific autophosphorylation of P100gag-mil in vitro, indicating that this activity is an intrinsic property of the viral protein. This substitution also suppresses two of the biological properties of MH2 which have previously been shown to be dependant upon the expression of v-mil, namely, the production of chicken myelomonocytic growth factor (cMGF) by v-myc-transformed chicken macrophages and the sustained proliferation of chicken neuroretina cells. These data strongly suggest that the biological properties of v-mil are mediated by the phosphorylation at serine/threonine residues of key cellular substrates. In contrast to the in vitro situation, both the mutant and wild-type proteins appear to be phosphorylated at the same sites and to the same extent in either transformed fibroblasts or macrophages. This, together with the fact that the sites phosphorylated in vivo and in vitro are essentially different indicate that most of the phosphate associated with P100gag-mil in transformed cells does not result from an obligate autophosphorylation event but from the phosphorylation by as yet uncharacterized cellular kinase(s).

Adenosine Triphosphate↗
PubMed1988

Truncation of the human EGF receptor leads to differential transforming potentials in primary avian fibroblasts and erythroblasts.

The transforming capacity of the normal and mutant human EGF receptor (EGFR) was investigated in primary chicken cells. In fibroblasts, both N- and C-terminal truncations resulted in a weak, additive oncogenic activity. However, not even double truncations caused a v-erbB-like phenotype. Upon EGF-binding, on the other hand, both normal and C-terminally truncated EGFRs resembled v-erbB in their fibroblast transforming potential. In erythroblasts, N-terminal truncation was sufficient to induce constitutive self-renewal, which was enhanced by deletion of 32 C-terminal amino acids but abolished by a larger truncation of 202 amino acids. In contrast to the normal EGFR, the receptor lacking 32 C-terminal amino acids resembled v-erbB in conferring erythropoietin independence for spontaneous differentiation to the transformed erythroblasts. Our results indicate that the C-terminal domain of the EGFR is non-essential in fibroblast transformation, but seems to be crucial for both self renewal induction and specificity of receptor function in erythroblasts.

Animals↗
PubMed1988

[Oncogenes and the origin of leukemia. Acute avian leukemia viruses].

Oncogenes have been intimately associated with the genesis of human neoplasms. A particularly useful system to study the mechanism of tumorigenesis is a small group of avian retroviruses that carry two oncogenes. These viruses causes acute leukemias and can transform hematopoietic cells in vitro. The mechanisms by which viral oncogenes affect the growth control and differentiation of their target cells is now understood in fair detail for two of these virus strains. In the avian erythroblastosis virus AEV, the v-erbB oncogene deregulates the growth control of erythroid precursors, while verbA blocks their terminal differentiation into erythrocytes. Based on the findings that v-erbB oncogene corresponds to a mutated growth factor receptor gene and that v-erbA corresponds to a mutated hormone receptor gene, models have been developed that explain the function of these two oncogenes on a molecular basis. The myelomonocytic leukemia virus MH2 acts by a completely different mechanism. In this case, the v-myc oncogene stimulates the proliferation of macrophage-like cells, while the v-mil gene stimulates them to produce their own growth factor, thus leading to autocrine growth. It will be interesting to determine whether the type of mechanisms of oncogene cooperativity elucidated for acute leukemia viruses are also operative during leukemogenesis in humans.

Alpharetrovirus↗
PubMed1988

An invariant asparagine residue belonging to a highly conserved domain in all protein kinases is instrumental in the protein kinase activity of the v-mil gene product.

P100gag-mil, the translation product of the v-mil oncogene of MH2 is a protein kinase specific of serine/threonine residues. We report here that the P100gag-mil encoded by the MH2-Hd isolate displays a considerably reduced kinase activity in vitro. Construction of chimeric viruses and sequencing revealed that the lesion responsible for this reduced activity results from a single point mutation converting an asparagine residue at position 720 in fully active P100gag-mil kinase into serine in the P100gag-mil of MH2-Hd. Since this asparagine residue together with an invariant aspartate residue bracket a highly conserved 6 amino-acid region in all known protein kinases as well as in phosphotransferases of bacterial origin, our results indicate that integrity of this region is essential to enzymatic function and support the notion that it could be directly involved in ATP binding or phosphate transfer from ATP to kinase substrates.

Animals↗
PubMed1988

A point mutation in the DNA binding domain of the v-myb oncogene of E26 virus confers temperature sensitivity for transformation of myelomonocytic cells.

We have molecularly cloned a mutant of the v-myb, ets-containing E26 avian leukemia virus which is temperature sensitive for the transformation of myeloid cells. Cells infected with this mutant, ts21E26, are immature at 37 degrees C and can be induced to differentiate into resting, macrophage-like cells when shifted to 42 degrees C. The sequence of ts21E26 reveals a single relevant nucleotide alteration resulting in a threonine to arginine change in the highly conserved, putative DNA binding v-myb portion of the p135gag-myb-ets protein. Surprisingly, a ts21E26 viral construct in which the v-ets gene domain was deleted was only weakly temperature sensitive, although temperature sensitivity was largely restored in another v-ets deletion mutant whose 3' terminal sequences were replaced with those from the AMV v-myb gene. These results suggest that the temperature sensitive lesion in v-myb of ts21E26 alters the DNA binding capacity of p135 at 42 degrees C and that the primary structure of the C-terminus of this protein has an influence on the activity of sequences that are further upstream.

Animals↗