Egg yolk phospholipids enriched with 1-O-octadecyl-2-oleoyl-sn-glycero-3-phospho-(N-palmitoyl) ethanolamine inhibit development of experimentally induced tumours.

Dietary phospholipids (PLs) and their derivatives have proved active in suppression of various health problems and conditions including cancer. In this work we compared the effect of dietary phospholipids from hen egg yolk enriched with N-acyl ether-phosphatidyl ethanolamine (NAEPE) termed bioactive phospholipids (BAP+ preparation) with PLs lacking NAEPE (BAP- preparation) on the growth of transformed cells in vitro and on the promotion and progression of experimental tumours in vivo. For the in vivo experiments we used the chicken model in which liver, lung, and kidney tumours arose via natural selection from single cells initiated by experimentally introduced somatic mutations caused by insertional mutagenesis. Mutagenized animals were fed BAP+ or BAP- diet in various regimens. We observed that BAP+ at low concentrations killed cells of various tumour cell lines in culture but did not compromise viability of non-transformed cells. Oral administration of the BAP+ preparation efficiently reduced progression of all tumour types. However, it did not significantly reduce the number of already initiated tumours and their growth when BAP+ was discontinued. Our data suggest that NAEPE combined with hen egg PLs significantly interferes with tumour progression, possibly through the inhibition of tumour cell viability.

DNA released by leukemic cells contributes to the disruption of the bone marrow microenvironment.

Reciprocal interactions between a tumor and its microenvironment control expansion of tumor cells. Here we show a specific type of interaction in which blasts of experimental leukemia destroy the bone marrow (BM) structures and kill stromal cells. The in vitro experiments showed that the cytotoxic agent released by leukemic cells is the fragmented DNA derived from their genome and occurring in nucleosome-like complexes. This DNA entered nuclei of BM or other cells and induced H2A.X phosphorylation at serine 139, similar to double-strand break-inducing agents. There was a correlation between large amounts of acquired DNA and death of recipient cells. Moreover, the DNA integrated into chromosomal DNA of recipient cells. Primary human acute myeloid leukemia cells also released fragmented DNA that penetrated the nuclei of other cells both in vitro and in vivo. We suggest that DNA fragments released from leukemic and also perhaps other types of tumor cells can activate DNA repair mechanisms or death in recipient cells of a tumor microenvironment, depending on the amount of the acquired DNA. This can impair DNA stability and viability of tumor stromal cells, undermine homeostatic capacity of tumor microenvironment and facilitate tumor progression.

Melanocyte fate in neural crest is triggered by Myb proteins through activation of c-kit.

The c-myb proto-oncogene and its oncogenic derivative v-mybAMV encode transcriptional regulators engaged in the commitment of hematopoietic cells. While the c-Myb protein is important for the formation and differentiation of various progenitors, the v-MybAMV oncoprotein induces in chicks a progression and transformation of the single (monoblastic) cell lineage. Here we present the first evidence of cell fate-directing abilities of c-Myb and v-MybAMV proteins in avian neural crest (NC), where both proteins determine melanocytogenesis. The increased concentration of c-Myb induces progression into dendritic melanocytes and differentiation. The v-myb oncogene converts essentially all NC cells into melanocytes and causes their transformation. Both Myb proteins activate in NC cells expression of the c-kit gene and stem cell factor c-Kit signaling--one of the essential pathways in melanocyte development. These observations suggest that the c-myb-c-kit pathway represents a common regulatory scheme for both hematopoietic and neural progenitors and establishes a novel experimental model for studies of melanocytogenesis and melanocyte transformation.

Transcription factor c-Myb is involved in the regulation of the epithelial-mesenchymal transition in the avian neural crest.

Multipotential neural crest cells (NCCs) originate by an epithelial-mesenchymal transition (EMT) during vertebrate embryogenesis. We show for the first time that the key hematopoietic factor c-Myb is synthesized in early chick embryos including the neural tissue and participates in the regulation of the trunk NCCs. A reduction of endogenous c-Myb protein both in tissue explants in vitro and in embryos in ovo, prevented the formation of migratory NCCs. A moderate over-expression of c-myb in naive intermediate neural plates triggered the EMT and NCC migration probably through cooperation with BMP4 signaling because (i) BMP4 activated c-myb expression, (ii) elevated c-Myb caused accumulation of transcripts of the BMP4 target genes msx1 and slug, and (iii) the reduction of c-Myb prevented the BMP4-induced formation of NCCs. The data show that in chicken embryos, the c-myb gene is expressed prior to the onset of hematopoiesis and participates in the formation and migration of the trunk neural crest.

The leucine zipper region of Myb oncoprotein regulates the commitment of hematopoietic progenitors.

The development of blood cells proceeds from pluripotent stem cells through multipotent progenitors into mature elements belonging to at least 8 different lineages. The lineage choice process during which stem cells and progenitors commit to a particular lineage is regulated by a coordinated action of extracellular signals and transcription factors. Molecular mechanisms controlling commitment are largely unknown. Here, the transcription factor v-Myb and its leucine zipper region (LZR) are identified as regulators of the commitment of a common myeloid progenitor and progenitors restricted to the myeloid lineage. It is demonstrated that wild-type v-Myb with the intact LZR directs development of progenitors into the macrophage lineage. Mutations in this region compromise commitment toward myeloid cells and cause v-Myb to also support the development of erythroid cells, thrombocytes, and granulocytes, similar to the c-Myb protein. In agreement with that, the wild-type v-Myb induces high expression of myeloid factors C/EBP beta, PU.1, and Egr-1 in its target cells, whereas SCL, GATA-1, and c-Myb are more abundant in cells expressing the v-Myb LZR mutant. It is proposed that Myb LZR can function as a molecular switch, affecting expression of lineage-specifying transcription factors and directing the development of hematopoietic progenitors into either myeloid or erythroid lineages.

An ex vivo model to study v-Myb-induced leukemogenicity.

The v-myb(AMV) oncogene transforms myelomonocytic cells in vitro and induces acute monoblastic leukemia in chickens. We analyzed the activity of the evolutionarily conserved PEST-like domain (P1 domain) for biochemical and biological activities of v-Myb in ex vivo cultures and in vivo. Deletion of the P1 domain did not affect v-Myb transcriptional activity, intracellular stability, or subcellular localization. However, it resulted in subtle yet important changes in biological activities. Although the mutant DeltaP1 v-Myb protein blocked the terminal differentiation of the monocyte/macrophage lineage as efficiently as the wild type (wt) in ex vivo cultures, it failed to induce the acute phase of monoblastic leukemia, with its fatal consequences, in vivo. Interestingly, in DeltaP1 v-myb-infected animals large numbers of monoblasts, comparable to those induced by wt v-myb, were present in the bone marrow but very few were found in the peripheral blood. The comparison of ex vivo wt- and DeltaP v-Myb bone marrow cells revealed several important features of v-Myb transformation: (i) the proliferation of transformed monoblasts is not an apparent consequence of the differentiation block with these processes being at least in part independent; (ii) the P1 domain is required for proliferation of v-Myb-mediated transformed monoblasts; (iii) the mechanism which renders transformed cells growth factor independent does not involve activation of an autocrine growth factor loop; and (iv) deletion of the P1 domain affects self-adhesion properties of v-myb-transformed monoblasts as well as their interaction with bone marrow stromal cells. These data indicate that the DeltaP1 v-myb mutant and ex vivo bone marrow cell cultures represent a valuable tool for studies on the mechanisms of leukemia formation.

Ultrastructural analysis of v-myb oncogene product cooperation with components of avian cell nuclear matrix.

The cooperation of the v-Myb oncoprotein with extracted nuclear matrix of avian haematopoietic cells expressing the v-myb oncogene was studied by means of immunoelectron microscopy. The nuclear matrix was extracted by a gentle method of detergent treatment at moderate ionic strength and visualized either in ultrathin LR White sections, in unembedded resin-free sections, and in addition by the aqueous spreading technique. Using anti-Myb polyclonal antibody we have shown interaction of the v-Myb protein product with extracted nuclear matrix. This oncoprotein, however, was easily released from the structure by a detergent as well as by DNAase treatment and ammonium sulphate extraction. Prefixation of structures before detergent treatment prevented this extraction. The v-Myb protein marker was distributed in clusters or associated with fibrillar structures in most cases. Single markers decorating these fibrillar or less dense structures were also detected.

Interaction of V-Myb oncoprotein with spread chromatin of avian haematopoietic cells.

Interactions of v-Myb oncoprotein with spread chromatin of avian LSCC-BM2 cells expressing v-myb oncogene were studied by means of immunoelectron microscopy. The application of this technique using anti-Myb polyclonal antibody combined with the Miller type spreading for visualisation of chromatin revealed the presence of Myb protein on stretched chromatin fibres. Intense labelling was apparent on the chromatin dispersed by hypotonic treatment, where the label was present frequently in clusters, although individual marks along the fibrillar molecules were also found. The combination of hypotonic and detergent treatment resulted in better dispersal of chromatin, more frequent detection of active transcription units, but also in removal of some proteins from chromatin fibres. The labelling of chromatin with anti-Myb antibody was substantially reduced in this case and was dependent on detergent concentration used. The marker was found less frequently on chromatin fibres usually present in clusters on remaining protein structures. Our findings confirmed direct interaction of v-Myb protein with chromatin structure. This interaction is apparently affected by detergent treatment.

The Myb leucine zipper is essential for leukemogenicity of the v-Myb protein.

The AMV v-Myb oncoprotein causes oncogenic transformation of myelomonocytic cells in vivo and in vitro. Its transforming capacity is strictly dependent upon the N-terminal DNA binding domain, the central transactivation region, and on the C-terminal domain containing a putative leucine zipper motif. Here we show that the v-MybL3,4A mutant, in which Leu325 and Leu332 of the leucine zipper have been replaced by alanines, failed to induce leukemia in virus infected chicken. This demonstrates that the leucine zipper domain is indispensable for v-myb induced leukemogenesis in vivo. v-MybL3,4A was, however, still able to transform myelomonocytic cells from chicken bone marrow in vitro. Yet, while v-mybL3,4A transformed cells were impaired in growth at 37 degrees C, they failed to grow at 42 degrees C, the physiological body temperature of avian species. This might explain the loss of v-MybL3,4A leukemogenic potential in vivo. We also demonstrate that the v-Myb leucine zipper domain interacts in vitro with two host cell proteins, p26 and p28. This interaction is compromised in v-MybL3,4A indicating that binding of v-Myb to p26 and p28 might be important for the leukemogenic potential of v-Myb.

V-myb oncogene and c-myb proto-oncogene expression in avian cells: morphological changes of the cells and topographic localization of myb proteins.

Morphological changes of avian cells expressing the v-myb oncogene or c-myb proto-oncogene were studied by means of electron microscopy. Expression of both genes lead to distinct morphological changes of these cells. The nucleus of LSCC-BM2 cells espressing v-myb gene was of normal size but usually of irregular shape. It contained large unravelled nucleoli with typical interstices in some cells. Small nucleolar structures were also localized in the periphery of nuclear membrane. Nuclear envelope revealed reduced perinuclear space between two membranes. LSCC-BK3 cells expressing the c-myb gene were characterized by distinctly enlarged nucleus, in most cases of irregular shape. It contained only one nucleolus markedly enlarged, often unravelled, with apparent interstitial area. Nucleoli with nucleolonemas were observed in some cells. Nuclear envelope formed by two obscure membranes showed reduced perinuclear space. Topographic localization of v-Myb and c-Myb protein products was not basically different, both being detected in the nucleus of avian cells. v-Myb and c-Myb markers were distributed mostly in clusters, usually associated with interchromatin granules, but some marker was associated also with the nuclear membrane. Both Myb products were never detected in nucleolar structures of avian cells. Morphological changes of avian cells expressing myb genes and topographic localization of Myb proteins in these cells were different from those found in the insect cells expressing myb genes. The observed differences are discussed.

Okazaki fragments, a constant component of avian myeloblastosis virus core-bound 7 S DNA.

We have shown that the unusual CsCl-buoyant density and velocity sedimentation properties of the isolated host 7 S DNA species associated with the core fraction of avian myeloblastosis virus (AMV) are made mainly by tight association of RNA pieces prevalently joined to the single-stranded portion of this material. It was shown indirectly on sedimentation patterns of [methyl-3H]thymidine and [14C]uridine double-labelled and glyoxylated total AMV DNA, and directly in phosphorylation experiments with T4 polynucleotide kinase performed on the single-stranded portion of AMV DNA that the RNA-DNA link in AMV DNA is of a covalent nature and that the 5'-terminal end of DNA at the RNA-DNA junction is occupied by all four common deoxyribonucleotides. This first evidence of the presence of Okazaki fragments in 7 S AMV DNA clearly indicates that this DNA does not represent a randomly fragmented host DNA included by chance into virions but special fragments of host DNA having the properties of DNA replicative structures with possible consequences for some viral function(s) including those involved in virus-cell interactions.

Avian nephroblastomas induced by a retrovirus (MAV-2) lacking oncogene. III. Presence of defective MAV-2 proviruses in tumour DNA.

Using Southern hybridization with a series of probes derived from the MAV provirus we searched for structurally changed MAV-2 proviruses in the DNA from MAV-2-induced nephroblastomas. Anomalous fragments of MAV-2 provirus were found in all samples analysed in more detail. Comparison of the sizes of anomalous fragments detected by different probes in the same sample indicates that the fragments, at least in a majority of cases, do not represent recombinant proviruses (which might contain transduced oncogene) but deleted proviruses only. Various types of proviral deletions occur in different nephroblastoma clones, and no preferred type of deletion was found. The defects in MAV-2 sequences appear in infected chickens de novo, because defective proviruses also occur in tumours of chickens that have been infected with the plaque-purified MAV-2 preparations and, in addition, most of the proviral defects found prevent the virus from replicating and from being transmitted by infection. The regular occurrence of defective proviruses in tumour DNA supports the concept that the proviral defects are involved in oncogenesis. Hypotheses on the substance of this involvement are discussed.

Avian nephroblastomas induced by a retrovirus (MAV-2) lacking oncogene. II. Search for common sites of proviral integration in tumour DNA.

To demonstrate the existence of a common site of integration in independent tumour clones, restriction mapping of the vicinity of integrated MAV-2 proviruses in nephroblastoma DNA was performed, using Southern hybridization with an MAV-2 specific probe U3(pAT). The results have shown that (1) nephroblastomas are of semiclonal origin. (2) Nephroblastoma cells contain an average of 5 clonally located integrated proviruses per diploid genome; they do not contain any detectable amount of non-integrated proviruses. (3) In the DNA from independent nephroblastoma clones, there appear at an increased frequency Tth111I fragments of 14.6 and 17.8 kb that hybridize with the U3(pAT) probe. Considering a random selection of integration sites, such coincidence is of little probability. Thus we suppose that these fragments represent a common site(s) of integration with an MAV-2 proviral insert. Two hypotheses concerning possible mechanisms of nephroblastoma induction are discussed: proto-oncogene insertional activation and anti-oncogene insertional inactivation.