Michal Dvorak

Myb-interacting Protein, ATBF1, Represses Transcriptional Activity of Myb Oncoprotein

Using the yeast two-hybrid system, the transcription factor ATBF1 was identified as v-Myb- and c-Myb-binding protein. Deletion mutagenesis revealed amino acids 2484–2520 in human ATBF1 and 279–300 in v-Myb as regions required for in vitro binding of both proteins. Further experiments identified leucines Leu325 and Leu332 of the Myb leucine zipper motif as additional amino acid residues important for efficient ATBF1-Myb interaction in vitro. In co-transfection experiments, the full-length ATBF1 was found to form in vivo complexes with v-Myb and inhibit v-Myb transcriptional activity. Both ATBF1 2484–2520 and Myb 279–300 regions were required for the inhibitory effect. Finally, the chicken ATBF1 was identified, showing high degree of amino acid sequence homology with human and murine proteins. Our data reveal Myb proteins as the first ATBF1 partners detected so far and identify amino acids 279–300 in v-Myb as a novel protein-protein interaction interface through which Myb transcriptional activity can be regulated.

c-Jun involvement in vitamin E succinate induced apoptosis of reticuloendotheliosis virus transformed avian lymphoid cells

Previous studies have shown that treatment of avian reticuloendotheliosis virus-transformed RECC-UTC4-1 (C4-1) lymphoblastoid cells with 10 μg/ml (18.8 μM) of RRR-α-tocopheryl succinate (vitamin E succinate, VES) for 3 days induced approximately 50% of the cells to undergo apoptosis. Elevated and prolonged expression of c-jun mRNA and protein was temporally correlated with VES-induced cell death. Data presented in this paper show that the elevated and prolonged expression of c-jun message and protein are not accounted for by enhanced stability, and show the involvement of c-Jun in VES-induced apoptosis in this lymphoblastoid cell type. C4-1 cells infected with a virus carrying a dominant, negatively acting mutant form of c-Jun, supjun-1, exhibited: (i) 71% reduction in VES-induced apoptosis, (ii) a 2.0 – 2.5-fold decrease in wildtype, endogenous c-Jun expression, and (iii) a 2.4 – 2.6-fold reduction in AP-1 binding activity. Additionally, cells co-treated with VES plus RRR-α-tocopherol, exhibited a 70% reduction in apoptosis, a marked reduction in c-Jun expression and a 1.6-fold reduction in AP-1 binding activity. These studies suggest that c-Jun plays a crucial role in VES-induced apoptosis in C4-1 cells, and add to our understanding of mechanisms of action involved in VES-mediated tumor cell growth inhibition.

Glycol porphyrin derivatives as potent photodynamic inducers of apoptosis in tumor cells.

The design and synthesis of glycol-functionalized porphyrins that contain one to four low molecular weight glycol chains that are linked via ether bonds to the meta-phenyl positions of meso-tetraphenylporphyrin and the comparison of fluorinated and nonfluorinated para derivatives are reported. The cellular uptake and photodynamic activity significantly depend on terminal groups of the glycol substituent. Hydroxy glycol porphyrins, in contrast with methoxy glycol porphyrins, show efficient intracellular transport and a high induction of apoptosis in tumor cell lines in vitro . Furthermore, the ethylene glycol chain at the meta position exhibits a superior efficacy that leads to the permanent ablation of human breast carcinoma (MDA-MB-231) in nude mice. In addition, fluorination enhanced the photosensitizing potential of para-phenyl derivatives. The analysis of the cell-death mechanism revealed that glycol-functionalized porphyrins represent novel nonmitochondrially localized photosensitizers that have a profound ability to induce apoptosis in tumor cells that act upstream of caspase activation. The strong interaction with a tumor marker (sialic acid) indicates the preferential association of these compounds with tumor cells.

Novel Porphyrin Conjugates with a Potent Photodynamic Antitumor Effect: Differential Efficacy of Mono- and Bis-β-cyclodextrin Derivatives In Vitro and In Vivo

Abstract In the present study we investigated the photosensitizing properties of two novel mono- and bis-cyclodextrin tetrakis (pentafluorophenyl) porphyrin derivatives in several tumor cell lines and in BALB/c mice bearing subcutaneously transplanted syngeneic mouse mammary carcinoma 4T1. Both studied sensitizers were localized mainly in lysosomes and were found to induce cell death by triggering apoptosis in human leukemic cells HL-60. In 4T1 and other cell lines both apoptotic and necrotic modes of cell death occurred depending on drug and light doses. Mono-cyclodextrin porphyrin derivative P(β-CD)1 exhibited stronger in vitro phototoxic effect than bis-cyclodextrin derivative P(β-CD)2. However, in vivo P(β-CD)2 displayed faster tumor uptake with maximal accumulation 6 h after application, leading to complete and prolonged elimination of subcutaneous tumors within 3 days after irradiation (100 J cm−2). In contrast, P(β-CD)1 uptake was slower (48 h) and the reduction of tumor mass was only transient, reaching the maximum at the 12 h interval when a favorable tumor-to-skin ratio appeared. Thus, P(β-CD)2 represents a new photosensitizing drug displaying fast and selective tumor uptake, strong antitumor activity and fast elimination from the body.

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

Abstract.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.

Identification of Potential Human Oncogenes by Mapping the Common Viral Integration Sites in Avian Nephroblastoma

Gene deregulation is a frequent cause of malignant transformation. Alteration of the gene structure and/or expression leading to cellular transformation and tumor growth can be experimentally achieved by insertion of the retroviral genome into the host DNA. Retrovirus-containing host loci found repeatedly in clonal tumors are called common viral integration sites (cVIS). cVIS are located in genes or chromosomal regions whose alterations participate in cellular transformation. Here, we present the chicken model for the identification of oncogenes and tumor suppressor genes in solid tumors by mapping the cVIS. Using the combination of inverse PCR and long terminal repeat-rapid amplification of cDNA ends technique, we have analyzed 93 myeloblastosis-associated virus type 2-induced clonal nephroblastoma tumors in detail, and mapped >500 independent retroviral integration sites. Eighteen genomic loci were hit repeatedly and thus classified as cVIS, five of these genomic loci have previously been shown to be involved in malignant transformation of different human cell types. The expression levels of selected genes and their human orthologues have been assayed in chicken and selected human renal tumor samples, and their possible correlation with tumor development, has been suggested. We have found that genes associated with cVIS are frequently, but not in all cases, deregulated at the mRNA level as a result of proviral integration. Furthermore, the deregulation of their human orthologues has been observed in the samples of human pediatric renal tumors. Thus, the avian nephroblastoma is a valid source of cancer-associated genes. Moreover, the results bring deeper insight into the molecular background of tumorigenesis in distant species.

p38 MAPK plays an essential role in apoptosis induced by photoactivation of a novel ethylene glycol porphyrin derivative

In this study, we provide evidence that photostimulation of various cancer cells preloaded with a new photosensitizing compound, tetrakis-meso-(4-ethyleneglycol-2,3,5,6-tetrafluorophenyl) porphyrin (PORF-TEG), results in rapid activation of the cell death machinery. PORF-TEG, although primarily localized in lysosomes, induces mitochondria-driven apoptosis. The induction of apoptosis is accompanied by immediate and sustained activation of p38 mitogen-activated protein kinase (MAPK) and transient activation of c-Jun N-terminal kinase (JNK). Conversely, the inhibition of p38 by PD 169316 or SB202190 and by the p38α dominant-negative mutant as well as the deletion of the p38α gene (MEFs-KO) protected cells from apoptosis, whereas inhibition of JNK did not. Activation of the p38 signaling pathway occurs upstream of caspase activation. In addition, preincubation of cells with scavengers of reactive oxygen species attenuated p38 and caspase activation and increased cell survival, thus connecting reactive oxygen species formation with the activation of the p38 pathway. Later events included degradation of Bcl-2, activation of tBid, and cleavage of Bad and Mcl-1. The data suggest a key role for p38 MAPK in PORF-TEG-photoinduced apoptosis.

The twist gene is a common target of retroviral integration and transcriptional deregulation in experimental nephroblastoma

The genes involved in the transformation of kidney blastema cells were searched for in avian nephroblastomas induced by the MAV2 retrovirus. The twist gene was identified as a common site of provirus integration in tumor cells. Twist was rearranged by the MAV2 provirus in three out of 76 independent nephroblastoma samples. The MAV2 integration sites were localized within 40 nucleotides of the twist 5′UTR region, right upstream from the ATG initiation codon. The integrated proviruses were deleted at their 5′ends. As a consequence, twist transcription became controlled by the retroviral 3′LTR promoter and was strongly upregulated, more than 200 times. In addition, 2–100 times elevated twist transcription was also detected in the majority of other nephroblastoma samples not containing MAV2 in the twist locus. We propose that chicken nephroblastoma originates from a single blastemic cell in which the MAV retrovirus, through its integration, has deregulated specific combinations of genes controlling proliferation and differentiation. The activation of the twist gene expression appears to contribute to tumorigenesis, as there is an in vivo positive selection of tumor cell clones containing the twist gene hyperactivated by MAV2 sequences inserted within the twist promoter.

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

Abstract.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.

Impact of chicken thrombopoietin and its receptor c-Mpl on hematopoietic cell development

OBJECTIVE The primary objective of this study was to identify and clone the first nonmammalian thrombopoietin (TPO), chicken TPO, and its receptor c-Mpl for the purpose of characterizing their activities both in vitro and in vivo. MATERIALS AND METHODS Chicken TPO was cloned using the methods of reverse transcriptase polymerase chain reaction and rapid amplification of cDNA ends. Northern blotting and RNAse protection assays were employed to analyze the levels of RNA expression in a panel of tissues and cell lines. To study cell surface expression of c-Mpl, polyclonal antibodies were prepared against bacterially derived c-Mpl. Both baculovirus-derived recombinant TPO and retrovirally expressed TPO and c-Mpl were used for the in vivo experiments. RESULTS Both chicken TPO and its receptor c-Mpl were identified and cloned. Expression of chicken TPO was restricted to only the liver and spleen, while c-mpl was expressed in the bone marrow, lung, and spleen. In vitro experiments with sorted multipotent chicken bone marrow-derived progenitors demonstrated that TPO plays a role in the commitment of these cells to the thrombocytic lineage. Furthermore, TPO in cooperation with stem cell factor also supports proliferation of multipotent progenitors. In experimental animals, the intravenous application of recombinant chicken TPO or overexpression of TPO and c-mpl via retroviral infection lead to erythroblastosis and thromboblastosis. CONCLUSION The characterized chicken thrombopoietin and its receptor c-Mpl will be valuable tools to further study thrombocytic differentiation and hematopoietic stem cell development. Moreover, the introduced experimental model of the chicken bipotent thrombo-/erythropoietic-progenitor can be used to identify key regulators of cell fate determination.