Petr Bartunek

The impact of c-met/scatter factor receptor on dendritic cell migration.

Dendritic cells (DC) are professional antigen‐presenting cells that possess both migratory properties and potent T cell stimulatory activity, and that allow the uptake of antigenic material inperipheral tissues and its subsequent presentation in the T cell areas of lymphoid organs. Thus motility represents a central property that is required for DC function. Here we report on the expression of the receptor tyrosine kinase c‐met in DC. c‐Met is the high affinity receptor for scatter factor (SF)/hepatocyte growth factor, and ligand‐activated c‐met exhibits mitogenic, morphogenic andmotogenic activity in vivo and in vitro. c‐Met is signaling competent in DC since it is effectively tyrosine phosphorylated in response to SF ligand. It is demonstrated here that ligand‐activated c‐met regulates DC adhesion to the extracellular matrix component laminin but leaves antigen presenting function unaffected. Importantly, in ear sheet explant experiments activationof c‐met by ligand induces emigration of cutaneous DC (Langerhans cell, LC) from skin, but SF is not a chemoattractant factor for DC. Our results suggest an important role of the c‐met/SF system in DC/LC migration.

[4] Avian hematopoietic cell culture: In vitro model systems to study oncogenic transformation of hematopoietic cells

Publisher Summary This chapter describes the avian hematopoietic cell culture. It reviews the various techniques that are available to transform hematopoietic progenitors from several lineages using specific oncogenes or their combinations. The chapter also presents in vitro model systems to study oncogenic transformation of hematopoietic cells. Hematopoietic cells are produced throughout the lifetime of an individual from a small set of stem cells. The progeny of these stem cells make decisions involving self-renewal and differentiation to give rise to the cells of the erythroid, myeloid, and lymphoid lineages. These cells either proliferate to expand a certain compartment, or differentiate along a lineage-specific pathway. Through a delicate balance between proliferation and differentiation, the numbers of the cells within various lineages are controlled and homeostasis is maintained. Avian hematopoietic cells provide a unique model system to study the normal and abnormal hematopoiesis, because of their long in vitro life span (30-50 generations), normal and oncogene-transformed avian hematopoietic cells can be grown into mass cultures. The chapter also describes the techniques developed for the growth and in vitro differentiation of normal chicken hematopoietic cells.

Molecular cloning, expression and regulation of the avian tubby-like protein 1 (tulp1) gene

The tubby-like protein 1 (tulp1) gene is a member of the tubby multigene family which includes tub, tulp1, tulp2 and tulp3. Human and mouse tulp1 genes were cloned and mutations in tulp1 have been implicated in retinitis pigmentosa in man. Here we report on the cDNA cloning of the chicken tulp1 homologue and its protein product deduced from the nucleotide sequence. The chicken Tulp1 protein comprises 358 amino acids with a calculated molecular mass of 40 kDa. The overall structure of Tub and Tulp proteins, exemplified by the highly conserved C-terminal domain of 255 amino acids and the signature motif KLACE, is also preserved in chicken Tulp1. Phylogenetic analysis demonstrates that chicken tulp1 cDNA and protein are closely related to human and mouse tulp1. In addition, chicken tulp1 mRNA is abundantly expressed in retina similar to tulp1 expression in human and mouse. Two tulp1-specific transcripts of 2 and 3 kb in size were identified that showed differential regulation during embryonic and postnatal development. Finally, tulp1 mRNA was found to be expressed in chicken erythroid cells and upregulated by ligand-activated thyroid hormone receptor (TR alpha/c-erbA).

GAR22: A novel target gene of thyroid hormone receptor causes growth inhibition in human erythroid cells

OBJECTIVE Thyroid hormone receptors (TRs) are ligand-dependent transcription factors with a major impact on erythroid cell development. Here we investigated TR activity on red cell gene expression and identified TR target genes. The impact of the TR target gene GAR22 (growth arrest-specific 2 [GAS2]-related gene on chromosome 22) on red cell differentiation was determined. MATERIALS AND METHODS Stem cell factor/erythropoietin (SCF/EPO)-dependent red cell progenitors were differentiated in vitro in the presence or absence of thyroid hormone. Hormone-induced changes in gene expression were measured by a genome-wide approach with DNA microarrays. Ectopic expression of the TR target gene GAR22 was used to determine its impact on red cell differentiation. RESULTS Ligand-activated TR effectively accelerated red cell progenitor differentiation in vitro concomitantly with inducing growth arrest. We demonstrate that activated TR-induced specific gene expression patterns of up- or downregulated genes, including distinct clusters associated with accelerated differentiation in response to treatment. Mining for T3-induced genes identified basic transcription element binding protein 1/Krüppel-like factor 9 (BTEB1/KLF9) and GAR22 as TR target genes. BTEB1/KLF9 is a known TR target gene while GAR22, initially identified as a putative tumor suppressor, represents a novel TR target gene. We demonstrate that ectopic GAR22 expression in red cell progenitors lengthens the cell cycle and causes growth inhibition, but leaves red cell gene expression unaffected. CONCLUSION This study identifies GAR22 as a novel and direct TR target gene. Our results suggest that hormone-induced GAR22 might represent an important trigger of growth inhibition induced by thyroid hormone in red cell progenitors.

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.

MOLECULAR CLONING, EXPRESSION AND EVOLUTIONARY ANALYSIS OF THE AVIAN TYROSINE KINASE JAK1

The Janus protein tyrosine kinases (JAK) constitute a protein family that plays a pivotal role in signalling of a large number of cytokine receptors. The cDNA of the chicken homologue of JAK1 was cloned and its nucleotide sequence determined. Chicken JAK1 protein comprises 1150 amino acids as deduced from its cDNA sequence with a calculated molecular mass of 133kDa. The overall structure of JAK proteins exemplified by the JAK homology domains JH1-JH7 is also preserved in chicken JAK1. Additionally, phylogenetic analysis demonstrates that chicken JAK1 is more closely related to mammalian JAK1 than to those of fish, exhibiting 80%, 79% and 63% identity in amino acid sequence to human, mouse and zebrafish JAK1, respectively. JAK1 proteins were found to be most conserved in the kinase (JH1) and pseudokinase (JH2) domains. This data is supported by Southern hybridization studies of ZOO blots. Chicken JAK1 shows a ubiquitous expression pattern and is transcribed as a 5.5kb mRNA in various tissues and cell types. JAK1 expression was particularly high in lymphoid cells.

Glycol porphyrin derivatives and temoporfin elicit resistance to photodynamic therapy by different mechanisms

The development of drug resistance is a major problem which often occurs during anticancer chemotherapies. Photodynamic therapy (PDT) has been studied as an alternative treatment modality for drug-resistant tumors, however the question of resistance to PDT and potential cross-resistance with chemotherapy has yet to be fully answered. To investigate the mechanism of resistance to PDT, we developed an in vitro experimental model system in a mouse mammary carcinoma cell line 4T1. We used two ethylene glycol derivatives of tetraphenylporphyrin, and tetraphenylchlorin derivative, temoporfin, as photosensitizers (PS). PDT-resistant clones were obtained by exposure to a set concentration of PS followed by irradiation with increasing light doses. PDT resistance to soluble glycol porphyrins was mediated mainly by increased drug efflux through ABCB1 (P-glycoprotein) as we demonstrated by specific ABCB1 knockdown experiments, which in turn rescued the sensitivity of resistant cells to PDT. In contrast, resistance raised to temoporfin, which is generally more lipophilic than glycol porphyrins, elicited mechanism based on sequestration of the drug to lysosomes. The resistance that is acquired from a particular PS could be overcome by using a different PS, which is not susceptible to the same mechanism(s) of resistance. Elucidation of the underlying mechanisms in various types of resistance might facilitate improvements in PDT treatment design.

DISP3, a Sterol-Sensing Domain-Containing Protein that Links Thyroid Hormone Action and Cholesterol Metabolism

In the body, the brain is the most cholesterol-rich organ. Despite this, remarkably little is known about the mechanisms in the brain that regulate cholesterol homeostasis. Due to the blood-brain barrier, plasma lipoproteins are unable to traverse, and instead cholesterol must be synthesized de novo from within the central nervous system. Thyroid hormone receptors, activated in response to thyroid hormone (T(3)), are known to modulate the level of serum cholesterol via complex regulatory pathways. By screening for T(3)-regulated genes we have identified Disp3, a sterol-sensing domain-containing protein that is related to the Dispatched family of proteins. Analysis by RT-PCR and immunohistochemistry demonstrated that DISP3 is predominately expressed in specific cell types of the brain, retina, and testis. Using the model of hyperthyroidism in vivo, we observed the modulation of Disp3 expression in the retina. Furthermore, in vitro analysis of Disp3 expression in cells treated with T(3) revealed both positive and negative regulation. DISP3 localizes within the endoplasmic reticulum and was further found to colocalize with cholesterol. Ectopic expression of DISP3 in fibroblasts resulted in elevated cholesterol levels combined with an altered cholesterol distribution. Given that DISP3 is highly expressed in Purkinje cells, hippocampal neurons, and retinal ganglion cells and that its overexpression results in increased cholesterol levels, it is tempting to postulate that DISP3 may contribute to cholesterol homeostasis in neural cell types. Taken together, we propose that DISP3 represents a new molecular link between thyroid hormone and cholesterol metabolism.

The Expression of c-Myb Correlates with the Levels of Rhabdomyosarcoma-specific Marker Myogenin

The transcription factor c-Myb is required for modulation of progenitor cells in several tissues, including skeletal muscle and its upregulation is observed in many human malignancies. Rhabdomyosarcomas (RMS) are a heterogeneous group of mesodermal tumors with features of developing skeletal muscle. Several miRNAs are downregulated in RMS, including miR-150, a negative regulator of c-Myb expression. Using the C2C12 myoblast cell line, a cellular model of skeletal muscle differentiation, we showed that miR-150 controls c-Myb expression mainly at the level of translation. We hypothesized that a similar mechanism of c-Myb regulation operates in RMS tumors. We examined expression of c-Myb by immunohistochemistry and revealed c-Myb positivity in alveolar and embryonal tumors, the two most common subgroups of RMS. Furthermore, we showed direct correlation between c-Myb production and myogenin expression. Interestingly, high myogenin levels indicate poor prognosis in RMS patients. c-Myb could, therefore, contribute to the tumor phenotype by executing its inhibitory role in skeletal muscle differentiation. We also showed that c-Myb protein is abundant in migratory C2C12 myoblasts and its ectopic expression potentiates cell motility. In summary, our results implicate that metastatic properties of some RMS subtypes might be linked to c-Myb function.