Jarmila Králová

Porphyrin−Cyclodextrin Conjugates as a Nanosystem for Versatile Drug Delivery and Multimodal Cancer Therapy

The porphyrin-cyclodextrin conjugates were prepared and tested for selective and effective multifunctional drug delivery and therapy. The porphyrin receptor system combines efficient binding of the selected drug to the cyclodextrin cavity and photosensitizing properties of the porphyrin moiety with high accumulation of the whole complex in cancer tissue. The combined effect of chemotherapy and photodynamic therapy is demonstrated by in vitro and in vivo studies.

Molecular cloning and expression of the human and mouse homologues of the Drosophila dachshund gene

Abstract Recent genetic analysis of the Drosophila dachshund (dac) gene has established that dac encodes a novel nuclear protein that is involved in both eye and leg development. In the Drosophila eye, dac expression appears to be controlled by the product of the eyeless/Pax6 gene. In order to analyze the Pax6 pathway in vertebrates we have isolated and characterized the cDNA and genomic clones corresponding to the human and mouse homologues of Drosophiladac. A full-length human cDNA encoding dachshund (DACH) encodes the 706 amino acids protein with predicted molecular weight of 73 kDa. A 109 amino acid domain located at the N-terminus of the DACH showed significant sequence and secondary structure homologies to the ski/sno oncogene products. Northern blot analysis found human DACH predominantly in adult kidney, heart, and placenta, with less expression detected in the brain, lung, skeletal muscle and pancreas. A panel of human cell lines was studied and most notably a large proportion of neuroblastomas expressed DACH mRNA. Mouse Dach encodes a protein of 751 amino acids with predicted molecular weight of 78 kDa that is 95% identical to the human DACH. RNase protection analysis showed the highest Dach mRNA expression in the adult mouse kidney and lung, whereas lower expression was detected in the brain and testis. RT/PCR analysis readily detected Dach mRNA in the adult mouse cornea and retina. Dach mRNA expression in the mouse E11.5 embryo was observed primarily in the fore and hind limbs, as well as in the somites.

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.

GATA-1 and c-myb crosstalk during red blood cell differentiation through GATA-1 binding sites in the c-myb promoter

GATA-1 and c-Myb transcription factors represent key regulators of red blood cell development. GATA-1 is upregulated and c-myb proto-oncogene expression is downregulated when red cell progenitors differentiate into erythrocytes. Here we have employed a culture system, that faithfully recapitulates red blood cell differentiation in vitro, to follow the kinetics of GATA-1 and c-myb expression. We show that c-myb proto-oncogene expression is high in progenitors and effectively downregulated at the time when nuclear GATA-1 accumulates and cells differentiate into erythrocytes. Additionally, we identified two GATA-1 binding sites within the c-myb promoter and demonstrate that GATA-1 protein binds to these sites in vitro. Furthermore, GATA-1 represses c-myb expression through one of the GATA-1 binding sites in transient transfection experiments and this requires FOG-1. Thus, our study provides evidence for a direct molecular link between GATA-1 activity and c-myb proto-oncogene expression during terminal red cell differentiation.

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.

Optical sensing of sulfate by polymethinium salt receptors: colorimetric sensor for heparin

Polymethinium salts based on substituted malondialdehyde have been prepared; the salt with PEG substitution showed high selectivity for sulfate anions and heparin in aqueous medium at physiological condition; intracellular imaging of heparin-rich subcellular compartments was achieved with our polymethinium novel receptor for cancer cell lines.

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.

Role of ER Stress Response in Photodynamic Therapy: ROS Generated in Different Subcellular Compartments Trigger Diverse Cell Death Pathways

We have analyzed the molecular mechanisms of photoinduced cell death using porphyrins with similar structure differing only in the position of the ethylene glycol (EG) chain on the phenyl ring. Meta- and para-positioned EG chains targeted porphyrins to different subcellular compartments. After photoactivation, both types of derivatives induced death of tumor cells via reactive oxygen species (ROS). Para derivatives pTPP(EG)4 and pTPPF(EG)4 primarily accumulated in lysosomes activated the p38 MAP kinase cascade, which in turn induced the mitochondrial apoptotic pathway. In contrast, meta porphyrin derivative mTPP(EG)4 localized in the endoplasmic reticulum (ER) induced dramatic changes in Ca2+ homeostasis manifested by Ca2+ rise in the cytoplasm, activation of calpains and stress caspase-12 or caspase-4. ER stress developed into unfolded protein response. Immediately after irradiation the PERK pathway was activated through phosphorylation of PERK, eIF2α and induction of transcription factors ATF4 and CHOP, which regulate stress response genes. PERK knockdown and PERK deficiency protected cells against mTPP(EG)4-mediated apoptosis, confirming the causative role of the PERK pathway.