T-lymphopoiesis is Severely Compromised in Ubiquitin-Green Fluorescent Protein Transgenic Mice.

Tagging cells of experimental organisms with genetic markers is commonly used in biomedical research. Insertion of artificial gene constructs can be highly beneficial for research as long as this tagging is functionally neutral and does not alter the tissue function. The transgenic UBC-GFP mouse has been recently found to be questionable in this respect, due to a latent stem cell defect compromising its lymphopoiesis and significantly influencing the results of competitive transplantation assays. In this study, we show that the stem cell defect present in UBC-GFP mice negatively affects T-lymphopoiesis significantly more than B-lymphopoiesis. The production of granulocytes is not negatively affected. The defect in T-lymphopoiesis causes a low total number of white blood cells in the peripheral blood of UBC-GFP mice which, together with the lower lymphoid/myeloid ratio in nucleated blood cells, is the only abnormal phenotype in untreated UBCGFP mice to have been found to date. The defective lymphopoiesis in UBC-GFP mice can be repaired by transplantation of congenic wild-type bone marrow cells, which then compensate for the insufficient production of T cells. Interestingly, the wild-type branch of haematopoiesis in chimaeric UBC-GFP/wild-type mice was more active in lymphopoiesis, and particularly towards production of T cells, compared to the lymphopoiesis in normal wild-type donors.

Iron Overload Causes Alterations of E-Cadherin in the Liver.

Iron overload causes tissue damage in the liver, but its initial effects at the molecular and cellular level are not well understood. Epithelial cadherin (E-cad) is a major adhesion protein in adherens junctions and is associated with several signal transduction pathways. Dysfunction of E-cad causes instability of adherens junctions, which leads to cell invasion, cell migration, and carcinogenesis. We found in liver samples from iron-overloaded mice that the apparent molecular mass of E-cad was reduced from 125 to 115 kDa in sodium dodecyl sulphate polyacrylamide gel electrophoresis under reducing conditions and immunoblotting, and that the cellular expression of E-cad was decreased in immunohistochemistry. The mRNA level of E-cad, however, did not change significantly, suggesting that the alterations are posttranslational. Interestingly, incubation of control liver extracts with Fe2+ alone also produced the same mobility shift. Neither an oxidant nor an antioxidant influenced this shift in vitro, suggesting that reactive oxygen species, which are generated by iron and known to cause damage to macromolecules, are not involved. Treatment of the 115 kDa E-cad with deferoxamine, an iron chelator, thus removing Fe2+, shifted the molecular mass back to 125 kDa, demonstrating that the shift is reversible. The observation also implies that the alteration that causes the mobility shift is not due to transcriptional control, deglycosylation, and proteolysis. This reversible mobility shift of E-cad has not been previously known. The alteration of E-cad that causes the mobility shift might be an initial step to liver diseases by iron overload.

Decreased hemojuvelin protein levels in mask mice lacking matriptase-2-dependent proteolytic activity.

Matriptase-2, a membrane protein encoded by the Tmprss6 gene, is a negative regulator of hepcidin expression. Although matriptase-2 has been proposed to cleave membrane hemojuvelin, we have recently found decreased hemojuvelin protein levels in Tmprss6 -/- mice. The purpose of this study was to confirm this observation by determining hemojuvelin protein levels in another strain of mice with disrupted Tmprss6 gene, and to determine the effect of matriptase-2 deficiency on the expression of other membrane proteins participating in the bone morphogenetic protein signal transduction. Mask mice, which lack the proteolytic domain of matriptase-2, displayed decreased liver hemojuvelin protein content, while Id1 mRNA level, an indicator of hemojuvelin-dependent signal transduction, was increased. Protein levels of bone morphogenetic protein receptors Alk3 and Acvr2a were unchanged, and transferrin receptor 2 and neogenin protein levels were slightly decreased. The results confirm that the loss of matriptase-2 increases bone morphogenetic protein-dependent signaling, while paradoxically decreasing liver hemojuvelin protein content. The regulation of transferrin receptor 2 protein levels by transferrin saturation was not affected in mask mice. How the loss of matriptase-2 proteolytic activity leads to decreased hemojuvelin protein levels is at present unclear.

Availability of haematopoietic niches for transplanted stem cells.

Following transplantation, donor haematopoietic stem cells (HSCs) must reach specific parts of haematopoietic stroma tissue known as stem cell niches to become engrafted and to start blood cell production. Regularly, they have to compete with the host's HSCs for a limited number of niches. The exact mechanisms of HSC engraftment as well as of niche "opening" to incoming HSCs by conditioning treatments are not well-known yet. Significant and stable engraftment of syngeneic donor HSCs can be achieved in untreated mice only after transplantation of very large numbers of marrow cells. Engraftment can be largely facilitated by the stem cell mutations reducing numbers of the host HSCs. Pre-transplantation manipulations of the host haematopoietic tissue enhance engraftment depending on how much they damage HSCs. Ionizing radiation appears to be the most effective in this respect despite proliferative quiescence of a majority of HSCs. The review summarizes major achievements in deciphering biological principles of the HSCs and their engraftment after transplantation obtained in experimental research studying murine haematopoiesis.

In vivo growth of mantle cell lymphoma xenografts in immunodeficient mice is positively regulated by VEGF and associated with significant up-regulation of CD31/PECAM1.

Mantle cell lymphoma (MCL) is an aggressive lymphoma subtype with dismal prognosis. New treatments are needed to improve outcome of relapsed/ refractory disease. Recently, several drugs targeting at least partially the process of angiogenesis have been successfully tested in the therapy of MCL. Molecular mechanisms that regulate MCL-induced angiogenesis and that might represent potential new druggable targets remain, however, incompletely understood. We established two mouse models of human MCL by subcutaneous xenotransplantation of JEKO-1 and HBL-2 cell lines into immunodeficient mice. Histological analyses of xenografts confirmed their neovascularization. The growth of xenografts was significantly suppressed by single-agent therapy with bevacizumab, monoclonal antibody targeting vascular endothelial growth factor (VEGF). Subsequently, we analysed expression of 94 angiogenesis related genes in ex vivo isolated JEKO-1 and HBL-2 cells compared to in vitro growing cells using TaqMan low-density arrays. The most up-regulated genes in both JEKO-1 and HBL-2 xenografts were genes encoding platelet/endothelial cell-adhesion molecule (CD31/PECAM1), VEGF receptor 1 (FLT1), hepatocyte growth factor (HGF), angiogenin (ANG) and transcription factor PROX1. The most downregulated genes in both JEKO-1 and HBL-2 xenografts were midkine (MDK) and ephrine B2 (EPHB2). In summary, our results demonstrate an important role of angiogenesis in the biology of MCL and provide preclinical evidence of potent anti-MCL activity of bevacizumab. In addition, gene expression profiling of 94 angiogenesis-related targets revealed several in vivo up-regulated and down-regulated transcripts. The most differentially expressed target in both MCL tumours was CD31/PECAM1. Whether any of these molecules might represent a potential druggable target in MCL patients remains to be elucidated.

Multi-level disruption of the extrinsic apoptotic pathway mediates resistance of leukemia cells to TNF-related apoptosis-inducing ligand (TRAIL).

Disruption of apoptotic pathways belongs to commonly reported molecular mechanisms that underlie cancer drug resistance. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL, Apo2L) is a cytokine of the TNF family with selective anti-tumor activity and minimal toxicity toward healthy tissues. Primary leukemia cells are, however, largely intrinsically resistant to TRAIL-induced apoptosis. In this study we analyzed molecular differences between TRAIL-resistant K562 cell line and TRAIL-sensitive K562 clones. We demonstrate that TRAIL-sensitive K562 cells differ from the TRAIL-resistant cell line by cell surface downregulation of TRAIL decoy receptor 1, upregulation of both TRAIL death receptors, enhanced assembly and improved functioning of the death-inducing signaling complex, and increased cytoplasmic protein expression of CASP8 and key proapoptotic BCL2 members BID, BIM, BAD and BAK. The molecular basis of the intrinsic leukemia cell TRAIL resistance thus appears a consequence of the multi-level disruption of the extrinsic apoptotic pathway. The results of this study also suggest that the leukemia TRAIL-resistance is functional, leaving a possibility of overcoming the resistance by preexposure of the leukemia cells to potent TRAIL sensitizers, e.g. BH3-mimetics.

5-azacitidine in aggressive myelodysplastic syndromes regulates chromatin structure at PU.1 gene and cell differentiation capacity.

Epigenetic 5-azacitidine (AZA) therapy of high-risk myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML) represents a promising, albeit not fully understood, approach. Hematopoietic transcription factor PU.1 is dynamically regulated by upstream regulatory element (URE), whose deletion causes downregulation of PU.1 leading to AML in mouse. In this study a significant group of the high-risk MDS patients, as well as MDS cell lines, displayed downregulation of PU.1 expression within CD34+ cells, which was associated with DNA methylation of the URE. AZA treatment in vitro significantly demethylated URE, leading to upregulation of PU.1 followed by derepression of its transcriptional targets and onset of myeloid differentiation. Addition of colony-stimulating factors (CSFs; granulocyte-CSF, granulocyte-macrophage-CSF and macrophage-CSF) modulated AZA-mediated effects on reprogramming of histone modifications at the URE and cell differentiation outcome. Our data collectively support the importance of modifying the URE chromatin structure as a regulatory mechanism of AZA-mediated activation of PU.1 and induction of the myeloid program in MDS.

Hepcidin expression in the liver of mice with implanted tumour reacts to iron deficiency, inflammation and erythropoietin administration.

Cancer is known to be an important cause of anaemia due to several factors including iron deficiency and inflammation. Hepcidin, a key regulator of iron metabolism, is up-regulated by iron and inflammatory stimuli such as interleukin 6, and decreased by iron deficiency, enhanced erythropoiesis and hypoxia. It is supposed to play a crucial role in changes of iron metabolism in anaemia of chronic disease, which is characterized by sequestering iron in macrophages and decreasing its availability for red blood cell production. To study the effect of tumour growth on hepcidin expression, we implanted human melanoma cells into mice and studied the changes of the amount of liver hepcidin mRNA by real-time PCR. We observed development of anaemia, which correlated with the size of the tumour. Hepcidin expression significantly decreased with the anaemia development, but in late stages we observed an increase of its expression together with an increase of mRNA for interleukin 6. However, the increase of hepcidin expression could be inhibited by exogenous erythropoietin administration. In our model of tumour growth, hepcidin expression reflected anaemia development and iron deficiency, erythropoietin administration and inflammation, and we suppose that it could therefore serve as a useful marker of these clinical situations common in cancer patients and play a role in the pathogenesis of cancer-associated anaemia.

Hepcidin downregulation by repeated bleeding is not mediated by soluble hemojuvelin.

Hepcidin is a key regulator of iron homeostasis, while hemojuvelin is an important component of the hepcidin regulation pathway. It has been recently proposed that soluble hemojuvelin, produced from hemojuvelin by the protease furin, decreases hepcidin expression. The aim of the presented study was to examine the downregulation of hepcidin by chronic bleeding in hemojuvelin-mutant mice. Male mice with targeted disruption of the hemojuvelin gene (Hjv-/- mice) and wild-type littermates were maintained on an iron-deficient diet and subjected to weekly phlebotomies for 7 weeks. Gene expression was examined by real-time PCR. In wild type mice, repeated bleeding decreased hepcidin mRNA by two orders of magnitude. In Hjv-/- mice, basal hepcidin expression was low; however, repeated bleeding also decreased hepcidin mRNA content by an order of magnitude. Phlebotomies reduced hepatic iron overload in Hjv-/- mice by 80 %. Liver and muscle furin mRNA content was not significantly changed. No effect on hepatic Tmprss6 mRNA content was observed. Results from the study indicate that soluble hemojuvelin is not the sole factor responsible for hepcidin downregulation. In addition, the presented data suggest that, under in vivo conditions, tissue hypoxia does not transcriptionally regulate the activity of furin or TMPRSS6 proteases.

Hepcidin expression in adipose tissue increases during cardiac surgery.

Hepcidin, a key regulator of iron metabolism, plays a crucial role in the pathogenesis of anemia of chronic disease. Although it is produced mainly in the liver, its recently described expression in adipose tissue has been shown to be enhanced in massive obesity due to chronic low-grade inflammation. Our objective was to study the changes in hepcidin expression in adipose tissue during acute-phase reaction. We measured hepcidin mRNA expression from isolated subcutaneous and epicardial adipose tissue at the beginning and at the end of the surgery. The expression of mRNAs for hepcidin and other iron-related genes (transferrin receptor 1, divalent metal transporter 1, ferritin, ferroportin) were measured by real-time RT-PCR. Hepcidin expression significantly increased at the end of the surgery in subcutaneous but not in epicardial adipose tissue. Apart from the increased levels of cytokines, the parameters of iron metabolism showed typical inflammation-induced changes. We suggest that acute inflammatory changes could affect the regulation of hepcidin expression in subcutaneous adipose tissue and thus possibly contribute to inflammation-induced systemic changes of iron metabolism.

The late-stage foetal liver microenvironment is essential for later sensitivity of B-lymphopoiesis to suppression by oestrogens.

B-lymphopoiesis in FL differs notably from that of adult B-lymphopoiesis in being resistant to suppression by oestrogens due to the lack of expression of oestrogen receptors in B-cell progenitors and precursors. We have transplanted middle-stage FL cells (E14.5) to adult male mice and demonstrated that B-lymphopoiesis derived from FL cells remained resistant to suppression by exogenous oestrogen for several months compared to adult BM cells. This significant difference strongly suggests that the latestage FL environment exerts an inductive action on the haematopoietic stem cells and is mandatory for later sensitivity of B-lymphopoiesis to suppression by oestrogens. The results also provide the first in vivo functional confirmation of a differential responsiveness of FL- and adult BM-derived B-lymphopoiesis to suppression by oestrogens.

Risedronate has no adverse effects on mouse haematopoiesis.

Bisphosphonates are commonly used for treatment of osteoporosis. They inhibit osteoclast activity and thus bone resorption. It was shown that they also affect some other cell types including tumour and endothelial cells. The effects of risedronate on bone marrow microenvironment were not studied yet. As endothelial cells are integral part of bone marrow microenvironment, it is important to know whether prolonged administration of bisphosphonates does not affect haematopoietic stem cells and bone marrow haematopoiesis. We fed mice two weeks with risedronate. We found no effect of risedronate treatment on bone marrow stem cells using the method of congenic bone marrow repopulation. Risedronate administration in the dose which is considered to be comparable to a dose of risedronate used for treatment of osteoporosis in women seems to be safe in terms of effects on mouse haematopoiesis.

Cell death signalling pathways in the pathogenesis and therapy of haematologic malignancies: overview of therapeutic approaches.

Malignant diseases, including haematologic malignancies, are associated with defects in the cell death mechanism. These defects are not only important for the growth advantage of the malignant clone, but when understood can be used for specific therapeutic targeting of malignant cells while sparing normal cells. The promising groups of agents that trigger, directly or indirectly, apoptosis of haematologic cancer cells are reviewed in this article. Some of the agents have recently been approved for therapy, some are under the clinical evaluation in various phases of clinical trials and some are tested under the experimental laboratory conditions.

Protein kinase inhibitors.

Since protein kinases have been found to be implicated in many diseases, first of all malignancies, they are considered as promising therapeutic targets. Many protein kinase inhibitors have been designed by now. These molecules have a low molecular weight and most of them bind to protein kinases competing with ATP for the ATP-binding site. Some protein kinase inhibitors currently undergo clinical trials or have already been successfully introduced into treatment as exemplified by Bcr-Abl, c-kit and PDGFR inhibitor imatinib mesylate (Gleevec), flavopiridol and roscovitine, inhibitors of cyclin-dependent kinases, or erlotinib and gefitinib inhibiting EGFR. Discovery of these molecules seems to begin a new era in medicine, especially oncology. Targeting protein kinases represents a promising approach and gives us new hopes of effective non-invasive cancer treatment.

Expression of actin isoforms in human auricular cartilage.

The objective of this study was to determine whether human auricular chondrocytes can also express alpha-- -smooth muscle actin. Immunohistochemistry using monoclonal antibodies for alpha-smooth actin, muscle-specific actin, beta-actin, S-100 protein, CD34, and desmin was performed on samples of human ear cartilage obtained from 20 individuals during a partial resection of the ear for different reasons. Moreover, the RT-PCR analysis of actin isoforms in auricular chondrocytes was performed. Approximately 60 % of the chondrocytes of the ear cartilage expressed alpha-smooth muscle actin as demonstrated by immunohistochemistry in all the examined samples. Actin-positive chondrocytes occurred in both external subperichondrial layers of the auricular cartilage. This finding was confirmed by the RT-PCR technique. The knowledge of this fact could help us to better understand the chondrocyte changes occurring during the healing and transplantation of auricular cartilage. The question of whether it is necessary to refer to these predominating cells in ear cartilage as myochondrocytes is considered. This is the first report of an unusual immunophenotype and contractile potential for human auricular chondrocytes.

Protein kinases, their function and implication in cancer and other diseases.

Protein phosphorylation is known to play an important role in various cellular processes such as cell division, metabolism, survival and apoptosis. It is driven by specific enzymes, tyrosine and serine-threonine protein kinases. Human protein kinases constitute a complicated system with intricate internal and external interactions. The complexity and sophistication of the system implies its vulnerability. Alterations in functions of these enzymes may launch series of pathological changes within the cell and as a result cause diseases. Protein kinases have been shown to be involved in various pathological processes, first of all malignancies. Deregulation of different protein kinases has been found in chronic myelogenous leukaemia, gastrointestinal stromal tumours, various other sarcomas and cancers as well as non-malignant disorders. Therefore, they are regarded as important effectors in human pathology and represent prospective therapeutic targets.

Effect of lipopolysaccharide and bleeding on the expression of intestinal proteins involved in iron and haem transport.

Haem carrier protein 1 (Hcpl) is a component of the haem-iron uptake pathway in the small intestine. Using quantitative real-time PCR, we examined the expression of Hcp1 and other intestinal iron-transporting proteins in male C57BL/6 mice with experimentally altered iron homeostasis. Intestinal Hcp1 mRNA content was not significantly changed by iron overload (600 mg/kg); however, it was increased to 170 % of controls 72 h after withdrawal of 0.7 ml of blood; the same treatment increased intestinal Cybrd1 mRNA to 900 % of controls. LPS treatment (1 mg/kg, 6 h) decreased intestinal Hcp1 mRNA content to 66 % of controls and Flvcr mRNA content to 65 % of controls, while Cybrd1 mRNA, Dmt1 mRNA and Fpn1 mRNA decreased to 6 %, 43 % and 32 %, respectively. In 129SvJ mice with targeted disruption of the hemojuvelin (Hfe2) gene, which display very low expression of liver hepcidin, Cybrd1 mRNA content increased to 1040 %, Dmt1 mRNA content to 200 % and Fpn1 mRNA to 150 % when compared to wild-type mice; changes in Hcp1, Abcg2 and Flver mRNA content were only minor. Overall, these results suggest that, during inflammation, the intestinal haem-iron uptake pathway is not as strongly transcriptionally downregulated as the non-haem iron uptake pathway. A decrease in circulating hepcidin increases the expression of proteins participating in non-haem iron uptake, but has no significant effect on Hcp1 mRNA content.

Cell death signalling pathways in the pathogenesis and therapy of haematologic malignancies: overview of apoptotic pathways.

Apoptosis, a Greek descriptive term for falling leaves or petals, plays an important role in the progression of many diseases. Apoptosis is essential for the development and survival of multi-cellular organisms. Malignant diseases, including haematologic malignancies, are associated with defects in the cell death mechanism. These defects are not only important for the growth advantage of malignant clones, but when understood can be used for specific therapeutic targeting of malignant cells while sparing normal cells. The cellular and molecular mechanisms of apoptosis have been extensively demonstrated and are reviewed in this article. In this part of the review we focus on basic details of the apoptosis pathways, key players of the receptor-mediated apoptosis, and molecules involved in the cross-talk between individual apoptosis pathways and apoptosis regulation.

Hepcidin mRNA levels in mouse liver respond to inhibition of erythropoiesis.

Hepcidin, a key regulator of iron metabolism, decreases intestinal absorption of iron and its release from macrophages. Iron, anemia, hypoxia, and inflammation were reported to influence hepcidin expression. To investigate regulation of the expression of hepcidin and other iron-related genes, we manipulated erythropoietic activity in mice. Erythropoiesis was inhibited by irradiation or posttransfusion polycythemia and stimulated by phenylhydrazine administration and erythropoietin. Gene expression of hepcidin and other iron-related genes (hemojuvelin, DMT1, ferroportin, transferrin receptors, ferritin) in the liver was measured by the real-time polymerase chain reaction. Hepcidin expression increased despite severe anemia when hematopoiesis was inhibited by irradiation. Suppression of erythropoiesis by posttransfusion polycythemia or irradiation also increased hepcidin mRNA levels. Compensated hemolysis induced by repeated phenylhydrazine administration did not change hepcidin expression. The decrease caused by exogenous erythropoeitin was blocked by postirradiation bone marrow suppression. The hemolysis and anemia decrease hepcidin expression only when erythropoiesis is functional; on the other hand, if erythropoiesis is blocked, even severe anemia does not lead to a decrease of hepcidin expression, which is indeed increased. We propose that hepcidin is exclusively sensitive to iron utilization for erythropoiesis and hepatocyte iron balance, and these changes are not sensed by other genes involved in the control of iron metabolism in the liver.