Osteogenic Potential of the Transcription Factor c-MYB.

The transcription factor c-MYB is a well-known marker of undifferentiated cells such as haematopoietic cell precursors, but recently it has also been observed in differentiated cells that produce hard tissues. Our previous findings showed the presence of c-MYB in intramembranous bones and its involvement in the chondrogenic steps of endochondral ossification, where the up-regulation of early chondrogenic markers after c-myb overexpression was observed. Since we previously detected c-MYB in osteoblasts, we aimed to analyse the localisation of c-MYB during later stages of endochondral bone formation and address its function during bone matrix production. c-MYB-positive cells were found in the chondro-osseous junction zone in osteoblasts of trabecular bone as well as deeper in the zone of ossification in cells of spongy bone. To experimentally evaluate the osteogenic potential of c-MYB during endochondral bone formation, micromasses derived from embryonic mouse limb buds were established. Nuclear c-MYB protein expression was observed in long-term micromasses, especially in the areas around nodules. c-myb overexpression induced the expression of osteogenic-related genes such as Bmp2, Comp, Csf2 and Itgb1. Moreover, alizarin red staining and osteocalcin labelling promoted mineralised matrix production in c-myb-overexpressing cultures, whereas downregulation of c-myb by siRNA reduced mineralised matrix production. In conclusion, c-Myb plays a role in the osteogenesis of long bones by inducing osteogenic genes and causing the enhancement of mineral matrix production. This action of the transcription factor c-Myb might be of interest in the future for the establishment of novel approaches to tissue regeneration.

Cyanobacterial ultrastructure in light of genomic sequence data.

Cyanobacteria are physiologically and morphologically diverse photosynthetic microbes that play major roles in the carbon and nitrogen cycles of the biosphere. Recently, they have gained attention as potential platforms for the production of biofuels and other renewable chemicals. Many cyanobacteria were characterized morphologically prior to the advent of genome sequencing. Here, we catalog cyanobacterial ultrastructure within the context of genomic sequence information, including high-magnification transmission electron micrographs that represent the diversity in cyanobacterial morphology. We place the image data in the context of tabulated protein domains-which are the structural, functional, and evolutionary units of proteins-from the 126 cyanobacterial genomes comprising the CyanoGEBA dataset. In particular, we identify the correspondence between ultrastructure and the occurrence of genes encoding protein domains related to the formation of cyanobacterial inclusions. This compilation of images and genome-level domain occurrence will prove useful for a variety of analyses of cyanobacterial sequence data and provides a guidebook to morphological features.

[Techniques to study transendothelial migration in vitro]. / Moznosti studia transendoteliální migrace in vitro.

The most dangerous aspect of cancer is the metastatic spread to other parts of the body. Cancer cells frequently use circulation to spread to secondary locations. By entering the blood-stream (in a process called intravasation) and by crossing the vessel walls at the metastatic sites (extravasation) tumor cells disseminate to distal organs and eventually form life  threatening metastases. Crossing the vessel walls (transendothelial migration) is a vital step of metastatic cascade and the elucidation of mechanisms involved in transendothelial migration might inspire new strategies of targeted antimetastatic therapy. There are several methods to study transendothelial migration in living models (in vivo). Although they offer complex physiological microenvironment, they are expensive and technically demanding, therefore not widely used. As an alternative, sophisticated techniques to investigate transendothelial migration in vitro have been developed. They are generally more available and feasible, but there is still considerable variability in the difficulty of performance, the requirements for specialized devices, accuracy of in vivo simulation and relevance for oncological applications. The classification, various modifications, pros and cons of in vitro techniques for studying transendothelial migration are summarized in this review.

[The use of flow cytometry for analysis of the mitochondrial cell death]. / Vyuzití prutokové cytometrie pro analýzu mitochondriální bunecné smrti.

Apoptosis is type I programmed cell death, a process that is essential for development and tissue homeostasis. It is a prevalent form of cell death and it proceeds via two signaling pathways - external (receptor pathway) triggered by death receptors and intrinsic (mitochondrial) apoptotic pathway with major involvement of mitochondria. Mitochondria are important cellular organelles producing energy stored in molecules of adenosine triphosphate that are essential for cell survival. The mitochondrial cell death is characterized by permeabilization of the mitochondrial outer membrane and dissipation of the transmembrane potential. Mitochondria are electronegative organelles and depolarization of the mitochondrial membrane is important for the release of proapoptotic signals. Aberrant control of the mitochondrial cell death might contribute to several diseases including cancer. Mitochondria are also a source of reactive oxygen species, Ca2+ ions and other proteins that affect processes important for the initiation and progression of tumors independently of apoptosis. Current studies focus on research of mitochondrial membrane potential and reactive oxygen species modulating various signaling pathways within the cell, their importance in carcinogenesis, and in treatment of oncological patients. Monitoring of the apoptotic markers, such as the mitochondrial membrane potential (MMP), and the level of reactive oxygen species in samples of oncological patients has a predictive value for the output of treatment protocols.

Roscovitine-induced apoptosis of H1299 cells depends on functional status of p53.

Roscovitine, an inhibitor of cyclin-dependent kinases, is promising anticancer agent. Its antiproliferative and cytotoxic effects can be mediated by the p53 signaling pathway. To define the role of p53 in roscovitine-induced cell response, we prepared H1299/p53 cell lines inducibly expressing specific variants of p53 (p53wt and hotspot R175H, temperature-dependent P98A, A159V, S215G, Y220C, Y234C mutants). In the presence of roscovitine, each cell line variant behaved in specific way reflecting activity of the p53 protein. Roscovitine decreased production of the cell cycle inhibitor p21 and induced apoptosis. This effect was the most efficient in cells expressing p53wt protein with full activity. The cell expressing partially and conditionally active p53 mutants responded to roscovitine less efficiently. The cells expressing p53 mutants A159V and Y234C were very sensitive to roscovitine but their response was clearly temperature-dependent. The cells expressing P98A, S215G and Y220C p53 mutants exhibited only weak sensitivity to roscovitine and underwent apoptosis in low frequency. In principle, each td p53 mutant responded to roscovitine in distinct way. We showed clearly that the impact of roscovitine on H1299 cells depends on functional status of p53 they produce. This suggests that patients with tumors exhibiting specific p53 variants can benefit from the roscovitine therapy.

v-Myb suppresses phorbol ester- and modifies retinoic acid-induced differentiation of human promonocytic U937 cells.

The c-myb protooncogene as well as its transforming derivate, the v-myb oncogene code for transcription factors. They regulate transcription of specific target genes thus controlling proliferation, differentiation and apoptosis of hematopoietic cells. Up-regulation of the c-myb expression or rearrangement/amplification of the myb locus are often involved in leukemogenesis. Enforced myb expression blocks differentiation of various leukemic cell lines. Human promonocytes U937 can be induced to differentiate to monocyte/macrophage-like cells using phorbol esters or to granulocytes using retinoic acid. In order to investigate transforming capability of v-myb, we expressed the v-myb oncogene of avian myeloblastosis virus in U937 cells. We found that v-Myb efficiently suppressed formation of macrophages upon treatment with phorbol ester. Some features of granulocytic differentiation of retinoic acid-treated U937 cells were affected by the v-Myb protein as well. These results document that v-Myb is significantly involved in control of myeloid differentiation.

Transcription factor c-Myb inhibits breast cancer lung metastasis by suppression of tumor cell seeding.

Metastasis accounts for most of cancer-related deaths. Paracrine signaling between tumor cells and the stroma induces changes in the tumor microenvironment required for metastasis. Transcription factor c-Myb was associated with breast cancer (BC) progression but its role in metastasis remains unclear. Here we show that increased c-Myb expression in BC cells inhibits spontaneous lung metastasis through impaired tumor cell extravasation. On contrary, BC cells with increased lung metastatic capacity exhibited low c-Myb levels. We identified a specific inflammatory signature, including Ccl2 chemokine, that was expressed in lung metastatic cells but was suppressed in tumor cells with higher c-Myb levels. Tumor cell-derived Ccl2 expression facilitated lung metastasis and rescued trans-endothelial migration of c-Myb overexpressing cells. Clinical data show that the identified inflammatory signature, together with a MYB expression, predicts lung metastasis relapse in BC patients. These results demonstrate that the c-Myb-regulated transcriptional program in BCs results in a blunted inflammatory response and consequently suppresses lung metastasis.

Retinoic acid receptor alpha suppresses transformation by v-myb.

Retinoic acid (RA) is capable of inducing the differentiation of various myelomonocytic cell lines. During this differentiation process, the levels of c-myb expression decline, suggesting that the RA receptor (RAR) may act in part by down-regulating this proto-oncogene. We have now investigated whether the RAR can also inhibit the function of Myb proteins themselves. We have found that transcriptional activation of a Myb-responsive reporter gene can be inhibited by RA in a human monocytic cell line. This inhibition could not be overcome by the expression of exogenous Myb. The RAR did not interfere with DNA binding by Myb proteins in vitro, suggesting that the functional inhibition occurs at the level of transcriptional activation. To determine the biological relevance of the inhibition of Myb proteins by the RAR, we have used v-myb-transformed monoblasts. These cells differentiate into macrophages in the presence of phorbol ester (tetradecanoyl phorbol acetate [TPA]) but are normally unresponsive to RA treatment. The introduction of an inducible, exogenous RAR alpha into v-myb-transformed monoblasts permitted an RA-dependent differentiation into macrophage-like cells similar to those induced by TPA. These results demonstrate that transformation by v-myb is recessive to RAR alpha and imply that many types of non-RA-responsive leukemia cells may become responsive following the introduction of the RAR.

Incidence of lysogenic, colicinogenic and siderophore-producing strains among human non-pathogenic Escherichia coli.

The current incidence of Escherichia coli strains in healthy humans capable of producing the inhibitory exoproducts, such as temperate bacteriophages, corpuscular or HMW (high-molar mass) and proteinaceous or LMW (low-molar mass) colicins and siderophores was determined. Fifty-three E. coli strains were collected from the colons of 53 healthy human volunteers in Brno (Czechia) and tested for spontaneous and induced production of inhibitory exoproducts in a cross-test against each other. Of the strains tested, 37.7% produced bacteriophages, 41.5% produced from one to several LMW colicins, 11.3% formed HMW colicins and 15.1% (eight strains) produced exocellular siderophores different from enterochelin. Of these, seven strains formed aerobactin and one strain formed an untyped siderophore. E. coli strains differ greatly in the incidence of colicinogeny and lysogeny from its closest systemic relatives in the genus Escherichia and therefore should not be regarded as a model bacterium in this respect.

[Regulation of osteoclast activity: a new approach in the therapy of bone diseases]. / Regulace aktivity osteoklastu: prostor pro terapii nekterých onemocnení kostí.

Bone remodelling is process of constant resorption and formation of a bone. Osteoclasts are the cells responsible for bone resorption. Deregulation of osteoclast differentiation, activity or function can cause severe diseases, such as osteoporosis, osteopetrosis or rheumatoid arthritis. Advances in molecular biology of osteoclasts and osteoimmunology open new approaches for the specific and efficient therapy.

c-Myb prevents TPA-induced differentiation and cell death in v-Myb transformed monoblasts.

Myelomonocytic cells transformed by v-Myb or altered forms of c-Myb do not contain the full-length c-Myb protein found in most immature hematopoietic cells. To determine if c-Myb was a dominant inhibitor of v-Myb, we have induced the synthesis of full-length c-Myb in monoblasts transformed by v-Myb. We found that although some morphological changes occurred, the presence of both c-Myb and v-Myb was compatible with cell growth. However, the response to phorbol ester (TPA) was significantly altered by c-Myb. Monoblasts transformed by v-Myb can be induced to differentiate into macrophages by treatment with TPA. This process is accompanied by a significant amount of cell death. However, when c-Myb was made TPA-inducible in these cells, TPA-induced differentiation into macrophages was blocked and cell death was prevented. These results demonstrate a significant difference in the biological effects of v-Myb and c-Myb in transformed myelomonocytic cells.

By-pass of TPA-induced differentiation and cell cycle arrest by the c-Myb DNA-binding domain.

Monoblasts transformed by v-Myb can be induced to differentiate into macrophages by treatment with phorbol ester (TPA). This differentiation occurs during both the G1 and the G2 phases of the cell cycle and is accompanied by cell cycle arrest. The introduction of a protein consisting of the three repeats (3R) of the c-Myb DNA-binding domain permits the by-pass of this phorbol ester-induced differentiation and cell cycle arrest. In particular, monoblasts which express the 3R protein progress through both the G1/S and G2/M transitions in the presence of phorbol ester. However, the 3R protein contains no detectable transcriptional activation domain. These results demonstrate that the c-Myb DNA-binding domain can regulate the cell cycle without functioning as a direct transcriptional activator.

Retinoid X receptor suppresses transformation by the v-myb oncogene.

The v-myb oncogene of avian myeloblastosis virus causes acute monoblastic leukemia in vivo and transforms myelomonocytic cells in culture. Retinoids are potent regulators of proliferation and differentiation in various cell types, and they can initiate differentiation in certain types of leukemic cells. However, the BM2 v-myb-transformed chicken monoblastic cell line is resistant to retinoic acid treatment. We found that overexpression of the retinoid X receptor confers sensitivity of BM2 cells to retinoic acid, resulting in induction of growth arrest and terminal differentiation. In contrast, the frequency of apoptosis was not affected by the retinoid X receptor in this cell type. We also demonstrated that suppression of transformation by v-Myb results from the negative effect of retinoid X receptor on v-Myb transactivation function, similar to that previously described for the retinoic acid receptor. The retinoid X receptor-induced inhibition of transactivation by v-Myb seems to be enhanced by a cell type-specific factor(s), which is not required by retinoic acid receptor.

Mouse Incisor Stem Cell Niche and Myb Transcription Factors.

Dental hard tissues are formed particularly by odontoblasts (dentin) and ameloblasts (enamel). Whereas the reparation of dentin is often observed, enamel does not regenerate in most species. However, in mouse incisor, a population of somatic stem cells in the cervical loop is responsible for the incisor regeneration. Understanding of the specificities of these cells is therefore of an interest in basic research as well as regenerative therapies. The Myb transcription factors are involved in essential cellular processes. B-Myb is often linked to the stem cell phenotype, and c-Myb expression marks undifferentiated and proliferating cells such as the stem cells. In the presented study, temporo-spatial expression of B-Myb and c-Myb proteins was correlated with localisation of putative somatic stem cells in the mouse incisor cervical loop by immunohistochemistry. B-Myb expression was localised mostly in the zone of transit-amplifying cells, and c-Myb was found in the inner enamel epithelium, the surrounding mesenchyme and in differentiated cells. Taken together, neither B-Myb nor c-Myb was exclusively present or abundant in the area of the incisor stem cell niche. Their distribution, however, supports recently reported novel functions of c-Myb in differentiation of hard tissue cells.

Role of c-Myb in chondrogenesis.

The Myb locus encodes the c-Myb transcription factor involved in controlling a broad variety of cellular processes. Recently, it has been shown that c-Myb may play a specific role in hard tissue formation; however, all of these results were gathered from an analysis of intramembranous ossification. To investigate a possible role of c-Myb in endochondral ossification, we carried out our study on the long bones of mouse limbs during embryonic development. Firstly, the c-myb expression pattern was analyzed by in situ hybridization during endochondral ossification of long bones. c-myb positive areas were found in proliferating as well as hypertrophic zones of the growth plate. At early embryonic stages, localized expression was also observed in the perichondrium and interdigital areas. The c-Myb protein was found in proliferating chondrocytes and in the perichondrium of the forelimb bones (E14.5-E17.5). Furthermore, protein was detected in pre-hypertrophic as well as hypertrophic chondrocytes. Gain-of-function and loss-of-function approaches were used to test the effect of altered c-myb expression on chondrogenesis in micromass cultures established from forelimb buds of mouse embryos. A loss-of-function approach using c-myb specific siRNA decreased nodule formation, as well as downregulated the level of Sox9 expression, a major marker of chondrogenesis. Transient c-myb overexpression markedly increased the formation of cartilage nodules and the production of extracellular matrix as detected by intense staining with Alcian blue. Moreover, the expression of early chondrogenic genes such as Sox9, Col2a1 and activity of a Col2-LUC reporter were increased in the cells overexpressing c-myb while late chondrogenic markers such as Col10a1 and Mmp13 were not significantly changed or were downregulated. Taken together, the results of this study demonstrate that the c-Myb transcription factor is involved in the regulation and promotion of endochondral bone formation.

Dicistronic selection for nuclear proteins in living animal cells.

We present an expression/selection system designed for the purification of cell lines inducibly expressing genes coding for unselectable proteins by using dicistronic selection for the cell surface marker CD4. This system enabled us to establish and purify c-myb expressing variants of the v-myb transformed chicken monoblast cell line BM2 with high efficiency.

Tolerated variations in a genome: the case of closely related Bacillus phages PZA, phi 29 and phi 15--a review.

Restriction-site analysis was used to estimate the relationship of bacteriophages PZA, phi 29 and phi 15. Complete nucleotide sequences of PZA and luminal diameter 29 genomes were compared and tolerated variations were assessed. Most of the base-pair changes are silent nucleotide substitutions in the third position of codons but amino acid changing substitutions are also observed. The terminal portions of the phage genomes diverged faster than their central parts. Gene mutations in phage PZA were induced by hydroxylamine and their frequency was compared with the evolutionary mutability.

Determination of optimal conditions for detection of acute myeloid leukemia t(8;21) and t(15;17) translocations using RT-PCR.

Prognosis of patients with acute myeloid leukemia (AML) is not satisfactory. Long time remission can be achieved only in 25-40% children with AML. The aim of the study is to improve diagnosis of AML in cases characterized by specific chromosomal translocations t(8;21) and t(15;17). These translocations can be efficiently detected using reverse transcriptase-polymerase chain reaction (RT-PCR). To determine optimal conditions for detection of the t(8;21) and t(15;17) translocations in human cells, we performed a detail study of experimental conditions for RT-PCR on human cell lines NB-4 and KASUMI-1. For detection of t(8;21) using Pfx polymerase, the optimal PCR conditions included magnesium ion concentration in the range of 0.6 to 1 mM, 0.4 microM primer concentration, 5 degrees C annealing temperature and 1 microl of template cDNA. For detection of t(15;17) using the Pfx polymerase, the optimal conditions included 1 mM magnesium ion concentration, 0.6 microM primer concentration, 63 degrees C-66 degrees C annealing temperature and 2 microl of template cDNA. The results proved to be valid for clinical diagnostics of these chromosomal aberrations in blood and/or bone marrow samples of AML patients.

Cytotoxic effects of colicins E1 and E3 on v-myb-transformed chicken monoblasts.

Colicins show a considerable cytostatic activity, which is much less known and understood than their killing activity targeting bacteria of the Enterobacteriaceae family. In this communication, the cytotoxic effects of colicins E1 and E3 on v-myb-transformed chicken monoblasts BM2 are presented. We detected clear reduction of the viable cell number induced by colicins E1 and E3, occurring without apparent changes in cell cycle profiles. The level of inhibition was proportional to the colicin concentration within the limits of 0.5-1.25 microg/ml. This result documents that colicins produced by Enterobacteriaceae exert their cytotoxic effects on leukemic cells.

A possible cross-talk of retinoic acid- and TPA-driven myeloid differentiation pathways.

Retinoic acid (RA) and phorbol esters are important regulators of cellular proliferation and differentiation processes which are capable of arresting growth and inducing differentiation of various leukemic cells. We have studied and compared the effects of these agents on the line of v-myb oncogene-transformed chicken monoblasts BM2. We found that although there are differences in the differentiation kinetics and cell cycle stop points, the differentiation pathways induced by RA and phorbol esters seem to be significantly interconnected.