Differentiation during hematopoiesis leads to the generation of many cell types with specific functions. At various stages of maturation, the cells may change pathologically, leading to diseases including acute leukemias (ALs). Expression levels of regulatory molecules (such as the IKZF, GATA, HOX, FOX, NOTCH and CEBP families, as well as SPI-1/PU1 and PAX5) and lineage-specific molecules (including CD2, CD14, CD79A, and BLNK) may be compared between pathological and physiological cells. Although the key steps of differentiation are known, the available databases focus mainly on fully differentiated cells as a reference. Precursor cells may be a more appropriate reference point for diseases that evolve at immature stages. Therefore, we developed a quantitative real-time polymerase chain reaction (qPCR) array to investigate 90 genes that are characteristic of the lymphoid or myeloid lineages and/or are thought to be involved in their regulation. Using this array, sorted cells of granulocytic, monocytic, T and B lineages were analyzed. For each of these lineages, 3-5 differentiation stages were selected (17 stages total), and cells were sorted from 3 different donors per stage. The qPCR results were compared to similarly processed AL cells of lymphoblastic (n=18) or myeloid (n=6) origins and biphenotypic AL cells of B cell origin with myeloid involvement (n=5). Molecules characteristic of each lineage were found. In addition, cells of a newly discovered switching lymphoblastic AL (swALL) were sorted at various phases during the supposed transdifferentiation from an immature B cell to a monocytic phenotype. As demonstrated previously, gene expression changed along with the immunophenotype. The qPCR data are publicly available in the LeukoStage Database in which gene expression in malignant and non-malignant cells of different lineages can be explored graphically and differentially expressed genes can be identified. In addition, the LeukoStage Database can aid the functional analyses of next-generation sequencing data.
- MeSH
- akutní bifenotypická leukemie genetika imunologie patologie MeSH
- B-lymfocyty imunologie patologie MeSH
- buněčná diferenciace genetika MeSH
- buněčný rodokmen genetika MeSH
- čipová analýza tkání MeSH
- hematopoéza genetika MeSH
- imunofenotypizace MeSH
- lidé MeSH
- nádorové proteiny biosyntéza MeSH
- regulace genové exprese u leukemie MeSH
- T-lymfocyty imunologie patologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Autophagy is essential for successful white adipocyte differentiation but the data regarding the timing and relevance of autophagy action during different phases of adipogenesis are limited. We subjected 3T3-L1 preadipocytes to a standard differentiation protocol and inhibited the autophagy within time-limited periods (days 0-2; 2-4; 4-6; 6-8) by asparagine or 3-methyladenine. In the normal course of events, both autophagy flux and the mRNA expression of autophagy related genes (Atg5, Atg12, Atg16, beclin 1) is most intensive at the beginning of differentiation (days 0-4) and then declines. The initiation of differentiation is associated with a 50% reduction of the mitochondrial copy number on day 2 followed by rapid mitochondrial biogenesis. Preadipocytes and differentiated adipocytes differ in the mRNA expression of genes involved in electron transport (Nufsd1, Sdhb, Uqcrc1); ATP synthesis (ATP5b); fatty acid metabolism (CPT1b, Acadl); mitochondrial transporters (Hspa9, Slc25A1) and the TCA cycle (Pcx, Mdh2) as well as citrate synthase activity. Autophagy inhibition during the first two days of differentiation blocked both phenotype changes (lipid accumulation) and the gene expression pattern, while having no or only a marginal effect over any other time period. Similarly, autophagy inhibition between days 0-2 inhibited mitotic clonal expansion as well as mitochondrial network remodeling. In conclusion, we found that autophagy is essential and most active during an initial stage of adipocyte differentiation but it is dispensable during its later stages. We propose that the degradation of preadipocyte cytoplasmic structures, predominantly mitochondria, is an important function of autophagy during this phase and its absence prevents remodeling of the mitochondrial gene expression pattern and mitochondrial network organization.
- MeSH
- adipogeneze účinky léků genetika MeSH
- asparagin farmakologie MeSH
- autofagie účinky léků genetika MeSH
- buněčná diferenciace účinky léků genetika MeSH
- buňky 3T3-L1 MeSH
- mitochondrie účinky léků metabolismus MeSH
- myši MeSH
- tukové buňky cytologie účinky léků MeSH
- vývojová regulace genové exprese účinky léků genetika MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Survival and capability of cancer cells to form metastases fundamentally depend on interactions with their microenvironment. Secondary tumors originating from prostate carcinomas affect remodeling of bone tissue and can induce both osteolytic and osteocondensing lesions. However, particular molecular mechanisms responsible for selective homing and activity of cancer cells in bone microenvironment have not been clarified yet. Growth/differentiation factor-15 (GDF-15), a distant member of the TGF-beta protein family, has recently been associated with many human cancers, including prostate. We show that both pure GDF-15 and the GDF-15-containing growth medium of 1,25(OH)(2)-vitamin D(3)-treated prostate adenocarcinoma LNCaP cells suppress formation of mature osteoclasts differentiated from RAW264.7 macrophages and bone-marrow precursors by M-CSF/RANKL in a dose-dependent manner. GDF-15 inhibits expression of c-Fos and activity of NFkappaB by delayed degradation of IkappaB. Moreover, GDF-15 inhibits expression of carbonic anhydrase II and cathepsin K, key osteoclast enzymes, and induces changes in SMAD and p38 signaling. The lack of functional osteoclasts can contribute to accumulation of bone matrix by reduction of bone resorption. These results unveil new role of GDF-15 in modulation of osteoclast differentiation and possibly in therapy of bone metastases.
- MeSH
- buněčná diferenciace účinky léků MeSH
- buněčné linie MeSH
- časové faktory MeSH
- faktor stimulující kolonie makrofágů farmakologie MeSH
- femur cytologie MeSH
- inbrední kmeny myší MeSH
- izoenzymy metabolismus MeSH
- kalcitriol farmakologie MeSH
- karboanhydrasa II antagonisté a inhibitory MeSH
- kathepsin K antagonisté a inhibitory genetika MeSH
- kultivační média speciální farmakologie MeSH
- kyselá fosfatasa metabolismus MeSH
- lidé MeSH
- ligand RANK farmakologie MeSH
- makrofágy cytologie MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- nádory prostaty metabolismus MeSH
- NF-kappa B antagonisté a inhibitory MeSH
- osteoklasty metabolismus účinky léků MeSH
- protoonkogenní proteiny c-fos antagonisté a inhibitory MeSH
- růstový diferenciační faktor 15 farmakologie MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
Gametes of both sexes (sperm and oocyte) are highly specialized and differentiated but within a very short time period post-fertilization the embryonic genome, produced by the combination of the two highly specialized parental genomes, is completely converted into a totipotent state. As a result, the one-cell-stage embryo can give rise to all cell types of all three embryonic layers, including the gametes. Thus, it is evident that extensive and efficient reprogramming steps occur soon after fertilization and also probably during early embryogenesis to reverse completely the differentiated state of the gamete and to achieve toti- or later on pluripotency of embryonic cells. However, after the embryo reaches the blastocyst stage, the first two distinct cell lineages can be clearly distinguished--the trophectoderm and the inner cells mass. The de-differentiation of gametes after fertilization, as well as the differentiation that is associated with the formation of blastocysts, are accompanied by changes in the state and properties of chromatin in individual embryonic nuclei at both the whole genome level as well as at the level of individual genes. In this contribution, we focus mainly on those events that take place soon after fertilization and during early embryogenesis in mammals. We will discuss the changes in DNA methylation and covalent histone modifications that were shown to be highly dynamic during this period; moreover, it has also been documented that abnormalities in these processes have a devastating impact on the developmental ability of embryos. Special attention will be paid to somatic cell nuclear transfer as it has been shown that the aberrant and inefficient reprogramming may be responsible for compromised development of cloned embryos.
- MeSH
- blastocysta metabolismus MeSH
- buněčné jádro genetika MeSH
- chromatin genetika metabolismus patologie MeSH
- dediferenciace buněk MeSH
- embryonální vývoj genetika MeSH
- financování organizované MeSH
- genetické nemoci vrozené etiologie MeSH
- histony genetika MeSH
- klonování organismů škodlivé účinky MeSH
- lidé MeSH
- metylace DNA MeSH
- morula MeSH
- pluripotentní kmenové buňky MeSH
- přeprogramování buněk genetika MeSH
- savci MeSH
- stadium rýhování vajíčka metabolismus MeSH
- techniky jaderného přenosu normy škodlivé účinky MeSH
- totipotentní kmenové buňky MeSH
- vývojová regulace genové exprese MeSH
- zárodečné buňky metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- přehledy MeSH
Human embryonic stem cells (hES) are unique in their pluripotency and capacity for self-renewal. Therefore, we have studied the differences in the level of chromatin condensation in pluripotent and all-trans retinoic acid-differentiated hES cells. Nuclear patterns of the Oct4 (6p21.33) gene, responsible for hES cell pluripotency, the C-myc (8q24.21) gene, which controls cell cycle progression, and HP1 protein (heterochromatin protein 1) were investigated in these cells. Unlike differentiated hES cells, pluripotent hES cell populations were characterized by a high level of decondensation for the territories of both chromosomes 6 (HSA6) and 8 (HSA8). The Oct4 genes were located on greatly extended chromatin loops in pluripotent hES cell nuclei, outside their respective chromosome territories. However, this phenomenon was not observed for the Oct4 gene in differentiated hES cells, for the C-myc gene in the cell types studied. The high level of chromatin decondensation in hES cells also influenced the nuclear distribution of all the variants of HP1 protein, particularly HP1 alpha, which did not form distinct foci, as usually observed in most other cell types. Our experiments showed that unlike C-myc, the Oct4 gene and HP1 proteins undergo a high level of decondensation in hES cells. Therefore, these structures seem to be primarily responsible for hES cell pluripotency due to their accessibility to regulatory molecules. Differentiated hES cells were characterized by a significantly different nuclear arrangement of the structures studied.
- MeSH
- buněčná diferenciace genetika účinky léků MeSH
- buněčné jádro genetika ultrastruktura MeSH
- buněčné linie MeSH
- chromozomální proteiny, nehistonové genetika metabolismus MeSH
- embryonální kmenové buňky metabolismus ultrastruktura MeSH
- financování organizované MeSH
- lidé MeSH
- pluripotentní kmenové buňky metabolismus ultrastruktura MeSH
- restrukturace chromatinu MeSH
- signální transdukce genetika MeSH
- trans-aktivátory metabolismus účinky léků MeSH
- tretinoin MeSH
- vazebná místa genetika MeSH
- Check Tag
- lidé MeSH
Analysis of c-myb gene down-regulation in differentiating C212 cells revealed that in proliferating cells, c-myb expression is high and ceases as the proliferation rate decreases. However, a low level of c-myb mRNA was detected in confluent non-proliferating differentiating cells for an extended period of time before it declined to an undetectable level. The time course of c-myb gene silencing in differentiating cells correlated with exposition of phosphatidylserine (PS) on the cell surface. Moreover, the interaction of exposed PS with exogenously added annexin V perturbed PS-mediated cell signaling and transiently up-regulated the declining c-myb expression. We, therefore, suggest that cell surface-exposed PS, which plays a role in the process of myotube formation, is also involved in the down-regulation of c-myb expression.
- MeSH
- annexin A5 metabolismus MeSH
- buněčná diferenciace MeSH
- buněčné linie MeSH
- DNA primery MeSH
- financování organizované MeSH
- fluorescenční protilátková technika MeSH
- fosfatidylseriny metabolismus MeSH
- messenger RNA genetika MeSH
- myši MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- protoonkogenní proteiny c-myb genetika MeSH
- sekvence nukleotidů MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
The p53 protein can control cell cycle progression, programmed cell death, and differentiation of many cell types. Ectopic expression of p53 can resume capability of cell cycle arrest, differentiation, and apoptosis in various leukemic cell lines. In this work, we expressed human p53 protein in v-Myb-transformed chicken monoblasts. We found that even this protein possessing only 53% amino acid homology to its avian counterpart can significantly alter morphology and physiology of these cells causing the G2-phase cell cycle arrest and early monocytic differentiation. Our results document that the species-specific differences of the p53 molecules, promoters/enhancers, and co-factors in avian and human cells do not interfere with differentiation- and cell cycle arrest promoting capabilites of the p53 tumor suppressor even in the presence of functional v-Myb oncoprotein. The p53-induced differentiation and cell cycle arrest of v-Myb-transformed monoblasts are not associated with apoptosis suggesting that the p53-driven pathways controlling apoptosis and differentiation/proliferation are independent.
- MeSH
- apoptóza genetika MeSH
- buněčná diferenciace fyziologie genetika MeSH
- buněčný cyklus genetika MeSH
- financování organizované MeSH
- G2 fáze genetika MeSH
- inhibitory růstu fyziologie genetika MeSH
- kur domácí MeSH
- lidé MeSH
- monocyty cytologie MeSH
- nádorový supresorový protein p53 fyziologie genetika MeSH
- onkogenní proteiny v-myb genetika MeSH
- proliferace buněk MeSH
- signální transdukce genetika MeSH
- transfekce MeSH
- transformované buněčné linie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
Maturation of blood cells depends on dramatic changes of expression profiles of specific genes. Although these changes have been extensively studied, their functional outcomes often remain unclear. In this study, we explored the identity and function of an unknown protein that was greatly overexpressed in v-myb-transformed BM2 monoblasts undergoing differentiation to macrophage-like cells. We identified this protein as vimentin, the intermediate filament protein. We show that an increased level of vimentin protein results from activation of the vimentin gene promoter occurring in monoblastic cells induced to differentiate by multiple agents. Furthermore, our studies reveal that the vimentin gene promoter is stimulated by Myb and Jun proteins, the key transcriptional regulators of myeloid maturation. Silencing of vimentin gene expression using siRNA markedly suppressed the ability of BM2 cells to form macrophage polykaryons active in phagocytosis and producing reactive oxygen species. Taken together, these findings document that up-regulation of vimentin gene expression is important for formation of fully active macrophage-like cells and macrophage polykaryons.
- MeSH
- 2D gelová elektroforéza MeSH
- buněčná diferenciace MeSH
- fibroblasty MeSH
- financování organizované MeSH
- geny jun genetika MeSH
- hematopoéza genetika MeSH
- hmotnostní spektrometrie MeSH
- křepelky a křepelovití MeSH
- kur domácí MeSH
- makrofágy cytologie fyziologie MeSH
- monocyty cytologie fyziologie MeSH
- onkogenní proteiny v-myb genetika MeSH
- promotorové oblasti (genetika) imunologie MeSH
- protoonkogenní proteiny c-jun fyziologie MeSH
- regulace genové exprese MeSH
- tetradekanoylforbolacetát MeSH
- transformované buněčné linie MeSH
- transkripční faktory genetika MeSH
- upregulace MeSH
- vimentin fyziologie genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
The p53 tumor suppressor protein is a transcription factor that mediates the cell's response to various kinds of stress by preventing cell division and/or inducing apoptosis. p53 gene mutations have been detected in nearly 50% of human cancers. These gene aberrations are mostly missense point mutations located predominantly in the central DNA-binding domain. In addition to the classical inactivating mutations, there are also dominant-negative, gain-of-function, temperature-sensitive, and cold-sensitive, discriminating, superactive p53 mutations, and some mutations that do not inactivate p53 activity. Several approaches have been developed for detection and analyses of p53 mutations: first, immunochemical methods have been developed to detect p53 protein levels; second, molecular analyses targeting changes in DNA structure are utilized; and third, functional assays are used to explore the biological properties of the p53 protein. Functional analysis of separated alleles in yeast targets the transactivation capability of the p53 protein expressed in yeast cells. This method uses p53 mRNA isolated from cells and tissues to produce a p53 product by RT-PCR. This method has undergone continuous improvement and now serves as a powerful tool for distinguishing various functional types of p53 mutations. Understanding the exact impact of p53 mutation on its function is an important prerequisite for establishment of efficient anti-cancer therapies.
- MeSH
- buněčná diferenciace fyziologie účinky léků MeSH
- finanční podpora výzkumu jako téma MeSH
- forbolové estery farmakologie MeSH
- jaderné proteiny fyziologie genetika MeSH
- kur domácí MeSH
- monocyty cytologie fyziologie účinky léků MeSH
- myši MeSH
- onkogenní proteiny v-myb fyziologie MeSH
- trans-aktivátory fyziologie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
