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.

• Keywords
• Acute leukemia, B and T lymphocytes, Differentiation plasticity, Gene expression, Hematopoiesis, Lineage promiscuity,
• MeSH
• Leukemia, Biphenotypic, Acute genetics   immunology   pathology   MeSH
• B-Lymphocytes immunology   pathology   MeSH
• Cell Differentiation genetics   MeSH
• Cell Lineage genetics   MeSH
• Tissue Array Analysis MeSH
• Hematopoiesis genetics   MeSH
• Immunophenotyping MeSH
• Humans MeSH
• Neoplasm Proteins biosynthesis   MeSH
• Gene Expression Regulation, Leukemic MeSH
• T-Lymphocytes immunology   pathology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Neoplasm Proteins 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.

• Keywords
• 3T3-L1 cells, Adipocytes, Autophagy, Differentiation, Mitochondria, Preadipocytes,
• MeSH
• Adipogenesis drug effects   genetics   MeSH
• Asparagine pharmacology   MeSH
• Autophagy drug effects   genetics   MeSH
• Cell Differentiation drug effects   genetics   MeSH
• 3T3-L1 Cells MeSH
• Mitochondria drug effects   metabolism   MeSH
• Mice MeSH
• Adipocytes cytology   drug effects   MeSH
• Gene Expression Regulation, Developmental drug effects   genetics   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Asparagine 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
• Cell Differentiation drug effects   MeSH
• Cell Line MeSH
• Time Factors MeSH
• Macrophage Colony-Stimulating Factor pharmacology   MeSH
• Femur cytology   MeSH
• Mice, Inbred Strains MeSH
• Isoenzymes metabolism   MeSH
• Calcitriol pharmacology   MeSH
• Carbonic Anhydrase II antagonists & inhibitors   MeSH
• Cathepsin K antagonists & inhibitors   genetics   MeSH
• Culture Media, Conditioned pharmacology   MeSH
• Tartrate-Resistant Acid Phosphatase MeSH
• Acid Phosphatase metabolism   MeSH
• Humans MeSH
• RANK Ligand pharmacology   MeSH
• Macrophages cytology   MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• Prostatic Neoplasms metabolism   MeSH
• NF-kappa B antagonists & inhibitors   MeSH
• Osteoclasts drug effects   metabolism   MeSH
• Proto-Oncogene Proteins c-fos antagonists & inhibitors   MeSH
• Growth Differentiation Factor 15 pharmacology   MeSH
• Dose-Response Relationship, Drug MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Macrophage Colony-Stimulating Factor MeSH
• Isoenzymes MeSH
• Calcitriol MeSH
• Carbonic Anhydrase II MeSH
• Cathepsin K MeSH
• Culture Media, Conditioned MeSH
• Tartrate-Resistant Acid Phosphatase MeSH
• Acid Phosphatase MeSH
• RANK Ligand MeSH
• NF-kappa B MeSH
• Proto-Oncogene Proteins c-fos MeSH
• Growth Differentiation Factor 15 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 metabolism   MeSH
• Cell Differentiation * MeSH
• Cell Line MeSH
• DNA Primers MeSH
• Fluorescent Antibody Technique MeSH
• Phosphatidylserines metabolism   MeSH
• RNA, Messenger genetics   MeSH
• Mice MeSH
• Reverse Transcriptase Polymerase Chain Reaction MeSH
• Proto-Oncogene Proteins c-myb genetics   MeSH
• Base Sequence MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Annexin A5 MeSH
• DNA Primers MeSH
• Phosphatidylserines MeSH
• RNA, Messenger MeSH
• Proto-Oncogene Proteins c-myb 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
• Blastocyst metabolism   MeSH
• Cell Differentiation MeSH
• Cell Nucleus genetics   MeSH
• Chromatin genetics   metabolism   pathology   MeSH
• Cell Dedifferentiation MeSH
• Embryonic Development genetics   MeSH
• Genetic Diseases, Inborn etiology   MeSH
• Histones genetics   MeSH
• Cloning, Organism adverse effects   MeSH
• Humans MeSH
• DNA Methylation MeSH
• Morula metabolism   MeSH
• Pluripotent Stem Cells MeSH
• Cellular Reprogramming genetics   MeSH
• Mammals MeSH
• Cleavage Stage, Ovum metabolism   MeSH
• Nuclear Transfer Techniques adverse effects   standards   MeSH
• Totipotent Stem Cells MeSH
• Gene Expression Regulation, Developmental MeSH
• Germ Cells metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Chromatin MeSH
• Histones 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
• Cell Differentiation drug effects   genetics   MeSH
• Cell Nucleus genetics   ultrastructure   MeSH
• Cell Line MeSH
• Chromosomal Proteins, Non-Histone genetics   metabolism   MeSH
• Embryonic Stem Cells metabolism   ultrastructure   MeSH
• Chromobox Protein Homolog 5 MeSH
• Humans MeSH
• Pluripotent Stem Cells metabolism   ultrastructure   MeSH
• Chromatin Assembly and Disassembly * MeSH
• Signal Transduction genetics   MeSH
• Trans-Activators drug effects   metabolism   MeSH
• Tretinoin pharmacology   MeSH
• Binding Sites genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chromosomal Proteins, Non-Histone MeSH
• Chromobox Protein Homolog 5 MeSH
• Trans-Activators MeSH
• Tretinoin 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
• Apoptosis genetics   MeSH
• Cell Differentiation genetics   physiology   MeSH
• Cell Cycle genetics   MeSH
• G2 Phase genetics   MeSH
• Growth Inhibitors genetics   physiology   MeSH
• Chickens MeSH
• Humans MeSH
• Monocytes cytology   MeSH
• Tumor Suppressor Protein p53 genetics   physiology   MeSH
• Oncogene Proteins v-myb genetics   MeSH
• Cell Proliferation * MeSH
• Signal Transduction genetics   MeSH
• Transfection MeSH
• Cell Line, Transformed MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Growth Inhibitors MeSH
• Tumor Suppressor Protein p53 MeSH
• Oncogene Proteins v-myb 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
• Electrophoresis, Gel, Two-Dimensional MeSH
• Cell Differentiation * MeSH
• Fibroblasts MeSH
• Genes, jun genetics   MeSH
• Hematopoiesis genetics   MeSH
• Mass Spectrometry MeSH
• Quail MeSH
• Chickens MeSH
• Macrophages cytology   physiology   MeSH
• Monocytes cytology   physiology   MeSH
• Oncogene Proteins v-myb genetics   MeSH
• Promoter Regions, Genetic genetics   MeSH
• Proto-Oncogene Proteins c-jun physiology   MeSH
• Gene Expression Regulation MeSH
• Tetradecanoylphorbol Acetate MeSH
• Cell Line, Transformed MeSH
• Transcription Factors genetics   MeSH
• Up-Regulation MeSH
• Vimentin genetics   physiology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Oncogene Proteins v-myb MeSH
• Proto-Oncogene Proteins c-jun MeSH
• Tetradecanoylphorbol Acetate MeSH
• Transcription Factors MeSH
• Vimentin 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
• Genes, p53 * MeSH
• Humans MeSH
• Mutation MeSH
• DNA Mutational Analysis methods   MeSH
• Tumor Suppressor Protein p53 analysis   genetics   physiology   MeSH
• Saccharomyces cerevisiae genetics   MeSH
• Tissue Distribution MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Tumor Suppressor Protein p53 MeSH

CREB-binding protein (CBP) regulates gene expression by binding to certain components of basal transcription machinery and by histone acetylation. In addition, it integrates various cellular signaling pathways through binding to multiple transcription factors, including the Myb proteins. We report in this study that CBP can partially suppress function of the v-Myb oncoprotein in leukemic cells. Although originally described as an activator of v-Myb function, we show that CBP can also act as a v-Myb suppressor. Ectopic expression of murine CBP in v-Myb-transformed chicken monoblasts reduced transcriptional activation abilities of the v-Myb protein and increased sensitivity to differentiation inducers such as phorbol ester or trichostatin A. In addition, exogenous CBP affected morphology of differentiated cells derived from BM2 monoblasts. These results indicate that cellular context is an important factor determining whether CBP will activate or suppress the protein it targets.

• MeSH
• Cell Differentiation drug effects   physiology   MeSH
• Phagocytosis physiology   MeSH
• Phorbol Esters pharmacology   MeSH
• Nuclear Proteins genetics   physiology   MeSH
• Chickens MeSH
• Hydroxamic Acids pharmacology   MeSH
• Monocytes cytology   drug effects   physiology   MeSH
• Mice MeSH
• Oncogene Proteins v-myb physiology   MeSH
• CREB-Binding Protein MeSH
• Trans-Activators genetics   physiology   MeSH
• Cell Line, Transformed MeSH
• Cell Transformation, Viral physiology   MeSH
• Avian Myeloblastosis Virus physiology   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Crebbp protein, mouse MeSH Browser
• Phorbol Esters MeSH
• Nuclear Proteins MeSH
• Hydroxamic Acids MeSH
• Oncogene Proteins v-myb MeSH
• CREB-Binding Protein MeSH
• Trans-Activators MeSH
• trichostatin A MeSH Browser