Východisko: Expresní microarrays představují vhodnou technologi i pro studium patologických procesů na genomické úrovni. Pro identifikace odlišně exprimovaných genů u pacientů s CML jsme použili plastikové microarrays, které kombinují hlavní přednosti nylonových membrán a skleněných microarrays. Pacienti a Metody. Leukocyty byly izolovány z periferní krve 6 pacientů s chronickou myeloidní leukemií v době stanovení diagnózy. U všech pacientů byl detekován BCR/ABL fúzní gen. Pro detekci expresních profilů byly použity Atlas Human Plastic 8K Microarrays (Clontech) s 8 327 genovými sondami. mRNA byla izolována z 45µg celkové RNA pomocí magnetických částic. Výsledky. Většina genů u pacientů vykazovala stejnou expresi jako kontrolní vzorek. U některých genů bylo možné detekovat podobné změny v expresi u všech (či většiny) pacientů např. zvýšená exprese: lactotransferrin, proteoglycan 2, elastase 2, MMP 9, defensin alpha 1,3,4, peptidoglycan recognition protein, cathepsin G, myeloperoxidase, proteinase 3; snížená exprese: CD68 antigen, cathepsin B, H factor, integrin-alpha X, interleukin 8, preB cell colony enhancing factor. Závěr. Naše studie odhalila variabilitu v expresi mnoha genů, ale i přesto jsme identifikovali skupinu genů s podobnými změnami v aktivitě. Tyto geny by mohly být asociovány s rozvojem onemocnění a budou podrobněji studovány.
Background: Expression microarrays provide a powerful technology for studying disease processes on a genomic scale. We used plastic microarrays for identification of differentially expressed genes in chronic myeloid leukemia patients. Plastic arrays combine the best properties of glass and nylon arrays.Patients and Methods: Leukocytes were isolated from peripheral blood of 6 CML patients at diagnosis. All patients were BCR/ABL fusion gene positive. For gene expression profiling we used Atlas Human Plastic 8K Microarrays (Clontech) with 8 327 gene probes. mRNA was isolated from 45µg of total RNA using magnetic bead method. Results: Majority of the patient genes showed the same transcription activity as in the control sample. In a few genes it was possible to observe similar significant changes of gene expression in all (most) patients e.g.up-regulation: lactotransferrin, proteoglycan 2, elastase 2, MMP9, defensin alpha 1,3,4, peptidoglycan recognition protein, cathepsin G, myeloperoxidase, proteinase 3; down-regulation: CD68 antigen, cathepsin B, H factor, integrin-alpha X, interleukin 8, preB cell colony enhancing factor. Conclusions: Although the study revealed variability of gene expression in many genes, we identified a number of genes with the similar expression regulation. The genes may be associated with the disease process and will be analysed in detail.
- MeSH
- Leukemia, Myelogenous, Chronic, BCR-ABL Positive diagnosis etiology genetics MeSH
- Protein Array Analysis methods instrumentation utilization MeSH
- Gene Expression genetics drug effects MeSH
- Financing, Organized MeSH
- Genetic Research MeSH
- Hematologic Neoplasms diagnosis etiology genetics MeSH
- Interleukins genetics immunology isolation & purification MeSH
- Cathepsins genetics immunology isolation & purification MeSH
- Medical Oncology methods trends MeSH
- Humans MeSH
- RNA diagnostic use genetics isolation & purification MeSH
- Oligonucleotide Array Sequence Analysis methods utilization MeSH
- Check Tag
- Humans MeSH
Immunoassays represent valuable and broadly used techniques for detection and quantification of proteins. Thanks to their high sensitivity, such techniques are powerful for analyzing growth factors, trophic factors, angiogenic factors, hormones, cytokines, chemokines, soluble receptors, and other proteins which play key roles in intercellular communication and operate as potent regulators of stem cell survival, proliferation, differentiation, or cell death. Multiplex immunological assays, in contrast to ELISA, offer simultaneous quantification of tens of proteins across multiple samples, and have been developed to save time, costs, and sample volumes. Among them, planar antibody microarrays and xMAP(®) bead-based assays have become particularly popular for characterization of proteins secreted by stem cells, as they are relatively easy, highly accurate, multiplex to a high degree and a broad spectrum of analytes can be measured. Here, we describe protocols for multiplex quantification of secreted proteins using Quantibody(®) microarrays (RayBiotech) and xMAP(®) assays (Luminex and its partners).
- MeSH
- Cell Culture Techniques MeSH
- Protein Array Analysis methods MeSH
- Cytokines metabolism MeSH
- Immunoassay methods MeSH
- Stem Cells physiology MeSH
- Culture Media, Conditioned metabolism MeSH
- Cells, Cultured MeSH
- Cell Communication * MeSH
- Intercellular Signaling Peptides and Proteins metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
A new clonal cell line, EM-G3, was derived from a primary lesion of human infiltrating ductal breast carcinoma. The line consisted of cuboidal cells with occasional appearance of more differentiated branched cells apparently involved in cell-to-cell communication. The EM-G3 cells, population doubling time 34 h, are dependent on the epidermal growth factor. Multicolor fluorescence in situ hybridization (mFISH) analysis demonstrated a stable diploid genome with several genetic changes. Immunocytochemical analysis of EM-G3 in vitro revealed positivity for keratins (K) K5, K14, K18, nuclear protein p63, epithelial membrane antigen (EMA) and other proteins indicative of a pattern of mammary epithelium bipotent progenitors. Detection of integrins alpha-6, beta-1, and protein CD44 by cDNA array also pointed to the character of basal/stem cells. In contrast, dominant cells in the human original tumor showed the luminal character (K18+, K19+, K5-, K14-, and p63-). However, cells with the immunocytochemical profile similar to that of cultured EM-G3 cells were found in minor clusters in the patient's tumor sections. The EM-G3 cells formed limited tumors in nu/nu mice. The cells in mouse tumors were organized in primitive ductal-like structures consisting of 1-3 large central luminal-like cells (EMA+) surrounded by peripheral myoepithelial-like cells (p63+/EMA-). The large central cells gradually disintegrated, forming a pseudolumen. Apparently, EM-G3 cells are able to partially differentiate in vivo as well as in vitro. Our results indicate that EM-G3 cells were derived from a premalignant population of common progenitors of luminal and myoepithelial cells that were immortalized in an early stage of tumorigenesis.
- MeSH
- Cell Differentiation MeSH
- Financing, Organized MeSH
- In Situ Hybridization, Fluorescence MeSH
- Immunohistochemistry MeSH
- Karyotyping MeSH
- Humans MeSH
- Mutation MeSH
- Mice, Nude MeSH
- Mice MeSH
- Cell Line, Tumor MeSH
- Neoplastic Stem Cells pathology MeSH
- Breast Neoplasms genetics pathology MeSH
- Oligonucleotide Array Sequence Analysis MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Female MeSH
- Animals MeSH
OBJECTIVE: Hematopoietic stem cells (enriched in fraction of CD34+ cells) have the ability to regenerate hematopoiesis in all of its lineages, and this potential is clinically used in transplanting bone marrow or peripheral blood stem cells. Our objective was to assemble a suitable method for evaluating gene expression in enriched populations of hematopoietic stem cells. We compared biologic properties of cells cultured ex vivo obtained using two different ways of immunomagnetic separation (positive selection of CD34+ cells and negative selection of Lin- cells) by means of a cDNA microarray technique. METHODS: CD34+ and Lin- cells were enriched from peripheral blood stem cell (PBSCs) grafts of patients with non-Hodgkin's lymphoma. Isolated cells were in the presence of cytokine PBSCs, Flt-3 ligand, interleukin-3, interleukin-6, and granulocyte colony-stimulating factor. At days 0, 4, 6, 8, 10, 12, and 14 cells were harvested and analyzed by cDNA microarrays. Total cell expansion, CD34+, colony-forming unit for granulocyte-macrophage and megakaryocytes expansion, vitality, and phenotype of cells were also analyzed. RESULTS: cDNA microarray analysis of cultured hematopoietic cells proved equivalence of the two enrichment methods for PBSC samples and helped us characterize differentiating cells cultured ex vivo. CONCLUSION: Our methodologic approach is helpful in characterizing cultured hematopoietic cells cultured ex vivo, but it is also suitable for more general purposes. Equivalence of CD34+ and Lin- selection methods from PBSC samples proved by cDNA microarray may have an implication for graft manipulation in an experimental setting of hematopoietic transplantation. Total cell expansion and colony formation and phenotype from CD34+ selected and from Lin- samples were comparable.
Neural crest (NC) is a transient embryonic tissue, whose cells are motile and multipotent until they reach their destination and differentiate according to microenvironmental cues into a variety of cell types. However, a subpopulation of these cells remains multipotent. They were found, among other locations, in a bulge of adult murine whisker follicle and were designated epidermal neural crest stem cells (EPI-NCSCs). The aim of this work is to ascertain whether the EPI-NCSCs could be isolated from human hair follicles as well. Due to their exceptional properties, they could represent potential candidates for stem cell therapy. The presented work focuses on the isolation and characterization of EPI-NCSCs from human skin. We obtained a population of cells that expressed markers of NC, NC progeny and general stem cell markers. After prolonged cultivation, the subpopulation of cells spontaneously differentiated into some of NC derivatives, i.e. neurons, smooth muscle cells and Schwann cell progenitors. Targeted differentiation with neuregulin 1 highly increased the number of Schwann cells in the culture. Human EPI-NCSCs could also grow under non-adherent conditions and form 3-dimensional spheres. Microarray analysis was performed and gene profile of human EPI-NCSCs was compared with the list of key genes of murine EPI-NCSCs and the list of genes up-regulated in newly induced NC cells. This revealed 94% and 88% similarity, respectively. All presented results strongly support the NCSC identity and multipotency of isolated human cells. These cells could thus be used in regenerative medicine, especially because of the easy accessibility of donor tissue.
- MeSH
- Cell Differentiation MeSH
- Neural Crest cytology metabolism MeSH
- Financing, Organized MeSH
- Immunohistochemistry MeSH
- Stem Cells cytology metabolism MeSH
- Cells, Cultured MeSH
- Humans MeSH
- Multipotent Stem Cells cytology metabolism MeSH
- Mice MeSH
- Neurons MeSH
- Reverse Transcriptase Polymerase Chain Reaction MeSH
- Schwann Cells MeSH
- Oligonucleotide Array Sequence Analysis MeSH
- Gene Expression Profiling MeSH
- Stem Cell Transplantation MeSH
- Vibrissae MeSH
- Hair Follicle cytology MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
MicroRNAs (miRNAs), important regulators of cellular processes, show specific expression signatures in different blood cell lineages and stages of hematopoietic stem cell (HSC) differentiation, indicating their role in the control of hematopoiesis. Because neonatal blood displays various features of immaturity, we might expect differential miRNA regulation. Herein, we determined miRNA expression profiles of umbilical cord blood (UCB) cell lineages and compared them to those of bone marrow (BM) and peripheral blood (PB) cell counterparts. Further, we determined mRNA expression profiles using whole-genome microarrays. An approach combining bioinformatic prediction of miRNA targets with mRNA expression profiling was used to search for putative targets of miRNAs with potential functions in UCB. We pointed out several differentially expressed miRNAs and associated their expression with the target transcript levels. miR-148a expression was suppressed in HSCs and its level inversely correlated with the previously verified target, DNA methyltransferase 3B, suggesting dependence of de novo DNA methylation in HSCs on miR-148a. Prolonged cell survival of UCB HSCs may be associated with low expression of miR-143 and miR-145 and up-regulation of their downstream targets (high expression of c-MYC and miR-17-92 and following repression of TGFBR2). In HSCs, we monitored significant up-regulation of eight miRNAs, which were previously verified as regulators of HOX genes. Further, miR-146b may be associated with immaturity of neonatal immune system because it is strongly up-regulated in UCB granulocytes and T lymphocytes compared to PB cell counterparts. Comparative analysis revealed 13 miRNAs significantly altered between UCB and BM CD34(+) cells. In UCB CD34(+) cells, we monitored up-regulation of miR-520h, promoting differentiation of HSCs into progenitor cells, and reduction of miR-214, whose expression might support HSC survival. In conclusion, UCB cells show specific miRNA expression patterns, indicating different regulation in these cells.
- MeSH
- Antigens, CD34 metabolism MeSH
- Cell Lineage genetics MeSH
- Adult MeSH
- Fetal Blood cytology metabolism MeSH
- Hematopoietic Stem Cells metabolism MeSH
- Blood Cells metabolism physiology MeSH
- Middle Aged MeSH
- Humans MeSH
- MicroRNAs genetics metabolism MeSH
- Infant, Newborn MeSH
- Oligonucleotide Array Sequence Analysis MeSH
- Aged MeSH
- Cluster Analysis MeSH
- Gene Expression Profiling MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Infant, Newborn MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Comparative Study MeSH
- Validation Study MeSH
Serrated adenocarcinoma (SAC) and colorectal carcinomas showing histological and molecular features of high-level of microsatellite instability (hmMSI-H) are both end points of the serrated pathway of colorectal carcinogenesis. Despite common features (right-sided location, CpG island methylation phenotype and BRAF mutation) there are no studies comparing the microRNA (miRNA) expression profiles in SACs and hmMSI-H. The microtranscriptome from 12 SACs and 8 hmMSI-H were analysed using Affymetrix GeneChip miRNA 3.0 arrays and differentially enriched functions involving immune response were observed from this comparison. miR-181a-2* was found significantly more expressed in hmMSI-H than in SAC and higher expression of this miRNA in microsatellite unstable colorectal cancer were corroborated by Real-Time PCR in an extended series (61 SAC, 21 hmMSI-H). An analysis of genes possibly regulated by miR-181a-2* was carried out and, amongst these, an inverse correlation of NAMPT with miR-181a-2* expression was observed, whereas, for TRAF1 and SALL1, additional regulation mechanisms involving CpG island methylation were observed. miR-181a-2* is associated with particular histological and molecular features of colorectal carcinomas within the serrated pathological pathway and might play a role in the immune responses of microsatellite instability carcinomas.
- MeSH
- CpG Islands MeSH
- Cytokines genetics metabolism MeSH
- TNF Receptor-Associated Factor 1 genetics metabolism MeSH
- Gene Ontology MeSH
- Carcinoma genetics metabolism physiopathology MeSH
- Colorectal Neoplasms genetics metabolism physiopathology MeSH
- Humans MeSH
- DNA Methylation MeSH
- MicroRNAs genetics metabolism MeSH
- Microsatellite Instability * MeSH
- Cell Line, Tumor MeSH
- Nicotinamide Phosphoribosyltransferase genetics metabolism MeSH
- Gene Expression Regulation, Neoplastic genetics MeSH
- Oligonucleotide Array Sequence Analysis MeSH
- Aged MeSH
- Transcription Factors genetics metabolism MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Spontaneous variation in appearance was studied in bacterial colonies of Serratia marcescens F morphotype(1): (i) A defined array of non-heritable phenotype variations does appear repeatedly; (ii) The presence of colonies of different bacterial species will narrow the variability toward the typical F appearance, as if such an added environmental factor curtailed the capacity of colony morphospace; (iii) Similarly the morphospace becomes reduced by random mutations leading to new, heritable morphotypes-at the same time opening a new array of variations typical for the mutant but not accessible directly from the original F morphospace. Results are discussed in context with biphasic model of early morphogenesis applicable to all multicellular bodies.
- Publication type
- Journal Article MeSH
- MeSH
- Blast Crisis drug therapy genetics pathology MeSH
- Leukemia, Myelogenous, Chronic, BCR-ABL Positive drug therapy genetics pathology MeSH
- Cytarabine administration & dosage MeSH
- Adult MeSH
- Granulocyte Colony-Stimulating Factor administration & dosage MeSH
- Financing, Organized MeSH
- Humans MeSH
- Mitoxantrone administration & dosage MeSH
- Multigene Family genetics MeSH
- Antineoplastic Combined Chemotherapy Protocols administration & dosage MeSH
- Gene Expression Regulation, Leukemic drug effects MeSH
- Oligonucleotide Array Sequence Analysis methods MeSH
- Gene Expression Profiling methods MeSH
- Check Tag
- Adult MeSH
- Humans MeSH
- Male MeSH
- Publication type
- Case Reports MeSH
