Simon M. Lin

Transcriptional profiling of the Sonic hedgehog response: A critical role for N-myc in proliferation of neuronal precursors

Cerebellar granule cells are the most abundant neurons in the brain, and granule cell precursors (GCPs) are a common target of transformation in the pediatric brain tumor medulloblastoma. Proliferation of GCPs is regulated by the secreted signaling molecule Sonic hedgehog (Shh), but the mechanisms by which Shh controls proliferation of GCPs remain inadequately understood. We used DNA microarrays to identify targets of Shh in these cells and found that Shh activates a program of transcription that promotes cell cycle entry and DNA replication. Among the genes most robustly induced by Shh are cyclin D1 and N-myc. N-myc transcription is induced in the presence of the protein synthesis inhibitor cycloheximide, so it appears to be a direct target of Shh. Retroviral transduction of N-myc into GCPs induces expression of cyclin D1, E2F1, and E2F2, and promotes proliferation. Moreover, dominant-negative N-myc substantially reduces Shh-induced proliferation, indicating that N-myc is required for the Shh response. Finally, cyclin D1 and N-myc are overexpressed in murine medulloblastoma. These findings suggest that cyclin D1 and N-myc are important mediators of Shh-induced proliferation and tumorigenesis.

Loss of patched and disruption of granule cell development in a pre-neoplastic stage of medulloblastoma.

Medulloblastoma is the most common malignant brain tumor in children. It is thought to result from the transformation of granule cell precursors (GCPs) in the developing cerebellum, but little is known about the early stages of the disease. Here, we identify a pre-neoplastic stage of medulloblastoma in patched heterozygous mice, a model of the human disease. We show that pre-neoplastic cells are present in the majority of patched mutants, although only 16% of these mice develop tumors. Pre-neoplastic cells, like tumor cells, exhibit activation of the Sonic hedgehog pathway and constitutive proliferation. Importantly, they also lack expression of the wild-type patched allele, suggesting that loss of patched is an early event in tumorigenesis. Although pre-neoplastic cells resemble GCPs and tumor cells in many respects, they have a distinct molecular signature. Genes that mark the pre-neoplastic stage include regulators of migration, apoptosis and differentiation, processes crucial for normal development but previously unrecognized for their role in medulloblastoma. The identification and molecular characterization of pre-neoplastic cells provides insight into the early steps in medulloblastoma formation, and may yield important markers for early detection and therapy of this disease.

Methods of Microarray Data Analysis

Methods of microarray data analysis III , Methods of microarray data analysis III , کتابخانه دیجیتال جندی شاپور اهواز

Heterogeneity of Flt3-Expressing Multipotent Progenitors in Mouse Bone Marrow

Mechanisms of lymphoid and myeloid lineage choice by hemopoietic stem cells remain unclear. In this study we show that the multipotent progenitor (MPP) population, which is immediately downstream of hemopoietic stem cells, is heterogeneous and can be subdivided in terms of VCAM-1 expression. VCAM-1+ MPPs were fully capable of differentiating into both lymphoid and myeloid lineages. In contrast, VCAM-1− MPPs gave rise to lymphocytes predominately in vivo. T and B cell development from VCAM-1− MPPs was 1 wk faster than that from VCAM-1+ MPPs. Furthermore, VCAM-1+ MPPs gave rise to common myeloid progenitors and VCAM-1− MPPs in vivo, indicating that VCAM-1− MPPs are progenies of VCAM-1+ MPPs. VCAM-1− MPPs, in turn, developed into lymphoid lineage-restricted common lymphoid progenitors. These results establish a hierarchy of developmental relationship between MPP subsets and lymphoid and myeloid progenitors. In addition, VCAM-1+ MPPs may represent the branching point between the lymphoid and myeloid lineages.

Impaired cell adhesion and apoptosis in a novel CLN9 Batten disease variant

We describe the ninth variant of neuronal ceroid lipofuscinosis (NCL) or Batten disease, due to defects in a putative new gene, CLN9. We therefore refer to the new variant as CLN9‐deficient. Two Serbian sisters and two German brothers are described. Their clinical history is characteristic for juvenile NCL. They show similar gene expression patterns. The existence of this variant is supported by the presence of curvilinear inclusions, fingerprint profiles, and granular osmiophilic deposits in neurons, lymphocytes, and conjunctival cells. Enzyme screening and sequencing of the coding regions of other NCL genes was negative. CLN9‐deficient cells have a distinctive phenotype. They have rounded cell bodies, have prominent nucleoli, attach poorly to the culture dish, and are sensitive to apoptosis but have increased growth rates. Gene expression of proteins involved in cell adhesion and apoptosis is altered in these cells. Sphingolipid metabolism is also perturbed. They have decreased levels of ceramide, sphingomyelin, lactosylceramide, ceramide trihexoside, and globoside and increased activity of serine palmitoyl transferase. Ann Neurol 2004

RNA-binding proteins to assess gene expression states of co-cultivated cells in response to tumor cells

BackgroundTumors and complex tissues consist of mixtures of communicating cells that differ significantly in their gene expression status. In order to understand how different cell types influence one anothers gene expression, it will be necessary to monitor the mRNA profiles of each cell type independently and to dissect the mechanisms that regulate their gene expression outcomes.ResultsIn order to approach these questions, we have used RNA-binding proteins such as ELAV/Hu, poly (A) binding protein (PABP) and cap-binding protein (eIF-4E) as reporters of gene expression. Here we demonstrate that the epitope-tagged RNA binding protein, PABP, expressed separately in tumor cells and endothelial cells can be used to discriminate their respective mRNA targets from mixtures of these cells without significant mRNA reassortment or exchange. Moreover, using this approach we identify a set of endothelial genes that respond to the presence of co-cultured breast tumor cells.ConclusionRNA-binding proteins can be used as reporters to elucidate components of operational mRNA networks and operons involved in regulating cell-type specific gene expression in tissues and tumors.

Molecular Profile and Partial Functional Analysis of Novel Endothelial Cell‐Derived Growth Factors that Regulate Hematopoiesis

Recent progress has been made in the identification of the osteoblastic cellular niche for hematopoietic stem cells (HSCs) within the bone marrow (BM). Attempts to identify the soluble factors that regulate HSC self‐renewal have been less successful. We have demonstrated that primary human brain endothelial cells (HUBECs) support the ex vivo amplification of primitive human BM and cord blood cells capable of repopulating non‐obese diabetic/severe combined immunodeficient repopulating (SCID) mice (SCID repopulating cells [SRCs]). In this study, we sought to characterize the soluble hematopoietic activity produced by HUBECs and to identify the growth factors secreted by HUBECs that contribute to this HSC‐supportive effect. Extended noncontact HUBEC cultures supported an eight‐fold increase in SRCs when combined with thrombopoietin, stem cell factor, and Flt‐3 ligand compared with input CD34+ cells or cytokines alone. Gene expression analysis of HUBEC biological replicates identified 65 differentially expressed, nonredundant transcripts without annotated hematopoietic activity. Gene ontology studies of the HUBEC transcriptome revealed a high concentration of genes encoding extracellular proteins with cell‐cell signaling function. Functional analyses demonstrated that adrenomedullin, a vasodilatory hormone, synergized with stem cell factor and Flt‐3 ligand to induce the proliferation of primitive human CD34+CD38−lin− cells and promoted the expansion of CD34+ progenitors in culture. These data demonstrate the potential of primary HUBECs as a reservoir for the discovery of novel secreted proteins that regulate human hematopoiesis.

MedlineR: an open source library in R for Medline literature data mining

SUMMARY We describe an open source library written in the R programming language for Medline literature data mining. This MedlineR library includes programs to query Medline through the NCBI PubMed database; to construct the co-occurrence matrix; and to visualize the network topology of query terms. The open source nature of this library allows users to extend it freely in the statistical programming language of R. To demonstrate its utility, we have built an application to analyze term-association by using only 10 lines of code. We provide MedlineR as a library foundation for bioinformaticians and statisticians to build more sophisticated literature data mining applications. AVAILABILITY The library is available from http://dbsr.duke.edu/pub/MedlineR.

Applications of Tree-Maps to hierarchical biological data

UNLABELLED A brief overview of Tree-Maps provides the basis for understanding two new implementations of Tree-Map methods. TreeMapClusterView provides a new way to view microarray gene expression data, and GenePlacer provides a view of gene ontology annotation data. We also discuss the benefits of Tree-Maps to visualize complex hierarchies in functional genomics. AVAILABILITY Java class files are freely available at http://mendel.mc.duke.edu/bioinformatics/ CONTACT mccon012@mc.duke.edu SUPPLEMENTARY INFORMATION For more information on TreeMapClusterView (see http://mendel.mc.duke.edu/bioinformatics/software/boxclusterview/), and http://mendel.mc.duke.edu/bioinformatics/software/geneplacer/).

Critical Assessment of Microarray Data Analysis: the 2001 challenge

UNLABELLED We initiated the Critical Assessment of Microarray Data Analysis (CAMDA) conference to stimulate and evaluate the development of advanced data analysis techniques for microarrays. A standard data set has been released for this data analysis challenge. The goal of this challenge is to assess the performance of different analytical methods and at the same time to determine how such methods should be evaluated. We hope this effort will catalyze the discussion of microarray data analysis among the research community of biologists, statisticians, mathematicians, and computer scientists. AVAILABILITY http://camda.duke.edu.