Tom Curran

Prediction of central nervous system embryonal tumour outcome based on gene expression

Embryonal tumours of the central nervous system (CNS) represent a heterogeneous group of tumours about which little is known biologically, and whose diagnosis, on the basis of morphologic appearance alone, is controversial. Medulloblastomas, for example, are the most common malignant brain tumour of childhood, but their pathogenesis is unknown, their relationship to other embryonal CNS tumours is debated, and patients’ response to therapy is difficult to predict. We approached these problems by developing a classification system based on DNA microarray gene expression data derived from 99 patient samples. Here we demonstrate that medulloblastomas are molecularly distinct from other brain tumours including primitive neuroectodermal tumours (PNETs), atypical teratoid/rhabdoid tumours (AT/RTs) and malignant gliomas. Previously unrecognized evidence supporting the derivation of medulloblastomas from cerebellar granule cells through activation of the Sonic Hedgehog (SHH) pathway was also revealed. We show further that the clinical outcome of children with medulloblastomas is highly predictable on the basis of the gene expression profiles of their tumours at diagnosis.

A zinc finger-encoding gene coregulated with c-fos during growth and differentiation, and after cellular depolarization

Egr-1 is an early growth response gene that displays fos-like induction kinetics in fibroblasts, epithelial cells, and lymphocytes following mitogenic stimulation. Sequence analysis of murine Egr-1 cDNA predicts a protein with three DNA binding zinc fingers. The human EGR1 gene maps to chromosome 5 (bands 5q23-31). Egr-1 mRNA increases dramatically during cardiac and neural cell differentiation, and following membrane depolarization both in vitro and in vivo. Thus, Egr-1 and c-fos are often coregulated with strikingly similar kinetics. These results, in conjunction with the Egr-1 primary structure, suggest that Egr-1 may function as a transcriptional regulator in diverse biological processes.

Stimulus-transcription coupling in neurons: role of cellular immediate-early genes

Excitation of neurons results in a series of finely orchestrated responses that occur over a time frame ranging from fractions of a second to hours or days. In the short term, stimulation evokes an array of biochemical and biophysical events that represent the execution of the neurophysiological phenotype of a particular cell. These processes, which contribute to the overall behavior of a neural circuit, do not require de novo protein synthesis. In contrast, stimulation is also linked to long-term phenotypic changes that require alterations in gene expression. Thus, one or more mechanisms must exist that couple cell-surface stimuli to the transcriptional regulatory apparatus of the neuron. In this article James Morgan and Tom Curran detail a stimulus-transcription coupling cascade, involving the products of the proto-oncogenes, c-fos and c-jun, that operates in many cell types including neurons.

Redox activation of Fos-Jun DNA binding activity is mediated by a DNA repair enzyme

The DNA binding activity of Fos and Jun is regulated in vitro by a post‐translational mechanism involving reduction‐oxidation. Redox regulation occurs through a conserved cysteine residue located in the DNA binding domain of Fos and Jun. Reduction of this residue by chemical reducing agents or by a ubiquitous nuclear redox factor (Ref‐1) recently purified from Hela cells, stimulates AP‐1 DNA binding activity in vitro, whereas oxidation or chemical modification of the cysteine has an inhibitory effect on DNA binding activity. Here we demonstrate that the protein product of the ref‐1 gene stimulates the DNA binding activity of Fos‐Jun heterodimers, Jun‐Jun homodimers and Hela cell AP‐1 proteins as well as that of several other transcription factors including NF‐kappa B, Myb and members of the ATF/CREB family. Furthermore, immunodepletion analysis indicates that Ref‐1 is the major AP‐1 redox activity in Hela nuclear extracts. Interestingly, Ref‐1 is a bifunctional protein; it also possesses an apurinic/apyrimidinic (AP) endonuclease DNA repair activity. However, the redox and DNA repair activities of Ref‐1 can, in part, be distinguished biochemically. This study suggests a novel link between transcription factor regulation, oxidative signalling and DNA repair processes in higher eukaryotes.

Reelin Is a Ligand for Lipoprotein Receptors

A signaling pathway involving the extracellular protein Reelin and the intracellular adaptor protein Disabled-1 (Dab1) controls cell positioning during mammalian brain development. Here, we demonstrate that Reelin binds directly to lipoprotein receptors, preferably the very low-density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2). Binding requires calcium, and it is inhibited in the presence of apoE. Furthermore, the CR-50 monoclonal antibody, which inhibits Reelin function, blocks the association of Reelin with VLDLR. After binding to VLDLR on the cell surface, Reelin is internalized into vesicles. In dissociated neurons, apoE reduces the level of Reelin-induced tyrosine phosphorylation of Dab1. These data suggest that Reelin directs neuronal migration by binding to VLDLR and ApoER2.

Scrambler and yotari disrupt the disabled gene and produce a reeler- like phenotype in mice

Formation of the mammalian brain requires choreographed migration of neurons to generate highly ordered laminar structures such as those in the cortices of the forebrain and the cerebellum. These processes are severely disrupted by mutations in reelin which cause widespread misplacement of neurons and associated ataxia in reeler mice,. Reelin is a large extracellular protein secreted by pioneer neurons that coordinates cell positioning during neurodevelopment,. Two new autosomal recessive mouse mutations, scrambler and yotari have been described that exhibit a phenotype identical to reeler. Here we report that scrambler and yotari arise from mutations in mdab1 (ref. 12), a mouse gene related to the Drosophila gene disabled ( dab ). Both scrambler and yotari mice express mutated forms of mdab1 messenger RNA and little or no mDab1 protein. mDab1 is a phosphoprotein that appears to function as an intracellular adaptor in protein kinase pathways. Expression analysis indicates that mdab1 is expressed in neuronal populations exposed to Reelin. The similar phenotypes of reeler, scrambler, yotari and mdab1 null mice indicate that Reelin and mDab1 function as signalling molecules that regulate cell positioning in the developing brain.

Identification and characterization of Ref-1, a nuclear protein that facilitates AP-1 DNA-binding activity.

Fos and Jun form a heterodimeric complex that regulates gene transcription by binding to the activator protein‐1 (AP‐1) DNA sequence motif. Previously, we demonstrated that the DNA‐binding activity of Fos and Jun is regulated in vitro by a novel redox (reduction‐oxidation) mechanism. Reduction of a conserved cysteine (cys) residue in the DNA‐binding domains of Fos and Jun by chemical reducing agents or by a nuclear redox factor stimulates DNA‐binding activity. Here, we describe purification and characterization of a 37 kDa protein (Ref‐1) corresponding to the redox factor. Although Ref‐1 does not bind to the AP‐1 site in association with Fos and Jun, it partially copurifies with a subset of AP‐1 proteins. Purified Ref‐1 protein stimulates AP‐1 DNA‐binding activity through the conserved Cys residues in Fos and Jun, but it does not alter the DNA‐binding specificity of Fos and Jun. Ref‐1 may represent a novel redox component of the signal transduction processes that regulate eukaryotic gene expression.

The genetic landscape of the childhood cancer medulloblastoma

Genomic analysis of a childhood cancer reveals markedly fewer mutations than what is typically seen in adult cancers. Medulloblastoma (MB) is the most common malignant brain tumor of children. To identify the genetic alterations in this tumor type, we searched for copy number alterations using high-density microarrays and sequenced all known protein-coding genes and microRNA genes using Sanger sequencing in a set of 22 MBs. We found that, on average, each tumor had 11 gene alterations, fewer by a factor of 5 to 10 than in the adult solid tumors that have been sequenced to date. In addition to alterations in the Hedgehog and Wnt pathways, our analysis led to the discovery of genes not previously known to be altered in MBs. Most notably, inactivating mutations of the histone-lysine N-methyltransferase genes MLL2 or MLL3 were identified in 16% of MB patients. These results demonstrate key differences between the genetic landscapes of adult and childhood cancers, highlight dysregulation of developmental pathways as an important mechanism underlying MBs, and identify a role for a specific type of histone methylation in human tumorigenesis.

Genomics Identifies Medulloblastoma Subgroups That Are Enriched for Specific Genetic Alterations

PURPOSE Traditional genetic approaches to identify gene mutations in cancer are expensive and laborious. Nonetheless, if we are to avoid rejecting effective molecular targeted therapies, we must test these drugs in patients whose tumors harbor mutations in the drug target. We hypothesized that gene expression profiling might be a more rapid and cost-effective method of identifying tumors that contain specific genetic abnormalities. MATERIALS AND METHODS Gene expression profiles of 46 samples of medulloblastoma were generated using the U133av2 Affymetrix oligonucleotide array and validated using real-time reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. Genetic abnormalities were confirmed using fluorescence in situ hybridization (FISH) and direct sequencing. RESULTS Unsupervised analysis of gene expression profiles partitioned medulloblastomas into five distinct subgroups (subgroups A to E). Gene expression signatures that distinguished these subgroups predicted the presence of key molecular alterations that we subsequently confirmed by gene sequence analysis and FISH. Subgroup-specific abnormalities included mutations in the Wingless (WNT) pathway and deletion of chromosome 6 (subgroup B) and mutations in the Sonic Hedgehog (SHH) pathway (subgroup D). Real-time RT-PCR analysis of gene expression profiles was then used to predict accurately the presence of mutations in the WNT and SHH pathways in a separate group of 31 medulloblastomas. CONCLUSION Genome-wide expression profiles can partition large tumor cohorts into subgroups that are enriched for specific genetic alterations. This approach may assist ultimately in the selection of patients for future clinical trials of molecular targeted therapies.

Reelin Is a Secreted Glycoprotein Recognized by the CR-50 Monoclonal Antibody

The neurological mouse mutant strain reeler displays abnormal laminar organization of several brain structures as a consequence of a defect in cell migration during neurodevelopment. This phenotype is a result of the disruption of reelin, a gene encoding a protein that has several structural characteristics of extracellular matrix proteins. To understand the molecular basis of the action of Reelin on neuronal migration, we constructed a full-lengthreelin clone and used it to direct Reelin expression. Here, we demonstrate that Reelin is a secreted glycoprotein and that a highly charged C-terminal region is essential for secretion. In addition, we demonstrate that an amino acid sequence present in the N-terminal region of Reelin contains an epitope that is recognized by the CR-50 monoclonal antibody. CR-50 was raised against an antigen expressed in normal mouse brain that is absent in reelermice. The interaction of CR-50 with its epitope leads to the disruption of neural cell aggregation in vitro. Here, we used CR-50 to precipitate Reelin from reticulocyte extracts programmed withreelin mRNA, from cells transfected withreelin clones, and from cerebellar explants. Thereelin gene product seems to function as an instructive signal in the regulation of neuronal migration.