John D. Norton

Early‐response gene signalling is induced by angiogenic oligosaccharides of hyaluronan in endothelial cells. Inhibition by non‐angiogenic, high‐molecular‐weight hyaluronan

The degradation products of hyaluronan are known to stimulate endothelial‐cell proliferation and to promote neovascularization associated with angiogenesis, whilst native high‐molecular‐weight hyaluronan is inhibitory to these processes. To investigate the cellular signalling pathways coupled to hyaluronan‐induced responses in angiogenesis, we have analyzed early‐response gene expression in vitro, in cultured bovine aortic endothelial cells. Angiogenic oligosaccharides of hyaluronan induced rapid transient up‐regulation of the immediate early genes c‐fos, c‐jun, jun‐B, Krox‐20 and Krox‐24. In contrast, native hyaluronan when used alone failed to elicit a significant change in expression of any of the genes tested, and when used in combination with angiogenic oligosaccharides of hyaluronan, gave a dose‐dependent inhibition of induced gene expression. However, prior addition of angiogenic hyaluronan, as little as one minute before addition of high‐molecular‐weight hyaluronan, abrogated this inhibition, suggesting that positive or negative responses associated with hyaluronan signalling are integrated at a very early stage following receptor binding. Conversely, prior addition of high‐molecular‐weight hyaluronan led to an irreversible block in gene expression and proliferative response. These data are consistent with native hyaluronan antagonizing the angiogenic response in part by blocking a signalling cascade at or immediately following ligand‐receptor interaction. Finally, we demonstrated that chronic exposure to oligosaccharides of hyaluronan is essential for cell proliferation, indicating that short‐term immediate early‐gene signalling is insufficient to elicit the proliferation of endothelial cells. Int. J. Cancer 71:251–256, 1997.

Id Helix-Loop-Helix Proteins Antagonize Pax Transcription Factor Activity by Inhibiting DNA Binding

ABSTRACT The Id subfamily of helix-loop-helix (HLH) proteins plays a fundamental role in the regulation of cellular proliferation and differentiation. The major mechanism by which Id proteins are thought to inhibit differentiation is through interaction with other HLH proteins and inhibition of their DNA-binding activity. However, Id proteins have also been shown to interact with other proteins involved in regulating cellular proliferation and differentiation, suggesting a more widespread regulatory function. In this study we demonstrate functional interactions between Id proteins and members of the Pax-2/-5/-8 subfamily of paired-domain transcription factors. Members of the Pax transcription factor family have key functions in regulating several developmental processes exemplified by B lymphopoiesis, in which Pax-5 plays an essential role. Id proteins bind to Pax proteins in vitro and in vivo. Binding occurs through the paired DNA-binding domain of the Pax proteins and results in the disruption of DNA-bound complexes containing Pax-2, Pax-5, and Pax-8. In vivo, Id proteins modulate the transcriptional activity mediated by Pax-5 complexes on the B-cell-specific mb-1 promoter. Our results therefore demonstrate a novel facet of Id function in regulating cellular differentiation by functionally antagonizing the action of members of the Pax transcription factor family.

The RNA binding protein Zfp36l1 is required for normal vascularisation and post-transcriptionally regulates VEGF expression

The Zfp36l1 gene encodes a zinc finger‐containing mRNA binding protein implicated in the posttranscriptional control of gene expression. Mouse embryos homozygous for a targeted mutation in the Zfp36l1 locus died mid‐gestation and exhibited extraembryonic and intraembryonic vascular abnormalities and heart defects. In the developing placenta, there was a failure of the extraembryonic mesoderm to invaginate the trophoblast layer. The phenotype was associated with an elevated expression of vascular endothelial growth factor (VEGF)‐A in the embryos and in embryonic fibroblasts cultured under conditions of both normoxia and hypoxia. VEGF‐A overproduction by embryonic fibroblasts was not a consequence of changes in Vegf‐a mRNA stability; instead, we observed enhanced association with polyribosomes, suggesting Zfp36l1 influences translational regulation. These data implicate Zfp36l1as a negative regulator of Vegf‐a gene activity during development. Developmental Dynamics 235:3144–3155, 2006.

Immunoprecipitation techniques for the analysis of transcription factor complexes

Interactions among transcription factors can be detected and analyzed by a variety of in vitro and in vivo approaches. In many studies, the existence of putative interactions among transcription factor partners is initially established from yeast two-hybrid screening and in vitro protein association analysis. The ability to detect candidate interacting proteins in coimmunoprecipitates from cell lysates provides an important criterion for establishing the authenticity of such protein interactions in vivo. This article describes methodology developed for detecting interactions between the helix-loop-helix protein, Id3, and the paired homeodomain protein, Pax5, and interactions involving the zinc finger transcription factor, CTCF. The importance of empirically establishing optimum conditions for cell lysis, selection of appropriate antibodies, conditions for immunoprecipitation, and detection of interacting partners are discussed.

Structural organisation and chromosomal mapping of the human Id-3 gene

The helix-loop-helix (HLH) family of transcription factors plays a central role in the regulation of cell growth, differentiation and tumourigenesis. Members of the Id (inhibitor of DNA binding) class of these nuclear proteins are able to heterodimerise with and thereby antagonise the functions of other transcription factors of this family. We report here on the genomic organisation of the human Id3 (HLH 1R21/heir1) gene. Comparison with the two other mammalian Id genes, Id1 and Id2, reveals a highly conserved protein coding gene organisation consistent with evolution from a common, ancestral Id-like gene. In addition, by using a yeast artificial chromosome (YAC) clone of Id3, we have fine-scale mapped the gene to chromosome band 1p36.1 by fluorescence in situ hybridisation (FISH) and, using the same FISH technique, we have detected heterogeneity in tumour-associated 1p36 chromosome translocations.

Screening for colorectal cancer: established and emerging modalities

It has been estimated that >95% of cases of colorectal cancer (CRC) would benefit from curative surgery if diagnosis was made at an early or premalignant polyp stage of disease. Over the past 10 years, most developed nation states have implemented mass population screening programs, which are typically targeted at the older (at-risk) age group (>50–60 years old). Conventional screening largely relies on periodic patient-centric investigation, particularly involving colonoscopy and flexible sigmoidoscopy, or else on the fecal occult blood test. These methods are compromised by either low cost-effectiveness or limited diagnostic accuracy. Advances in the development of diagnostic molecular markers for CRC have yielded an expanding list of potential new screening modalities based on investigations of patient stool (for colonocyte DNA mutations, epigenetic changes or microRNA expression) or blood specimens (for plasma DNA mutations, epigenetic changes, heteroplasmic mitochondrial DNA mutations, leukocyte transcriptome profile, plasma microRNA expression or protein and autoantibody expression). In this Review, we present a critical evaluation of the performance data and relative merits of these various new potential methods. None of these molecular diagnostic methods have yet been evaluated beyond the proof-of-principle and pilot-scale study stage and it could be some years before they replace existing methods for population screening in CRC.

Nucleotide sequence of the cDNA encoding human helix-loop-helix Id-1 protein: Identification of functionally conserved residues common to Id proteins

We have determined the cDNA sequence encoding a 154 amino acid human Id-1 helix-loop-helix protein. Comparison with the amino acid sequences of human and mouse Id-2 and Id-3 proteins, reveals conservation/divergence of several residues in the helix-loop-helix domain known to be important for heterodimerisation, together with a common casein kinase II phosphorylation site.

Immunohistochemical detection of mutant p53 protein in epithelial ovarian cancer using polyclonal antibody CMI: correlation with histopathology and clinical features.

Approximately 30-50% of cases of ovarian adenocarcinoma harbour mutations in the p53 tumour-suppressor gene associated with elevated levels of the protein detected by immunohistochemical staining. To investigate any relation between the presence of mutant p53 and clinicopathological features of disease, we examined a series of 50 cases of epithelial ovarian adenocarcinoma for expression of p53 by immunohistological staining on fixed, paraffin-embedded tissue sections using the polyclonal antibody CM1, and by direct nucleotide sequencing of polymerase chain reaction-amplified DNA from selected cases. Of the 50 cases examined, 28 (56%) were p53 positive and there was no significant correlation between p53 status and differentiation stage, clinical (FIGO) stage, multidrug resistance (mdr-1 P-glycoprotein) expression or response to treatment. However, we observed a statistically significant difference between the high prevalence of p53-positive serous tumours (18 out of 23) and the lower prevalence of p53-positive cases in mucinous tumours (3 of 12) suggesting that factors related to disease aetiology, associated with these histological subtypes, may determine the prevalence of functional inactivation of the p53 tumour-suppressor gene in ovarian adenocarcinoma.

A B cell specific immediate early human gene is located on chromosome band 1q31 and encodes an α helical basic phosphoprotein

We have determined the cDNA sequence of a human B cell specific, immediate early gene, designated 1R20, which is inducible in response to several B cell activation signals. The cDNA sequence predicts a 196 amino acid open reading frame comprising numerous highly basic residues and the predicted structure contains several potential alpha helical domains together with eight consensus protein phosphorylation sites. The 1R20 gene has been localised by fluorescence in situ hybridisation to chromosome band 1q31, a region known to be implicated in the pathogenesis of haemopoietic malignancies.

A novel immunoglobulin superfamily receptor (19A) related to CD2 is expressed on activated lymphocytes and promotes homotypic B-cell adhesion

A novel lymphocyte-specific immunoglobulin superfamily protein (19A) has been cloned. The predicted 335-amino-acid sequence of 19A represents a Type 1 membrane protein with homology with the CD2 family of receptors. A molecular model of the two predicted extracellular immunoglobulin-like domains of 19A has been generated using the crystal structure of CD2 as a template. In isolated lymphocytes, expression of 19A is induced by various activation stimuli, and enforced expression of the 19A gene promotes homotypic cell adhesion in a B-cell-line model. Collectively these data imply that the 19A protein plays a role in regulation of lymphocyte adhesion.