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Biomedical subjects

Robert G Ramsay

Publications and source records attributed to Robert G Ramsay.

14 recordsLinked to original sources
PubMed2007

Wnt-Frizzled signalling and the many paths to neural development and adult brain homeostasis.

The regulation of brain development and function is the result of complex cell-restricted and temporal expression profiles directed by signaling networks constantly imposing exquisite regulatory control on many genes at any one moment within a cell. The ultimate outcome is a genetically controlled balancing act where expression profiles of these hundreds of genes result in cellular proliferation, differentiation and the ultimate choice between long-term survival and apoptosis. During embryonic development there is a massive expansion of neurons and glia, which is balanced with programmed cell death as the brain matures and remodels. As developing brain cells differentiate, they migrate toward the region where they will ultimately seek out interactions with other cells and perform their specialized tasks. Although a number of signaling pathways have been shown to contribute to various processes allowing the maintenance of normal neurogenesis, the precise signaling machinery necessary for modulating the maintenance of both the neuroblast and differentiated neuronal population, and regulating transition between the two, is still being solved. Not surprisingly, the Wnt signaling pathway is important in regulating neural development but also appears to be involved in adult neurogenesis and some brain disorders. Here, we review key findings showing the pivotal nature of Wnt-Frizzled (FZD) signaling in neurogenesis as revealed by a number of molecular genetic studies using mice and other model organisms. We also review the current literature on the role of the Wnt pathway in the generation of brain cancers, particularly the most common primitive neuroectodermal tumors in childhood, neuroblastomas, and in neurodegenerative diseases such as Alzheimer's disease.

Adult↗
PubMed2006

Mutations in the MYB intron I regulatory sequence increase transcription in colon cancers.

Although MYB overexpression in colorectal cancer (CRC) is known to be a prognostic indicator for poor survival, the basis for this overexpression is unclear. Among multiple levels of MYB regulation, the most dynamic is the control of transcriptional elongation by sequences within intron 1. The authors have proposed that this regulatory sequence is transcribed into an RNA stem-loop and 19-residue polyuridine tract, and is subject to mutation in CRC. When this region was examined in colorectal and breast carcinoma cell lines and tissues, the authors found frequent mutations only in CRC. It was determined that these mutations allowed increased transcription compared with the wild type sequence. These data suggest that this MYB regulatory region within intron 1 is subject to mutations in CRC but not breast cancer, perhaps consistent with the mutagenic insult that occurs within the colon and not mammary tissue. In CRC, these mutations may contribute to MYB overexpression, highlighting the importance of noncoding sequences in the regulation of key cancer genes.

3' Untranslated Regions↗
PubMed2005

Hypothalamic 3',5'-cyclic adenosine monophosphate response element-binding protein loss causes anterior pituitary hypoplasia and dwarfism in mice.

The principal regulation of body growth is via a cascade of hormone signals emanating from the hypothalamus, by release of GHRH, which then directs the somatotroph cells of the pituitary to release GH into the blood stream. This in turn leads to activation of signal transducer and activator of transcription 5-dependent expression of genes such as IGF-I in hepatocytes, acid labile substance, and serine protease inhibitor 2.1, resulting in body growth. Here, using conditional cAMP response element binding protein (CREB) mutant mice, we show that loss of the CREB transcription factor in the brain, but not the pituitary, results in reduced postnatal growth consistent with dwarfism caused by GH deficiency. We demonstrate that although there appears to be no significant impact upon the expression of GHRH mRNA in CREB mutant mice, the amount of GHRH peptide is reduced. These findings show that CREB is required for the efficient production of GHRH in hypothalamus, in addition to its previously reported role in pituitary GH production and somatotroph expansion.

Animals↗
PubMed2005

Expression of stress response protein glucose regulated protein-78 mediated by c-Myb.

Glucose regulated protein-78, GRP78 has been implicated in the protection of tumor cells from cytotoxic damage and apoptosis. When protein profiles of colon cell lines were investigated we found remarkably high GRP78 expression in two cell lines. These cell lines express elevated levels of the transcription factor c-Myb due to genomic amplification of the c-myb locus and we hypothesized that c-Myb regulates GRP78 expression in colon cancer cells. The promoters of human and murine GRP78 and the related family member GRP94 were examined and potential c-Myb binding sites were identified and characterized. DNA binding studies with recombinant c-Myb and nuclear extracts together with ChIP assays on colon cell lines validated these sites. Endogenous GRP78 expression was further induced in these colon cells in response to Thapsigargin treatment, a potent inducer of the unfolded protein response. Transactivation studies with the human GRP78 promoter in colon cell lines showed reporter activity was dependent upon the presence of a conserved c-Myb binding site independent of sequences associated with the unfolded protein response. Finally, over-expression of c-Myb induced the endogenous GRP78 gene. These data suggest that amplification of c-myb in tumor cells may lead to robust GRP78 gene induction, which may in turn assist cells in survival under conditions of oxygen deprivation and nutrient stress.

Amino Acid Sequence↗
PubMed2005

Colon epithelial cell differentiation is inhibited by constitutive c-myb expression or mutant APC plus activated RAS.

Blocked differentiation is a hallmark of cancer cells and the restoration of differentiation programs in vivo is an actively pursued clinical aim. Understanding the key regulators of cyto-differentiation may focus therapies on molecules that reactivate this process. c-myb expression declines rapidly when human colon cancer epithelial cells are induced to differentiate with the physiologically relevant short-chain fatty acid, sodium butyrate. These cells show increased expression of alkaline phosphatase and cytokeratin 8. Similarly, murine Immorto-epithelial cells derived from wild-type colon cells also show c-myb mRNA declines when induced to differentiate with sodium butyrate. Immorto-cells harboring a single APC mutation are indistinguishable from wild-type cells with regard to differentiation, while addition of activated RAS alone markedly enhances differentiation. In marked contrast, complete differentiation arrest occurs when both APC and RAS are mutated. Expression of MybER, a 4-hydroxytamoxifen-activatable form of c-Myb, blocks differentiation in wildtype and APC mutant Immorto-cell lines as well as LIM1215 human colon carcinoma cells. These data identify two pathways of oncogenic change that lead to retarded epithelial cell differentiation, one involving the presence of a single APC mutation in conjunction with activated RAS or alternatively constitutive c-myb expression.

Adenomatous Polyposis Coli Protein↗
PubMed2005

Frizzled-7 receptor ectodomain expression in a colon cancer cell line induces morphological change and attenuates tumor growth.

Frizzled (FZD) receptors have a conserved N-terminal extracellular cysteine-rich domain that interacts with Wnts and co-expression of the receptor ectodomain can antagonize FZD-mediated signalling. Using the ectodomain as an antagonist we have modulated endogenous FZD7 signalling in the moderately differentiated colon adenocarcinoma cell line, SK-CO-1. Unlike the parental cell line, which grows as tightly associated adherent cell clusters, the FZD7 ectodomain expressing cells display a spread out morphology and grow as a monolayer in tissue culture. This transition in morphology was associated with decreased levels of plasma membrane-associated E-cadherin and beta-catenin, localized increased levels of vimentin and redistribution of alpha6 integrin to cellular processes in the FZD7 ectodomain expressing cells. The morphological and phenotype changes induced by FZD7 ectodomain expression in SK-CO-1 cells is thus consistent with the cells undergoing an epithelial-to-mesenchymal-like transition. Furthermore, initiation of tumor formation in a xenograft tumor growth assay was attenuated in the FZD7 ectodomain expressing cells. Our results indicate a pivotal role for endogenous FZD7 in morphology transitions that are associated with colon tumor initiation and progression.

Adenocarcinoma↗
PubMed2004

Suppressor screen in Mpl-/- mice: c-Myb mutation causes supraphysiological production of platelets in the absence of thrombopoietin signaling.

Genetic screens in lower organisms, particularly those that identify modifiers of preexisting genetic defects, have been used successfully to order components of complex signaling pathways. To date, similar suppressor screens have not been used in vertebrates. To define the molecular pathways regulating platelet production, we have executed a large-scale modifier screen with genetically thrombocytopenic Mpl(-/-) mice by using N-ethyl-N-nitrosourea mutagenesis. Here we show that mutations in the c-Myb gene cause a myeloproliferative syndrome and supraphysiological expansion of megakaryocyte and platelet production in the absence of thrombopoietin signaling. This screen demonstrates the utility of large-scale N-ethyl-N-nitrosourea mutagenesis suppressor screens in mice for the simultaneous discovery and in vivo validation of targets for therapeutic discovery in diseases for which mouse models are available.

Animals↗
PubMed2004

CREB function is required for normal thymic cellularity and post-irradiation recovery.

Recent generation of genetically modified Creb1 mutant mice has revealed an important role for CREB (cAMP responsive element binding protein) and the related proteins CREM (cAMP responsive element modulator) and ATF1 (activating transcription factor 1) in cell survival, in agreement with previous studies using overexpression of dominant-negative CREB (dnCREB). CREB and ATF1 are abundantly expressed in T cells and are rapidly activated by phosphorylation when T cells are stimulated through the T cell antigen receptor. We show that T cell-specific loss of CREB in mice, in combination with the loss of ATF1, results in reduced thymic cellularity and delayed thymic recovery following sublethal irradiation but no changes in T cell development or activation. These data show that loss of CREB function has specific effects on thymic T lymphocyte proliferation and homeostasis in vivo.

Activating Transcription Factor 1↗
PubMed2004

Colony-stimulating factor-1 promotes clonogenic growth of normal murine colonic crypt epithelial cells in vitro.

The intestinal epithelium is a continuously renewing tissue. In the colon, stem cells are maintained at the base of highly organized crypts, where they undergo asymmetric division and give rise to daughter cells that proliferate and migrate up the crypt as they differentiate, then become senescent and are finally shed into the intestinal lumen. The growth factor requirements of fetal and prenatal colon cells for colony formation and that influence the establishment of cell lines from Immorto-mouse (Charles River, Wilmington, MA) transgenic embryos were explored. Single cell suspensions were isolated and cultured in a large range of growth factor combinations and conditions to determine their growth properties in soft agar. We report an important advance in the culture of mouse colonocytes by using macrophage colony-stimulating factor (CSF-1) and granulocyte-macrophage colony-stimulating factor (GM-CSF). A substantial proportion of colonies grown under low oxygen tension in the presence of CSF-1 and GM-CSF express intestinal epithelial A33 antigen, have the expected gene expression profile, including c-fms and transcription factor c-myb, and show an appropriate epithelial cell morphology and undetectable CD45. Confocal microscopy on isolated crypts displays basolateral expression of c-Fms and E-cadherin on most epithelial cells. Fetal colon cultures from the Immorto-mouse with CSF-1 produced rapid outgrowth and readily established cell lines, in contrast to cultures without CSF-1. These observations have implications for the understanding of colon epithelial development and recovery following cytotoxic damage as well as providing a basis for the observation that some colon (and other epithelial) tumor cells respond to CSF-1 and GM-CSF.

Animals↗
PubMed2004

c-myb Heterozygous mice are hypersensitive to 5-fluorouracil and ionizing radiation.

Hypersensitivity to chemo- and radiotherapy employed during cancer treatment complicates patient management. Identifying mutations in genes that compromise tissue recovery would rationalize treatment and may spare hypersensitive patients undue tissue damage. Genes that govern stem cell homeostasis, survival, and progenitor cell maintenance are of particular interest in this regard. We used wild-type and c-myb knock-out mice as model systems to explore stem and progenitor cell numbers and sensitivity to cytotoxic damage in two radiosensitive tissue compartments, the bone marrow and colon. Because c-myb null mice are not viable, we used c-myb heterozygous mice to test for defects in stem-progenitor cell pool recovery following gamma-radiation and 5-fluorouracil treatment, showing that c-myb(+/-) mice are hypersensitive to both agents. While apoptosis is comparable in mutant and wild-type mice following radiation exposure, the crypt beds of c-myb(+/-) mice are markedly depleted of proliferating cells. Extrapolating from these data, we speculate that acute responses to cytotoxic damage in some patients may also be attributed to compromised c-myb function.

Animals↗
PubMed2003

Targeting c-Myb expression in human disease.

c-Myb is a transcription factor employed in the haematopoietic system and gastrointestinal tract to regulate the exquisite balance between cell division, differentiation and survival. In its absence, these tissues either fail to form, or show aberrant biology. Mice lacking a functional c-myb gene die in utero by day 15 of development. When inappropriately expressed, as is common in leukaemia and epithelial cancers of the breast, colon and gastro-oesophagus, c-Myb appears to activate gene targets of key importance to cancer progression and metastasis. These genes include cyclooxygenase-2 (COX-2), Bcl-2, BclX(L) and c-Myc, which influence diverse processes such as angiogenesis, proliferation and apoptosis. The clinical potential for blocking c-Myb expression in malignancies is based upon strong preclinical data and some trial-based evidence. The modest clinical experience to date has been with haematopoietic malignancies, but other disease classes may be amenable to similar interventions. The frontline agents to achieve this are nuclease-resistant oligodeoxynucleotides (ODNs), which are proving to be acceptable therapeutic reagents in terms of tolerable toxicities and delivery. Nevertheless, further effort must be focused on improving their efficacy, eliminating non-specific toxicity and optimising delivery. Optimisation issues aside, it would appear that anti-c-Myb therapies will be used with most success when combined with other agents, some of which will be established cytotoxic and differentiation-inducing drugs. This review will explore the future strategic use of ODNs in vivo, focusing on a wide spectrum of diseases, including several beyond the haematopoietic malignancies, in which c-Myb appears to play a role.

Animals↗
PubMed2002

Overexpression of wild-type p53 in lichen sclerosus adjacent to human papillomavirus-negative vulvar cancer.

Human papillomavirus is a risk factor for vulvar cancer, whereas human papillomavirus-negative late onset vulvar carcinoma is associated with the dermatologic condition, lichen sclerosus. Human papillomavirus E6 protein targets TP53 for degradation and by inference it has been assumed that human papillomavirus-negative vulvar cancer is dependent upon the acquisition of p53 somatic mutations and subsequent allelic loss. To investigate this, TP53 expression, loss of heterozygosity, and p53 genomic sequence were examined in 29 cases of human papillomavirus-negative vulvar carcinoma with adjacent lichen sclerosus. We examined 37 cases of lichen sclerosus without vulvar carcinoma, 10 cases of nongenital lichen sclerosus, and 12 cases of normal vulvar epithelium served as controls. TP53 was evident in 72% of vulvar carcinoma, 48% in epithelium adjacent to vulvar carcinoma, but was minimal in normal samples. When lichen sclerosus cases were selected to exclude samples with absolutely no TP53 expression through probable failed antigen retrieval or homozygous p53 loss the number of epithelial cells expressing TP53 increased progressively from nongenital lichen sclerosus to lichen sclerosus without vulvar carcinoma, then to lichen sclerosus with vulvar carcinoma (p<0.0001). These data suggest elevated TP53 is a feature of vulvar lichen sclerosus. Seventy-four percent of vulvar carcinoma had chromosome 17p-linked loss of heterozygosity, whereas 47% of adjacent lichen sclerosus featured loss of heterozygosity, but only 31% of vulvar carcinoma had p53 mutations, a frequency less than reported previously. Seven percent of adjacent lichen sclerosus had mutations, showing for the first time the presence of an identical mutation to the matched vulvar carcinoma. These data, however, implicate p53 mutations as a later event in vulvar carcinoma and in marked contrast to the original expectation, our loss of heterozygosity data are consistent with loss of another locus (not p53) on 17p operating as a tumor suppressor in lichen sclerosus destined to develop vulvar carcinoma.

Carcinoma, Squamous Cell↗
PubMedDate not supplied

Characterization of interactions between transcription factors and a regulatory region spanning nt -320 to -281 of the HIV-1 LTR in T-lymphoid and non-T-lymphoid cells.

HIV-1 gene expression is regulated by the interplay of transcription factors with multiple binding motifs present within the U3, R and U5 regions of the long terminal repeat (LTR). Here we report novel DNA binding complexes (termed 9a, 9b and 9c) between nuclear proteins from T-lymphoid and non-T-lymphoid cells and a region of the U3 LTR between nucleotides (nts) -320 to -281 in the HIV strain HXB2. Complex 9b bound a motif predicted to bind E-box or c-Myb proteins and a partially overlapping dyad symmetrical motif, and included basic helix-loop-helix proteins (E12, E47 or ITF-1) but surprisingly not c-Myb. Complex 9c, which bound to a pair of GATA sites, included GATA-3 and GATA-4 in Jurkat and MT-2 cells, respectively. We also demonstrate that the c-Myb/E-box and GATA sites form a bipartite motif required for the formation of complex 9a. Transient transfection experiments with T cells revealed that in the context of a minichromosome assembled full-length LTR, mutation of region -320 to -281 increased basal and PMA-stimulated LTR activity. These findings suggest that this region is an important component of the HIV-1 LTR required for response to different cellular transcription factors.

Binding Sites↗