Stem cell protein BMI-1 is an independent marker for poor prognosis in oligodendroglial tumours.

AIMS: The polycomb factor BMI-1 has recently been implicated in tumorigenesis of the central nervous system in several experimental animal models. However, the significance of BMI-1 in human glioma has not been investigated. Here we describe expression of the polycomb protein BMI-1 and its downstream targets p16(Ink4a) and MDM2 in both high- and low-grade human glioma. METHODS: Tumour samples were collected from 305 adult patients treated for primary grades 2-4 gliomas between 1980 and 2006 in Finland and Germany. BMI-1, p16 and MDM2 expression was evaluated using immunohistochemistry in representative paraffin-embedded tumour tissue. The significance of observed immunoreactivity, age at onset, gender, histopathological findings and proliferative index was analysed in univariate and multivariate survival models. RESULTS: BMI-1 was expressed in all histologic types of diffuse gliomas. We found a significant correlation (P = 0.007) between the frequency of BMI-1 immunoreactive tumour cells and poor survival in World Health Organization grades II-III oligodendrogliomas and oligoastrocytomas (n = 62). The median survival of patients grouped by low, intermediate or high frequency of BMI-1 immunoreactive tumour cells was 191 months, 151 months and 68 months, respectively. This association was also significant in the Cox multivariate regression model. Nuclear p16 immunopositivity predicted better survival in astrocytomas and an inverse correlation between p16 expression and the Ki-67 mitotic index was also observed. CONCLUSIONS: BMI-1 is found in all histological types of gliomas and the relative protein expression of BMI-1 is a novel independent prognostic marker in oligodendroglial tumours.

E6/E7 oncogenes increase and tumor suppressors decrease the proportion of self-renewing neural progenitor cells.

Many if not most tissues need a controlled number of stem cells to maintain normal function. Cancer can be seen as a process of disturbed tissue homeostasis, in which too many cells have or acquire too primitive identity. Here we measured how oncogenes and tumour suppressors affect the differentiation capacity, proportion and characteristics of progenitor cells in a model tissue. Neural progenitor cells (NPCs) were exposed to human papilloma virus E6, E7 or E6/E7 oncogenes, which degrade tumour suppressors p53 and pRb family members, respectively. E6/E7-expressing or p53-/- NPCs were able to differentiate, but simultaneously retained high capacity for self-renewal, proliferation, ability to remain multipotent in conditions promoting differentiation and showed delayed cell cycle exit. These functions were mediated through p53 and pRb family, and involved MEK-ERK signalling. Decreased amount of p53 increased self-renewal and proliferation, whereas pRb affected only proliferation. Our results suggest that the oncogenes increase whereas p53 and pRb family tumour suppressors decrease the number and proportion of progenitor cells. These findings provide one explanation how oncogenes and tumour suppressors control tissue homeostasis and highlight their importance in stem cell self- renewal, linked both to cancer and life-long tissue turnover.

Distribution of glial cell line-derived neurotrophic factor mRNA in human colon suggests roles for muscularis mucosae in innervation.

BACKGROUND/PURPOSE: Glial cell line-derived neurotrophic factor (GDNF) is a ligand for the receptor complex of GDNF family receptor alphas (GFRalphas) and Ret receptor tyrosine kinase, the product of a known Hirschsprung's disease gene. The aim of this study was to analyze the mRNA distribution of these genes in the developing human intestine to understand their roles in enteric innervation. METHODS: Cryosections of fetal and newborn stomach, ileum, and colon were hybridized in situ with S35-labeled cRNA probes to GDNF, Ret, GFRalpha-1 or GFRalpha-2. GDNF mRNA levels in fetal ileum and colon were compared by reverse transcription-polymerase chain reaction (PCR). RESULTS: GDNF mRNA expression was abundant in the muscularis mucosae of both fetal and newborn colon but was found neither in the neural plexuses nor in other regions of the intestine. Accordingly, by reverse transcription-PCR, GDNF mRNA level was many times higher in colon than ileum. Ret, GFRalpha-1 and GFRalpha-2 mRNA were expressed in the ganglionic cells of both myenteric and submucosal plexuses throughout the intestine. CONCLUSIONS: The highly restricted distribution of GNDF mRNA suggests an important role for muscularis mucosae in the development of human enteric nervous system. Ret, GFRalpha-1, and GFRalpha-2 most likely act as GDNF receptors in colon but may have alternative ligands in other enteric segments.

Mutation analysis of the glial cell line-derived neurotrophic factor gene in Parkinson's disease.

Glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for nigrostriatal dopaminergic, central cholinergic, and motoneurons. GDNF also prevents the neuronal loss in experimental animal models for Parkinson's disease (PD). We have now investigated the GDNF gene for possible mutations in a group of nonfamilial PD and other patients. By cleavase fragment length polymorphism (CFLP) analysis and direct sequencing of the full coding region of GDNF gene we found a novel GDNF sequence variant in 1 of 30 PD patients. The alteration does not change the predicted amino acid sequence and it was also found in 1 of 20 patients without PD, suggesting that it represents a polymorphism in the gene. No other sequence variations were found. We conclude therefore that mutations in the GDNF coding region are not commonly contributing to the pathogenesis of PD.

Hirschsprung's disease genes and the development of the enteric nervous system.

Hirschsprung's disease or aganglionic megacolon causes chronic, congenital obstipation at an incidence of 1 per 5000 live births. Two approaches have been vital to the present understanding of the pathogenesis and genetic background of the disease: disease linkage analyses and mouse models of aganglionic megacolon. Because the increasing number of transgenic or natural mouse strains with congenital megacolon has led to mutation screening in Hirschsprung's disease patients, almost every second patient could now receive a genetic explanation for his/her disease. The known disease genes include tyrosine kinase receptor Ret, endothelin receptor B and its ligand endothelin 3. In addition, mutations have been found in the gene encoding the glial cell line-derived neurotrophic factor, the ligand for Ret, but these may only have a modifier effect. The mouse models have also provided insight into the developmental mechanisms of the normal intestinal innervation. We combine here the present clinical data on the gene mutations in Hirschsprung's disease with the experimental molecular biology data, and formulate a hypothesis on the pathogenesis of this multigenic-multifactorial disease.

Glial-cell-line-derived neurotrophic factor is required for bud initiation from ureteric epithelium.

The shapes of different organs can be explained largely by two fundamental characteristics of their epithelial rudiments - the pattern of branching and the rate of proliferation. Glial-cell-line-derived neurotrophic factor (GDNF) has recently been implicated in the development of metanephric ureteric epithelium (Pichel, J. G., Shen, L., Sheng, H. Z., Granholm, A.-C., Drago, J., Grinberg, A., Lee, E. J., Huang, S. P., Saarma, M., Hoffer, B.J., Sariola, H. and Westphal, H. (1996). Nature 382, 73-76; Sánchez, M.P., Silos-Santiago, I., Frisén, J., He, B., Lira, S.A. and Barbacid, M. (1996). Nature 382, 70-73; Vega, Q.C., Worby, C.A., Lechner, M.S., Dixon, J.E. and Dressler, G.R. (1996). Proc. Nat. Acad. Sci. USA 93, 10657-10661). We have analysed the target cells of GDNF and the manner in which it controls ureteric development, and have compared it with other growth factors that have been associated with the regulation of branching morphogenesis, namely hepatocyte growth factor (HGF) and transforming growth factor-beta1 (TGFbeta1). We show that GDNF binds directly to the tips of ureteric bud branches, and that it has the ability to promote primary ureteric buds from various segments of Wolffian duct and to attract ureteric branches towards the source of GDNF. It increases cell adhesion, but is not obviously mitogenic for ureteric cells. The data indicate that GDNF is required primarily for bud initiation. Comparison of GDNF, HGF and TGFbeta1 suggests that the latter act later than GDNF, and may represent a partially redundant set of mesenchyme-derived growth factors that control ureteric development. Thus, GDNF is the first defined inducer in the embryonic metanephric kidney.

Cloning, mRNA distribution and chromosomal localisation of the gene for glial cell line-derived neurotrophic factor receptor beta, a homologue to GDNFR-alpha.

Glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for central dopaminergic neurons, motor neurons and several other populations of neurons in the central and peripheral nervous system. GDNF and its receptor complex of c-RET tyrosine kinase and a glycosyl-phosphatidylinositol linked protein GDNFR-alpha are of great interest due to their potential use in the therapy of Parkinson's and motoneuron diseases. We have cloned the human and rat cDNA sequences of GDNFR-beta, a new gene encoding for a 464 amino acid long homologue of GDNFR-alpha, and assign the locus of this new gene to human chromosome 8p21-22 and mouse chromosome 14D3-E1. Similarly to GDNFR-alpha, GDNFR-beta mediates GDNF-induced Ret autophosphorylation in transfected cells. By northern hybridisation we show that the transcript level of human GDNFR-beta mRNA is high in the adult brain, intestine and placenta and in fetal brain, lung and kidney. Studied by in situ hybridisation, GDNFR-beta mRNA shows in E17 rat embryo different distribution to that of GDNFR-alpha mRNA, especially, in adrenal gland, kidney and gut. In the developing nervous system, GDNFR-beta mRNA expression is restricted to certain neuronal populations, while GDNFR-alpha mRNA is widely expressed also in non-neuronal cells. The distinct tissue distribution of GDNFR-beta mRNA and its ability to mediate GDNF signal in transfected cells suggest a role in signal transduction of GDNF and, possibly, related neurotrophic factors in vivo.

Exclusion of the p75 neurotrophin receptor gene as a candidate gene for Meckel syndrome.

Nerve growth factor receptor p75 (NGFR) gene was investigated as a potential candidate gene in Meckel syndrome (MKS) because of its important role in embryonic development, chromosomal localization adjacent to the MKS locus and Meckel syndrome-resembling findings in knock-out mice phenotype. The sequence analysis of the coding region of the gene revealed one polymorphism but no potential disease mutation. Physical mapping of the critical chromosomal region finally showed that the NGFR gene lies outside the MKS locus.