Expression of SoxE and SoxD genes in human gliomas.

Members of group E and group D of the Sox gene family function as important transcriptional regulators of glial development in the central nervous system. Here, we have examined Sox gene expression in 60 human primary gliomas. Transcripts from each of the six group E and group D genes were expressed in gliomas of various types and malignancy grades, but with significant differences. SOX5, SOX9 and SOX10 were generally expressed at levels similar to or below those in adult brain tissue. In contrast, many oligodendrogliomas exhibited upregulation of SOX6, SOX8 and SOX13. Furthermore, loss of heterozygosity on chromosomal arms 1p and 19q was associated with significantly higher SOX8 mRNA levels. Low-grade astrocytomas, but not glioblastomas, also showed elevated SOX8 transcript levels. Taken together, the expression pattern of Sox genes in gliomas is heterogeneous and overall compatible with the less differentiated state of glioma cells as compared with their normal adult counterparts. Despite their restricted expression in astrocytes and oligodendrocytes during normal development, none of the Sox genes was selectively expressed in tumours of the oligodendroglial or astrocytic lineage. This is compatible with an origin of gliomas from neuroepithelial stem or precursor cells.

Hypermethylation and transcriptional downregulation of the CITED4 gene at 1p34.2 in oligodendroglial tumours with allelic losses on 1p and 19q.

Deletions of chromosomal arms 1p and 19q are frequent in oligodendroglial tumours and have been associated with sensitivity to radio- and chemotherapy as well as favourable prognosis. By using microarray-based expression profiling, we found that oligodendroglial tumours with 1p and 19q losses showed significantly lower expression of the CBP/p300-interacting transactivator with glutamic acid/aspartic acid-rich carboxyl-terminal domain 4 gene (CITED4) at 1p34.2 as compared to tumours without 1p and 19q losses. Mutational analysis showed no CITED4 mutations in gliomas. However, 1p and 19q losses as well as low expression of CITED4 transcripts were significantly associated with hypermethylation of the CITED4-associated CpG island. In line with the latter finding, treatment of CITED4 hypermethylated glioma cell lines with 5-aza-2'-deoxycytidine and trichostatine A resulted in a marked increase of the CITED4 transcript levels. Furthermore, CITED4 hypermethylation was significantly associated with longer recurrence-free and overall survival of patients with oligodendroglial tumours. Taken together, our results indicate that CITED4 is epigenetically silenced in the vast majority of oligodendroglial tumours with 1p and 19q deletions and suggest CITED4 hypermethylation as a novel prognostic marker in oligodendroglioma patients.

Functional characterization of the adrenoleukodystrophy protein (ALDP) and disease pathogenesis.

X-linked adrenoleukodystrophy (X-ALD) is the most common peroxisomal disorder characterized by abnormal accumulation of saturated very long chain fatty acids in tissues and body fluids with predominance in brain white matter and adrenal cortex. The clinical phenotype is highly variable ranging from the severe childhood cerebral form to asymptomatic persons. The responsible ALD gene encodes the adrenoleukodystrophy protein (ALDP), a peroxisomal integral membrane protein that is a member of the ATP-binding cassette (ABC) transporter protein family. The patient gene mutations are heterogeneously distributed over the functional domains of ALDP. The extreme variability in clinical phenotype, even within one affected family, indicates that besides the ALD gene mutations other factors strongly influence the clinical phenotype. To understand the cell biology and function of mammalian peroxisomal ABC transporters and to determine their role in the pathogenesis of X-ALD we developed a system for expressing functional ABC protein domains in fusion with the maltose binding protein. Wild type and mutant fusion proteins of the nucleotide-binding fold were overexpressed, purified, and characterized by photoaffinity labeling with 8-azido ATP or 8-azido GTP and a coupled ATP regenerating enzyme assay for ATPase activity. Our studies provide evidence that peroxisomal ABC transporters utilize ATP to become a functional transporter and that ALD gene mutations alter peroxisomal transport function. The established disease model will be used further to study the influence of possible disease modifier proteins on ALDP function.

Characterization and functional analysis of the nucleotide binding fold in human peroxisomal ATP binding cassette transporters.

The 70-kDa peroxisomal membrane protein (PMP70) and the adrenoleukodystrophy protein (ALDP) are half ATP binding cassette (ABC) transporters in the peroxisome membrane. Mutations in the ALD gene encoding ALDP result in the X-linked neurodegenerative disorder adrenoleukodystrophy. Plausible models exist to show a role for ATP hydrolysis in peroxisomal ABC transporter functions. Here, we describe the first measurements of the rate of ATP binding and hydrolysis by purified nucleotide binding fold (NBF) fusion proteins of PMP70 and ALDP. Both proteins act as an ATP specific binding subunit releasing ADP after ATP hydrolysis; they did not exhibit GTPase activity. Mutations in conserved residues of the nucleotidases (PMP70: G478R, S572I; ALDP: G512S, S606L) altered ATPase activity. Furthermore, our results indicate that these mutations do not influence homodimerization or heterodimerization of ALDP or PMP70. The study provides evidence that peroxisomal ABC transporters utilize ATP to become a functional transporter.

Clinical and genetic aspects of X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (ALD), a leukodystrophy characterized by abnormal accumulation of saturated very long chain fatty acids in brain white matter and adrenal cortex, is the most common inherited peroxisomal disorder. The biochemical defect is localized to the level of lignoceroyl-CoA synthesis, a step in the peroxisomal beta-oxidation of very long chain fatty acids. The responsible gene encodes a peroxisomal integral membrane protein of as yet unknown function which is a member of the ATP-binding cassette transporter protein superfamily. The patient gene mutations are heterogeneously distributed over the functional protein domains with a tendency to clustering in the nucleotide-binding fold. The mechanisms by which these mutations cause a loss of protein function is unknown. Diagnosis of patients and carriers, including prenatal testing, is mainly based on the clinical picture, the demonstration of increased levels of saturated very long chain fatty acids in tissues and body fluids as well as on DNA mutation analyses. There are at least six distinct clinical phenotypes ranging from the severe childhood cerebral form to asymptomatic persons. The various phenotypes commonly occur within the same kindred. Modifying genes and/or environmental factors may contribute to this phenomenon. At present, there is no proven therapy for the prevention or cure of the neurological disabilities. Several approaches are under investigation including diets, immunosuppression, bone marrow transplantation and gene therapy.

Genomic organization of the 70-kDa peroxisomal membrane protein gene (PXMP1).

The 70-kDa peroxisomal membrane protein (PMP70) is a member of a family of half-ATP-binding cassette (ABC) transporter proteins located in the human peroxisomal membrane. Other members include the PMP70-related peroxisomal membrane protein, the adrenoleukodystrophy protein (ALDP), and the adrenoleukodystrophy-related protein. The functions of ABC transporters in the peroxisomal membrane are poorly understood. Evidence from yeast and human mutants suggests that they are involved in the peroxisomal import of fatty acids and/or fatty acyl-CoAs into the organelle. We report the cloning and characterization of the human PMP70 structural gene (gene symbol: PXMP1) localized on human chromosome 1p21-p22. PXMP1 is approximately 65 kb in length, contains 23 exons, and is quite different in structure from the gene (ALD) that encodes the related protein, ALDP. We also analyzed the 5' flanking region of the human PXMP1 gene and the corresponding region of murine Pxmp-1. Both promoters have features of housekeeping genes, including a high GC content and multiple consensus Sp1 binding sequences. In more than 3 kb of Pxmp-1 5' flanking sequence we did not identify a consensus peroxisomal proliferator responsive element.