Astrocytoma derived short-term cell cultures retain molecular signatures characteristic of the tumour in situ.

The heterogeneity of tumours and uncertainties surrounding derived short-term cell cultures and established cell lines fundamentally challenge the research and understanding of tumour growth and development. When tumour cells are cultured, changes are inevitably induced due to the artificial growth conditions. Several recent studies have questioned how representative established cell lines or derived short-term cell cultures are of the tumour in situ. We have characterised gene expression changes induced by short-term culture in astrocytoma in order to determine whether derived short-term cell cultures are representative of the tumour in situ. In comparison to the majority of studies, paired biopsies and derived short-term cultures were investigated to reduce the effects of long-term culture and inter-tumour variability when comparing biopsies and derived cultures from tumours with the same histology from different individuals. We have used the Affymetrix GeneChip U133A to generate gene expression profiles of 6 paediatric pilocytic astrocytoma (PA) biopsies and derived short-term cell cultures and 3 adult glioblastoma multiforme (GBM) biopsies and derived short-term cultures. Significant differential gene expression is induced by short-term culture. However, when the biopsy and derived short-term cell culture samples were grouped according to tumour type (PA and GBM) a molecular signature of 608 genes showed significant differential expression between the groups. This gene cohort can distinguish PA and GBM tumours, regardless of the sample source, suggesting that astrocytoma derived short-term cultures do retain key aspects of the global tumour expression profile and are representative of the tumour in situ. Furthermore, these genes are involved in pathways and functions characteristic of adult GBM including VEGF signalling, hypoxia and TP53 signalling.

Molecular definition in a somatic cell hybrid of a specific 2:13 translocation breakpoint in childhood rhabdomyosarcoma.

A consistent, balanced, reciprocal chromosomal translocation t(2:13) (q35:q14) has been identified in alveolar rhabdomyosarcoma. Somatic cell hybrids have been constructed between rhabdomyosarcoma cell lines carrying the (2:13) translocation and mouse 3T3 cells. One hybrid cell line was shown to have retained the derivative (13:2) chromosome, but segregated the normal chromosome 13 and the derivative (2:13) chromosome. Using available DNA probes from human chromosome 13 we find that the loci for retinoblastoma, esterase D, p7D2, pG24E6.8 and pG14E1.9 lie distally to the 13q14 breakpoint, whereas those for p7F12, pHU10 and pG2E3.1 all lie proximally. Thus we have defined a region of 13q14 of approximately 28mB which contains the breakpoint associated with this rearrangement.

Methyl vinyl sulphone: a new class of Michael-type genotoxin.

Methyl vinyl sulphone (MVS) is a labile, Michael-reactive chemical, similar in structure to acrylamide (AA). Given that acrylamide is a reference mammalian mutagen and a rodent carcinogen, studies were undertaken to evaluate the potential genotoxicity of MVS. In common with AA, MVS was non-mutagenic to Salmonella but active as an aneugen to cultured mammalian cells. It is concluded that vinyl sulphones should be regarded as representative of a new class of genotoxic chemical whose mode of action is probably primarily dependent upon Michael reactivity to proteins.

A comparison of two in vitro mammalian cell cytogenetic assays for the detection of mitotic aneuploidy using 10 known or suspected aneugens.

Two in vitro cytogenetic assays were evaluated for their ability to detect aneugenic and polyploidy-inducing agents using a battery of 10 known or suspected aneugens supplied as part of the EEC 4th Environmental Research and Development Programme. The compounds tested were colchicine, vinblastine, chloral hydrate, thiabendazole, hydroquinone, thimerosal, cadmium chloride, econazole nitrate, pyrimethamine and diazepam. The cell division aberration assay employed a differential chromosome/spindle staining procedure to detect perturbations of the mitotic division apparatus. This assay was carried out in two pulmonary-derived Chinese hamster cell lines; the immortal DON:Wg3h culture and a low passage LUC2 culture. The second assay involved quantification of metaphase chromosomes, for which only the LUC2 cell line was used, due to the stability of its diploid karyotype. All the chemicals induced spindle disturbances in the immortal line. In addition, all the compounds except cadmium chloride yielded positive results in the LUC2 culture, although many were not as potent. In the low passage line, 8 of the compounds (colchicine, vinblastine, chloral hydrate, thiabendazole, thimerosal, econazole nitrate, pyrimethamine and diazepam) induced aneuploidy and/or tetraploidy. Cadmium chloride was negative in the chromosome enumeration assay and hydroquinone yielded inconclusive results. The study of cell division aberrations was much less time-consuming and technically complex than the counting of metaphase chromosomes. In addition, it provided a degree of mechanistic understanding of the mode of action of some aneugenic and polyploidy-producing agents. However, the enumeration of chromosomes provides a more definitive data set for the evaluation of a chemical's aneugenic potential.

Biology and genetics of malignant brain tumours.

Conventional therapies such as surgery, radiotherapy and, to a lesser extent, chemotherapy have produced significant increases in survival in patients with some types of brain tumours such as medulloblastoma. However, in many other types of brain tumour in both adults and children, the effect of these modalities has been more modest. A thorough understanding of the biology of malignant brain tumours is likely to provide the background for the development of new leads that might be amenable to therapeutic exploitation. This review examines some aspects of glioma biology that have been reported in the past 12 months, and which might be translated into clinical application.

Non-isotopic molecular cytogenetics in neuro-oncology.

The molecular genetic analysis of brain tumours has been the focus of considerable interest for a number of years. However, these studies have been largely directed towards understanding the fundamental biological processes involved in tumorigenesis and the techniques which have been used require considerable molecular biological skills. Unfortunately, there has not been the impetus to correlate basic biological studies with clinical or neuropathological features. The development of non-isotopic molecular cytogenetic in situ hybridization (ISH) techniques which can be applied to archival tumour material provides an opportunity to address a wide range of neuropathological questions at a genetic level. Identification of specific chromosomes has been made possible by the isolation of probes which recognize the highly repeated sequences present in the centromeric regions of individual chromosomes. Libraries of human chromosome-specific painting probes are also available. A range of probes which bind to the whole or part of specific single copy genes are becoming available. These can be detected with either fluorochromes with different emission colours or with enzymatic detection systems in either interphase nuclei derived from fresh, fixed and embedded tumour samples, touch preparations or smears (so-called 'interphase cytogenetics') as well as conventional metaphase spreads. Comparative genomic hybridization can be used to scan the entire genome for deletions or amplifications without any pre-existing information about the likely locations of these abnormalities or the availability of any specific DNA probes. These techniques can be used to identify aneuploidy or structural alterations in individual chromosomes and are likely to yield important information about the location of genes important in the pathogenesis of brain tumours and may also provide the basis for the refinement of diagnostic or prognostic criteria of these neoplasms.