Biomedical subjects

A P Cuthbert

Publications and source records attributed to A P Cuthbert.

25 records · Page 2Linked to original sources

PubMed1998

SURF1, encoding a factor involved in the biogenesis of cytochrome c oxidase, is mutated in Leigh syndrome.

Leigh Syndrome (LS) is a severe neurological disorder characterized by bilaterally symmetrical necrotic lesions in subcortical brain regions that is commonly associated with systemic cytochrome c oxidase (COX) deficiency. COX deficiency is an autosomal recessive trait and most patients belong to a single genetic complementation group. DNA sequence analysis of the genes encoding the structural subunits of the COX complex has failed to identify a pathogenic mutation. Using microcell-mediated chromosome transfer, we mapped the gene defect in this disorder to chromosome 9q34 by complementation of the respiratory chain deficiency in patient fibroblasts. Analysis of a candidate gene (SURF1) of unknown function revealed several mutations, all of which predict a truncated protein. These data suggest a role for SURF1 in the biogenesis of the COX complex and define a new class of gene defects causing human neurodegenerative disease.

Amino Acid Sequence↗

PubMed1996

The defect in the AT-like hamster cell mutants is complemented by mouse chromosome 9 but not by any of the human chromosomes.

X-ray sensitive Chinese hamster V79 cells mutants, V-C4, V-E5 and V-G8, show an abnormal response to X-ray-induced DNA damage. Like ataxia telangiectasia (AT) cells, they display increased cell killing, chromosomal instability and a diminished inhibition of DNA synthesis following ionizing radiation. To localize the defective hamster gene (XRCC8) on the human genome, human chromosomes were introduced into the AT-like hamster mutants, by microcell mediated chromosome transfer. Although, none of the human chromosomes corrected the defect in these mutants, the defect was corrected by a single mouse chromosome, derived from the A9 microcell donor cell line. In four independent X-ray-resistant microcell hybrid clones of V-E5, the presence of the mouse chromosome was determined by fluorescent in situ hybridization, using a mouse cot-1 probe. By PCR analysis with primers specific for different mouse chromosomes and Southern blot analysis with the mouse Ldlr probe, the mouse chromosome 9, was identified in all four X-ray-resistant hybrid clones. Segregation of the mouse chromosome 9 from these hamster-mouse microcell hybrids led to the loss of the regained X-ray-resistance, confirming that mouse chromosome 9 is responsible for complementation of the defect in V-E5 cells. The assignment of the mouse homolog of the ATM gene to mouse chromosome 9, and the presence of this mouse chromosome only in the radioresistant hamster cell hybrids suggest that the hamster AT-like mutant are homologous to AT, although they are not complemented by hamster chromosome 11.

Animals↗

PubMed1996

Identification of human tumour suppressor genes by monochromosome transfer: rapid growth-arrest response mapped to 9p21 is mediated solely by the cyclin-D-dependent kinase inhibitor gene, CDKN2A (p16INK4A).

Microcell transfer of intact normal human chromosomes into immortal mouse and hamster fibroblast cell lines has revealed growth suppressive activity associated with a small sub-set of the human complement. Here, we describe the results of a detailed study aimed at identifying the gene or genes responsible for the rapid growth-arrest response obtained with human chromosome-9. Initially, STS-PCR deletion mapping of segregants arising in monochromosome transfer experiments was used successfully to localize the active sub-chromosomal region to 9p21. Subsequent fine-structure deletion mapping of previously uniformative hybrid segregants, employing additional markers between D9S162 and D9S171, provided strong evidence that the cyclin-dependent kinase (cdk) inhibitor gene CDKN2A (p16INK4A) was solely responsible for the chromosome-9 effect; 9p21 microdeletions in a significant proportion of segregant clones were restricted to a single CDKN2A exon. Transfection experiments with CDKN2A and CDKN2B cDNA expression vectors, using mouse A9 cells and three human malignant melanoma cell lines as recipients, provided further evidence in support of this hypothesis. Collectively, our results indicate that expression of human CDKN2A (controlled either by its natural regulatory elements, or by a cytomegalovirus promoter) is incompatible with in vitro proliferation in immortalized rodent cells and in human melanoma cell lines. The rapidity of the growth inhibitory effects of CDKN2A was inconsistent with a mode of action involving induction of replicative cell senescence via telomerase repression, but was consistent with a mechanism based on cell cycle arrest through cdk inhibition. The study described here has generated a panel of microdeleted monochromosome-9 donor hybrids which may prove valuable in functional investigations aimed at identifying other important tumour suppressor genes located on human chromosome-9.

Animals↗

PubMed1995

Novel use of a selectable fusion gene as an "in-out" marker for studying genetic loss in mammalian cells.

Recent demonstrations of loss of heterozygosity in a wide variety of human cancers suggest that large multilocus genetic deletions (presumably including tumor suppressor genes) constitute a major class of genetic alteration in human carcinogenesis. Here we show that a bifunctional fusion gene (Hytk), suitable for both positive and negative selection, is an effective marker for studying genetic loss in mammalian cells with minimal interference from point-mutational changes. Studies with a transgenic V79 cell line in which a single functional copy of Hytk was stably inserted into the genome in a retroviral vector showed that loss of the marker (and presumably flanking cellular genetic material) could be induced efficiently by ionizing radiation (gamma-rays and fast neutrons) but only weakly by the powerful point-mutagen benzo[a]pyrene diol epoxide. In a first application of the system, we provide evidence that radiation-induced loss can occur through an indirect mechanism after a high-frequency event. Collectively, our results suggest that the Hytk marker should be a valuable tool for studying genome position effects on the tolerance of genetic loss in cultured human cells that represent different stages in clonal evolution and tumor progression.

3T3 Cells↗

PubMed1995

Mechanisms involved in the immortalization of mammalian cells by ionizing radiation and chemical carcinogens.

Immortalization is a prerequisite for the clonal evolution and malignant transformation of normal mammalian cells in culture. In order to gain a mechanistic insight into the genetics of carcinogen-induced cellular immortality, a cell culture assay has been developed based on the use of freshly explanted Syrian hamster dermal (SHD) fibroblasts. The relative efficacies of a variety of chemical and physical carcinogens at immortalizing SHD cells (against a zero background of spontaneous immortalization) were compared. Ionizing radiation and nickel chloride appeared to be more effective as immortalizing agents than powerful point mutagens, suggesting (but not proving) that clastogenic damage may be more significant in the immortalization process than point mutation. Frequencies of induced immortality (10(-6)-10(-7)/treated cell) were arguably consistent with a direct mutational mechanism involving a single genetic target. However, detailed cytogenetic characterization of a panel of newly immortalized cell lines revealed no non-random chromosomal alterations in the cells at the level of G-banding. Furthermore, additional experiments with the SHD system have provided confirmatory evidence that immortalization can occur as an indirect consequence of carcinogen exposure following an induced high frequency change in the treated population, rather than through direct targeted mutagenesis. Previous somatic cell genetic studies have suggested the possibility that a target gene for immortalization exists on the human and Chinese hamster X chromosomes. Here we provide strong evidence that the normal SHD X chromosome displays powerful senescence-inducing properties when introduced, by microcell transfer, into newly immortalized SHD recipients. These results suggest that induction of the immortal phenotype in SHD cells by carcinogens results primarily from functional inactivation of a senescence gene which may be X-linked. One possible mechanism for senescence gene inactivation consistent with our observations is through a sub-microscopic interstitial genetic deletion. However, the considerable efficacy of nickel (a human carcinogen) as an immortalizing agent at nonmutagenic doses raises the alternative possibility that immortalization may occur through an epigenetic mechanism.

Animals↗

PubMed1995

Construction and characterization of a highly stable human: rodent monochromosomal hybrid panel for genetic complementation and genome mapping studies.

Human:rodent somatic cell hybrids carrying a single, intact, selectable human chromosome are valuable both for functional somatic cell genetic analysis and genome mapping procedures. Here, we describe the construction and detailed molecular cytogenetic characterization of a panel of 23 stable hybrids, representing all 22 human autosomes plus the X-chromosome. Individual normal human chromosomes have been tagged with a selectable fusion gene (Hytk) introduced into the chromosome in a small (4.2 kbp) retroviral vector. Use of the Hytk marker permits both positive and negative ("in-out") selection to be applied to the human chromosome in any mammalian cell background. The panel includes 18 new hybrids isolated by direct microcell transfer from normal human diploid fibroblasts into mouse A9 cells.

Adult↗

PubMed1993

Cell immortalization as a key, rate-limiting event in malignant transformation: approaches toward a molecular genetic analysis.

Recent advances using somatic cell genetic approaches have provided a convincing body of evidence that the senescence of mammalian cells in culture is controlled by a small group of genes, one or more of which are functionally deleted in the process of immortalization. Microcell-mediated mono-chromosomal transfer methods should permit precise mapping of these genes to specific chromosomal regions. Cloning of senescence genes, using either conventional 'positional cloning' techniques or retroviral insertion mutagenesis, is now a realistic possibility. The leap in our understanding of the molecular genetic events driving the alternative cellular states of limited proliferative capacity and immortality, which such advances should precipitate, will finally permit the question of the role of cell immortalization in cancer to be addressed, and may open the door to the design of new modes of cancer therapy. In addition, the precise mechanism underlying the wide difference in transformability between human and rodent cells, which should also emerge from these investigations, is likely to make a significant contribution towards resolving the key issue of the relevance of rodent tumour induction assays in assessing the potential carcinogenicity of environmental chemicals.

Animals↗