(from the application): Because of the absence of telomerase activity in most normal human somatic cells, telomeres shorten when human cells proliferate in culture, eventually causing replicative senescence, a permanent state of growth arrest. It has been suggested that replicative senescence contributes to the properties of tissues in aging in vivo, but there are no current satisfactory models for studying this process in tissues in vivo. A cell transplantation model will be used to study the effects of telomere shortening in tissues, using human and bovine adrenocortical cells. Bovine cells form a good model for the behavior of human cells with respect to telomere biology and replicative senescence. Adrenocortical cells provide an appropriate cell type for this study because the cells form a discrete vascularized tissue structure after transplantation in immunodeficient (scid) mice. Two methods for producing replicative exhaustion in transplant tissues will be used. In the first, cells have autocrine stimulation of growth, by constitutive expression of insulin like growth factor-II, and also have regulated cell death by controlled expression of Pseudomonas exotoxin. In the second, a floxed SV40 T antigen gene drives cell proliferation but can be excised using Cre recombinase. In a third experiment cells in tissues will be placed directly into a senescence-like state by the expression of p14ARF (alternative reading frame of INK4A locus). If replicative exhaustion occurs as it does in culture cells are expected to show a G1 cell cycle arrest, a normal karyotype, without cell death. However, it is also possible that replicative exhaustion in tissues is not caused by replicative senescence but is caused by crisis, characterized by chromosomal instability, a cell cycle block with a 4N DNA content, accompanied by increased cell death. Microarray analysis of RNA from tissues with exhausted proliferative potential will provide information as to whether gene expression patterns are similar at replicative exhaustion in cell culture and in tissues, and whether expression patterns differ among the three different methods by which permanent growth arrest is achieved. These experiments will show whether replicative senescence is reached by telomere shortening or by other means in an experimental tissue structure. This model will provide an appropriate system for future studies of telomere biology in tissue aging and of the relationship of aging and cancer.
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