Hormones provide the stimulus for cell growth and synthesis in multicellular organisms. For certain cells, however, hormones can initiate a genetically programmed series of events inevitably resulting in cell death. This process is frequently invoked as embryos acquire the adult form during development. Malfunctions result in birth defects. In addition, many adult tissues retain sensitivity to specific hormones; withdrawal or supplementation of the hormone stimulus will cause glandular atrophy and regression. This process provides a means to kill cancers arising from hormonally-sensitive tissue. Our ability to determine the molecular mechanism by which hormones can drive cells to their death could have significant implications for the development of alternative cancer therapies and also allow us to better understand the cause of birth defects and to consider means to prevent their occurrance. In this project, we will utilize the regression of rat prostate tissue in response to androgen deprivation as a main model to characterize the changes in gene activity and cellular physiology which accompany the process of cell death: 1) Hormonally programmed cell death is an active process, requiring the ongoing expression of new gene products. Quantitative Northern and Western blot analysis will be used to show the pattern wherein a cascade sequence of genes (c-fos greater than c-myc greater than hsp-70) is induced in a variety of drying cells and tissues. This analysis will be performed concomitantly with in situ procedures to definitively link the expression of this cascade with the onset of cell death. 2) Activation of c-fos expression at the onset of cell death implies that abrupt changes in intracellular calcium ions levels play a role in this process. Calcium-channel blocking drugs will be utilized in an attempt to prevent or delay regression of hormonally sensitive tissues. In addition, calcium-channel activator drug will be utilized in an attempt to enhance tissue regression. We will also visually quantitate intracellular calcium ion content of dying cells in regressing prostate tissues with the use of a calcium-dependent fluorescent drug, FURA-2. 3) TRPM-2 is an evolutionarily conserved gene, expressed exclusively in atrophic and dying cells and tissues from a wide variety of species. With the use of recombinant DNA techniques, we will clone, sequence and functionally characterize this gene to determine whether its activation is the cause of cell death or whether it is induced in dying tissues in an attempt to avert this process. In either case, it is a fascinating marker of tissue degeneration and atrophy. Antibodies will be produced in order to determine its cellular localization and other physical characteristics of the TRPM-2 proteins product.
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