The proliferation and differentiation of normal prostate epithelial cells depend upon the action of the androgens, testosterone and dihydrotestosterone. Prostate cancer cells retain the ability to respond to androgens in the initial stages of cancer development but progressively become independent of exogenous androgens in advanced stages of the disease while maintaining the expression of functional androgen receptor (AR). We hypothesized that prostate cancer cells in the advanced stages of the disease acquire capability to synthesize androgens which activate AR in an intracrine manner. To test this hypothesis, we determined the expression of proteins and enzymes involved in cholesterol uptake, transport and its conversion into testosterone in androgen-dependent and androgen-independent prostate cancer cell lines. Established androgen independent prostate cancer cell lines, PC3 and DU145 cells, expressed mRNA and proteins for scavenger receptor type B 1 (SRB 1), steroidogenic acute regulatory (StAR) protein, metastatic lymph node 64 (MLN64), cytochrome P450 cholesterol side chain cleavage (CYP11), and other enzymes involved in androgen biosynthesis. Expression of all these proteins and enzymes was significantly higher in the androgen-independent derivative of LNCaP prostate cancer cells (C81) than in the androgen-dependent cell line (C33). In serum free cultures, the androgen independent cell line C81 secreted ~5 fold higher testosterone than C33 as determined in the conditioned media by specific immunoassays. These cells also converted radioactive cholesterol into testosterone which was identified by thin layer chromatography. To evaluate the effects of endogenous production of testosterone on the survival and growth of prostate cancer cells, C81 cells were treated with either aminoglutethimide to prevent conversion of cholesterol to pregnenolone or the AR antagonist, bicalutamide. Our results demonstrated growth inhibition ofC8l cells and a reduction in expression of the AR regulated genes, PSA and TMPRSS2 in aminoglutethimide and bicalutaniide treated cells which were reversed by exogenous androgens. These results indicate that prostate cancer cells in advanced stages of the disease synthesize androgens from cholesterol. These androgens activate AR in an intracrine maimer to regulate cell survival and proliferation. The ability of the prostate to synthesize androgens may be associated with progression to castration resistant prostate cancer and possibly represents a therapeutic target for advanced disease.

<p> Abstract: Determination is the process in which a stem cell commits to differentiation. The process of how a cell goes through determination is not well understood. Determination is important for proper regulation of cell turn-over in tissue and maintaining the adult stem cell population. Deregulation of determination or differentiation can lead to diseases such as several forms of cancer. In this study I will be using microarrays to identify candidate genes involved in determination by pulse induction of mouse erythroleukemia (MEL) cells with DMSO and looking at gene expression changes as the cells go through the early stages of erythropoiesis. The pulsed induction method I have developed to identify candidate genes is to induce cells for a short time (30 min, 2 hours, etc.) and allow them then to grow for the duration of their differentiation time (8 days). For reference, cells were also harvested at the time when the inducer is removed from the media. Results show high numbers of genes differentially expressed including erythropoiesis specific genes such as GATA1, globin genes and many novel candidate genes that have also been indicated as playing a role in the dynamic early signaling of erythropoiesis. In addition, several genes showed a pendulum effect when allowed to recover, making these interesting candidate genes for maintaining self-renewal of the adult stem cell population.</p>

Because Drosophila do not grow after initiation of metamorphosis, their final body size is determined by larval growth rate and duration of the larval growth phase. Drosophila metamorphosis is triggered by the steroid hormone ecdysone, which is produced in the prothoracic gland (PG). Ecdysone synthesis requires expression of the "Halloween" genes, which encode ecdysone biosynthetic enzymes. Growth rate is regulated by Insulin-like peptides, which are released from the insulin-producing cells (IPCs). Genetic ablation of the IPCs decreases growth rate and delays onset of metamorphosis, suggesting that ecdysone synthesis is induced by insulin signaling. Inhibiting PI3 Kinase (PI3K), the major effector of insulin signaling, in the PG similarly delays metamorphosis as a consequence of decreased ecdysone synthesis and decreased Halloween gene expression. In contrast, activating PI3K in the PG advances the onset of metamorphosis and increases Halloween gene expression. Here I report that increased insulin signaling, accomplished inhibiting the protein kinase A pathway in the IPCs increases insulin signaling and increases growth rate but also advances the onset of metamorphosis by increasing expression of at least one Halloween gene. Ecdysone synthesis is promoted by a second peptide hormone, PTTH, which activates Halloween gene expression via the Torso receptor followed by Ras and Raf in the PG. Null mutations in the transcription factor broad (br ) prevent torso transcription and thus prevent Halloween gene expression and metamorphosis. Here I identify Br as the mechanistic link between PI3K activity and Halloween gene expression. I found that PI3K activity is required for br expression by inhibiting the downstream kinase GSK-3. I provide evidence that three nuclear hormone receptors, βFTZ-F1, HR3 and E75, link GSK-3 activity with br expression: RNAi-mediated βFTZ-F1 or HR3 knockdown, or E75A overexpression, in the PG prevents br expression. I also found that ectopic Torso pathway activation, accomplished by expressing the constitutively active Rafgof , restores Halloween gene transcription to larvae lacking br or βFTZ-F1 , suggesting that these larvae fail to express Halloween genes because they fail to transcribe torso . These studies identify a potential molecular mechanism linking growth rate with competence to respond to the PITH metamorphic signal and thus initiate metamorphosis.

No description available.

In vertebrates, the heart is the first organ to form and function. The basic steps and molecular pathways involved in heart development are highly conserved. Myocardial progenitor-fated cells are among the first cells to migrate during gastrulation away from the primitive streak. These cells move bilaterally to populate the heart-forming region (HFR) in the anterior lateral plate mesoderm (ALPM). Once cells have reached the HFR, they receive the signals necessary to differentiate into myocardial progenitor cells. It is clear that the development of myocardial progenitor cells entails the migration of cells from the lateral embryonic margin to the ALPM. However, it is unclear whether cells are specified for a myocardial progenitor fate early in embryogenesis, a step that may promote their migration specifically to the ALPM, or whether the migration of cells to the ALPM alone is sufficient for differentiation into myocardial progenitor cells. A zebrafish mutant, grinch (grn), was indentified in which there is a defect in the development of myocardial progenitor cells. The mutation resulting in the grn phenotype was mapped to the gene encoding the G protein-coupled receptor Apelin receptor b (Aplnrb). I have used the aplnrb mutant embryo, as well as morpholino-mediated knockdown (morphant embryos) of aplnrb, and its paralog aplnra, to determine the function of Aplnr signaling in myocardial progenitor development. Results demonstrate that Aplnr signaling is necessary for the migration of cells from the lateral embryonic margin of the zebrafish embryo to the heart-forming region. Interestingly, this entails a novel cell-non-autonomous function for Aplnr signaling. Furthermore, both the only identified ligand for the receptor, Apelin, and the canonical mediators of Aplnr signaling, Gαi/o proteins, appear to be dispensable for this process. Loss of Aplnr signaling also appears to affect embryonic patterning of the early embryo through subtle perturbations of Nodal, Wnt, and Bmp signaling and attenuation of Nodal signaling can partially recapitulate the aplnr morphant cardiac phenotype. Taken together, my results suggest that Aplnr signaling plays a role in creating an environment that allows for the migration of cells to the heart-forming region, possibly through the regulation of early embryonic patterning.

In vertebrates, the heart is the first organ to form and function. The basic steps and molecular pathways involved in heart development are highly conserved. Myocardial progenitor-fated cells are among the first cells to migrate during gastrulation away from the primitive streak. These cells move bilaterally to populate the heart-forming region (HFR) in the anterior lateral plate mesoderm (ALPM). Once cells have reached the HFR, they receive the signals necessary to differentiate into myocardial progenitor cells. It is clear that the development of myocardial progenitor cells entails the migration of cells from the lateral embryonic margin to the ALPM. However, it is unclear whether cells are specified for a myocardial progenitor fate early in embryogenesis, a step that may promote their migration specifically to the ALPM, or whether the migration of cells to the ALPM alone is sufficient for differentiation into myocardial progenitor cells. A zebrafish mutant, grinch (grn), was indentified in which there is a defect in the development of myocardial progenitor cells. The mutation resulting in the grn phenotype was mapped to the gene encoding the G protein-coupled receptor Apelin receptor b (Aplnrb). I have used the aplnrb mutant embryo, as well as morpholino-mediated knockdown (morphant embryos) of aplnrb, and its paralog aplnra, to determine the function of Aplnr signaling in myocardial progenitor development. Results demonstrate that Aplnr signaling is necessary for the migration of cells from the lateral embryonic margin of the zebrafish embryo to the heart-forming region. Interestingly, this entails a novel cell-non-autonomous function for Aplnr signaling. Furthermore, both the only identified ligand for the receptor, Apelin, and the canonical mediators of Aplnr signaling, Gαi/o proteins, appear to be dispensable for this process. Loss of Aplnr signaling also appears to affect embryonic patterning of the early embryo through subtle perturbations of Nodal, Wnt, and Bmp signaling and attenuation of Nodal signaling can partially recapitulate the aplnr morphant cardiac phenotype. Taken together, my results suggest that Aplnr signaling plays a role in creating an environment that allows for the migration of cells to the heart-forming region, possibly through the regulation of early embryonic patterning.

Thesis (M.S.)--Baylor University, 2008. / Includes bibliographical references (p. 31-35)

Thesis (Ph.D.)--University of California, San Francisco, 2004. / Bibliography: leaves 136-147. Also available online.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1970. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (M.S.)--Bowling Green State University, 2009. / Document formatted into pages; contains vii, 44 p. : ill. Includes bibliographical references.