Of the thousands of dividing progenitor cells (PCs) generated daily in the adult brain only a very small proportion survive to become mature neurons through the process of neurogenesis. Identification of the mechanisms that regulate cell death associated with neurogenesis would aid in harnessing the potential therapeutic value of PCs. Apoptosis, or programmed cell death, is suggested to regulate death of PCs in the adult brain as overexpression of B-cell lymphoma 2 (Bcl-2), an anti-apoptotic protein, enhances the survival of new neurons. To directly assess if Bcl-2 is a regulator of apoptosis in PCs, this study examined the outcome of removal of Bcl-2 from the developing PCs in the adult mouse brain. Retroviral mediated gene transfer of Cre into adult floxed Bcl-2 mice eliminated Bcl-2 from developing PCs and resulted in the complete absence of new neurons at 30 days post viral injection. Similarly, Bcl-2 removal through the use of nestin-induced conditional knockout mice resulted in reduced number of mature neurons. The function of Bcl-2 in the PCs was also dependent on Bcl-2-associated X (BAX) protein, as demonstrated by an increase in new neurons formed following viral-mediated removal of Bcl-2 in BAX knockout mice. Together these findings demonstrate that Bcl-2 is an essential regulator of neurogenesis in the adult hippocampus.

Endocrine resistance is a significant clinical problem in the treatment of estrogen (E2) receptor positive breast cancers. There are two ER subtypes, ERα and ERβ, which promote and inhibit breast cancer cell proliferation respectively. While ER positive breast cancers typically express a high ratio of ERα to ERβ, the acquisition of antiestrogen resistance in vitro and in vivo is associated with increased relative expression of the ERβ. On some gene enhancers ERβ has been shown to function in opposition to the ERα in the presence of E2. Here we demonstrate that exposure to two different ERβ agonists results in decreased cell viability, and produced a marked reduction in G2/M phase in antiestrogen resistant breast cancer cell line in conjunction with altered cyclin D1, and cyclin E expression relative to E2. ERβ agonists also strongly downregulated Bcl-2 expression and recruited both ERs to the Bcl-2 and pS2 E2-response elements resulting in a reduction in mRNA transcripts from both of these genes. Bcl-2 reduction correlated with increased lipidation of LC3-I to LC3-II, indicative of increased autophagic flux. Although ERβ agonist treatment alone did not induce apoptosis, remarkably, the coaddition of ERβ agonist and the autophagy inhibitor, chloroquine, resulted in robust cell death. Lastly, in vivo studies demonstrate that preferential-ERβ agonists are not estrogenic in the uterus or mammary gland. Together, these observations suggest that combined therapies including an ERβ agonist and an autophagy inhibitor may provide the basis for a safe, novel approach to the treatment of antiestrogen-resistant breast cancers.

Preeclampsia and molar pregnancy are two devastating placental pathologies characterized by an immature proliferative trophoblast phenotype accompanied by excessive cell death. It is therefore of paramount importance to study the regulation of cell fate in the placenta, to gain a further understanding of the mechanisms that contribute to these diseases. In this dissertation we report that during normal placental development and in preeclampsia, Matador (Mtd), a pro-apoptotic member of the Bcl-2 family, has a dual function in regulating trophoblast cell proliferation and death. Importantly, we reveal a novel role of Mtd-L in promoting cyclin E1 expression and cell cycle progression. Of clinical importance, we also identify that both cyclin E1 and the CDK inhibitor p27, are increased in severe early onset preeclampsia. However, the inhibitory function of p27 in this pathology may be hampered due to its increased phosphorylation at Ser10, resulting in its nuclear export. Of equal importance, data presented demonstrate that placentae from severe early onset preeclampsia display a molecular profile distinct from late onset preeclampsia or intrauterine growth restricted pregnancies. In the final data chapter we demonstrate that Mtd is highly expressed in molar tissue, where it localizes to both apoptotic and proliferative cells. Our data suggests that an abundance of Mtd and cyclin E1 in conjunction with the low level of p27 may contribute to the hyperproliferative nature of the disorder. The body of work in this dissertation uncovers novel insights into the regulation of trophoblast cell fate. Importantly, the impact of Mtd on cyclin E1 to promote G1-S transition is a novel mechanism found to regulate trophoblast cell proliferation in normal and pathological placentation. Equally important is our identification of molecular differences between placental pathologies that may help to differentiate early and late onset preeclampsia, IUGR and molar pregnancy.

Preeclampsia and molar pregnancy are two devastating placental pathologies characterized by an immature proliferative trophoblast phenotype accompanied by excessive cell death. It is therefore of paramount importance to study the regulation of cell fate in the placenta, to gain a further understanding of the mechanisms that contribute to these diseases. In this dissertation we report that during normal placental development and in preeclampsia, Matador (Mtd), a pro-apoptotic member of the Bcl-2 family, has a dual function in regulating trophoblast cell proliferation and death. Importantly, we reveal a novel role of Mtd-L in promoting cyclin E1 expression and cell cycle progression. Of clinical importance, we also identify that both cyclin E1 and the CDK inhibitor p27, are increased in severe early onset preeclampsia. However, the inhibitory function of p27 in this pathology may be hampered due to its increased phosphorylation at Ser10, resulting in its nuclear export. Of equal importance, data presented demonstrate that placentae from severe early onset preeclampsia display a molecular profile distinct from late onset preeclampsia or intrauterine growth restricted pregnancies. In the final data chapter we demonstrate that Mtd is highly expressed in molar tissue, where it localizes to both apoptotic and proliferative cells. Our data suggests that an abundance of Mtd and cyclin E1 in conjunction with the low level of p27 may contribute to the hyperproliferative nature of the disorder. The body of work in this dissertation uncovers novel insights into the regulation of trophoblast cell fate. Importantly, the impact of Mtd on cyclin E1 to promote G1-S transition is a novel mechanism found to regulate trophoblast cell proliferation in normal and pathological placentation. Equally important is our identification of molecular differences between placental pathologies that may help to differentiate early and late onset preeclampsia, IUGR and molar pregnancy.

<p>Normal cells require input from extrinsic growth factors to control proliferation and survival. Recent studies have demonstrated that these same extrinsic signals also regulate cellular metabolism to ensure that metabolism adequately supports the demands of cell function, proliferation, and cell survival. The PI3K/Akt pathway is downstream of many growth factors and can promote both glucose metabolism and cell survival. Aberrant activation of the PI3K/Akt pathway is common in cancer, and its activation can contribute to the growth factor independence that is a hallmark of neoplastic cells. Metabolic demand is high in stimulated and leukemic T cells, and activation of Akt can increase glucose metabolism to meet these requirements. There is great interest in targeting the unique metabolism of cancer cells for cancer therapy, thus making an understanding of the interaction of metabolism and cell death essential. </p><p>Akt is also anti-apoptotic and can inhibit cell death by regulating members of the Bcl-2 family. Interestingly, the ability of Akt to prevent cell death is inextricably linked to its metabolic function. Several recent studies have demonstrated that glucose metabolism can affect Bcl-2 to family members to promote cell survival, but the role of Akt-dependent glucose metabolism in the regulation of Bcl-2 family members is not understood. Using a model of growth factor withdrawal-induced apoptosis, we show that Akt prevents cell death by maintaining glucose metabolism to regulate the Bcl-2 family members Puma and Mcl-1, and demonstrate the importance of this pathway in the survival of stimulated T lymphocytes and leukemia.</p><p>After growth factor withdrawal, active Akt suppressed Puma induction in abundant glucose, but Puma was rapidly upregulated in glucose-deficient conditions and was necessary and sufficient to promote efficient cell death. Importantly, glucose was not uniquely required, as provision of alternative mitochondrial fuels allowed Akt to suppress Puma and maintain survival. This metabolic regulation of Puma was mediated through partially p53-dependent transcriptional induction as well as control of Puma protein stability. </p><p>In addition to inhibiting Puma expression, active Akt prevented the loss of Mcl-1 after growth factor withdrawal by sustaining Mcl-1 protein synthesis in a glucose-dependent manner. Mcl-1 was essential for preventing Bim-induced apoptosis, as Akt could not inhibit Bim induction after growth factor deprivation. Slowing of Mcl-1 synthesis by inhibiting glucose metabolism reversed Mcl-1-mediated resistance of leukemic cells to the Bcl-2 inhibitor ABT-737. Importantly, Akt and glucose-reliant Mcl-1 expression required mTOR-dependent phosphorylation of 4EBP, and treatment with mTOR inhibitors also reversed ABT-737 resistance. </p><p>Together, this study demonstrates that Akt promotes cell survival by preventing metabolic checkpoints that stimulate Puma expression and stability and inhibit Mcl-1 synthesis, advancing our understanding of the links between metabolism and cell death. These studies highlight the importance of cellular metabolism--including a potential role for the alternative sugar fructose--in cancer cell survival that may provide a mechanistic understanding to drive development of metabolism-targeted cancer therapies.</p> / Dissertation

<p>Tissue homeostasis is controlled by the availability of growth factors, which sustain exogenous nutrient uptake and prevent apoptosis. Cancer cells, however, can express constitutively active oncogenic kinases such as BCR-Abl that promote these processes independent of extrinsic growth factors. When cells are deprived sufficient growth signals or when oncogenic kinases are inhibited, glucose metabolism decreases and cells activate the self-digestive process of autophagy, which clears damaged organelles and provides degradation products as an alternate fuel to support mitochondrial metabolism. Importantly, loss of growth signals can also lead to apoptosis mediated through Bcl-2 family proteins, and Bcl-2 has been reported to interfere with autophagy, potentially disrupting a key nutrient source just as glucose uptake becomes limiting. Since autophagy may support survival or lead to death depending on context, the role of this pathway in apoptosis-competent growth factor deprived cells remains unclear.</p><p>In this thesis, I examine the interactions of autophagy with Bcl-2 family proteins and apoptosis upon inhibition of growth signals in hematopoietic cells. In contrast to other studies, I found autophagy was rapidly induced in growth factor deprived cells regardless of Bcl-2 or Bcl-xL expression, and this led to increased production of fatty acids and amino acids for metabolism. While these data suggested autophagy may play a key role to support metabolism of growth factor deprived cells, provision of exogenous pyruvate or lipids as alternate fuel had little affect on cell survival. Instead, I found that autophagy modulated cell stress pathways and Bcl-2 family protein expression in a context specific fashion to impact cell fate.</p><p>My results show that autophagy's effect on cell survival is dependent on its level of induction within a cell. I observed that partial suppression of autophagy protects cells from stress and induction of pro-apoptotic Bcl-2 family expression, while complete inhibition of autophagy enhances stress and is pro-apoptotic. In experiments using shRNAi to partially suppress autophagy, I found increased survival upon growth factor deprivation in several different types of cells expressing anti-apoptotic Bcl-2 or Bcl-xL, indicating that autophagy promoted cell death in these instances. Cell death was not autophagic, but apoptotic, and relied on direct Chop-dependent transcriptional induction of the pro-apoptotic Bcl-2 family protein Bim. In contrast, complete acute disruption of autophagy through conditional Cre-mediated excision of the autophagy-essential gene Atg3 led to p53 phosphorylation, upregulation of p21 and the pro-apoptotic Bcl-2 family protein Puma, and rapid cell death of cells the presence or absence of growth factor. Importantly, transformed BCR-Abl-expressing cells had low basal levels of autophagy but were highly dependent on this process. Deletion of Atg3 or treatment with chemical autophagy inhibitors led to rapid apoptosis, and BCR-Abl expressing cells were unable to form leukemia in mice in without autophagy. Together, my data demonstrate a dual role for autophagy in cell survival or cell death and suggest that the level of autophagy in a cell is critical in determining its role in apoptosis and cell fate. Ultimately, these results may help to determine future approaches to modulate autophagy in cancer therapy.</p> / Dissertation

Purpose: To determine the expressions of bcl-2, p53, Ki-67 and PTEN on the basis of immunohistochemistry methods in upper urinary transitional cell carcinoma (TCC), and to correlate their presentations in specimens with clinical tumor stage, grade and patient survival. Material and Method: Paraffin-embedded primary upper urinary TCC specimens were divided into 2 groups for immunohistochemical study: Group 1 including 91 cases were treated with bcl-2, p53 and Ki-67 antibodies; group 2 including 93 specimens contained both tumor and benign tissues were treated with PTEN antibody. Semi- quantitatively, according to the amount of the stained cells, they were divided into 3 levels: level 1, scanty; level 2, focal; and level 3, diffuse. Association of immunoreactivity with tumor grade and stage was examined. Prognostic significance of tumor marker expression in patients¡¦ survival was accessed. Results: Group1: Of the 91 tumors most (98.9%) of the specimens showed level 1 bcl-2 expression and only 1 patient had level 2 expression. The p53 mutations were identified level 3 expression in 48.4% of the cases, followed by level 2 (26.4%) and level 1 (25.3%) identifications. The Ki-67 expression was recognized level 3 in 6 patients, level 2 in 21 and level 1 in 66 cases. Significant correlations were seen between p53 expression and tumor grading (p=0.004) and between immunostain of Ki-67 and clinical stage (p=0.031). The p53, bcl-2 and Ki-67 expressions in upper urinary tract TCC specimens were not a significant factor of patients¡¦ survival. Group 2: Of the tumors all cytoplasm has level 3 PTEN expressions and the nuclei, 18 (19.4%) showed scanty expression, 35 (37.6%) revealed focal expression, and diffuse expression was noted in 40 (43.0%) cases. Loss of PTEN expression in tumor nuclei was positively correlated with pathologic stage (p=0.019). Of the fibrocytes adjacent to tumor cells, the nuclei showed 24 (25.8%) scanty, 59 (63.4%) focal and 10 (10.8%) diffuse distribution of PTEN expressions. Poorly differentiated tumor (grade 3) specimens were correlated with loss of PTEN expression in fibrocytic nuclei adjacent to tumor (p=0.028). Most (58%) fibrocytic cytoplasm was scanty PTEN expression, followed by 23 (24.7%) diffuse and 16 (17.2%) focal immunostaining. PTEN expressions in upper urinary tract TCC specimens were not a significant factor of patients¡¦ survival. Conclusions: We examined 93 surgical specimens of upper urinary tract TCC for the expression of PTEN and 91 cases for bcl-2, p53 and Ki-67 by immunohistochemical stained. Correlation between tumor grading and p53 mutations and correlation between clinical stage and Ki-67 immunoreactivity were observed. Meanwhile, loss of PTEN expression in tumor nuclei of upper urinary TCC is correlated significantly with advanced tumor stage, and poorly differentiated tumor specimens were correlated with loss of PTEN expression in normal nuclei adjacent to tumor cells. However, no correlation between overall survival rate and tumor markers was identified. Thus, the detection of p53, bcl-2, Ki-67 and PTEN would be not enough for evaluation the prognosis of upper TCC.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 99-131).

Ovarian cancer is the fifth leading cause of cancer death among women in United States and has an alarming 1.4% (1 in 71) lifetime risk. The lack of overt symptoms and the absence of a reliable screening test to detect ovarian cancer result in over 70% of women being diagnosed after the disease has spread beyond the ovary resulting in a poor prognosis. A key characteristic of ovarian cancer is the ability of tumor cells to evade apoptosis, or programmed cell death contributing to the limitless replicative potential, which is a hallmark of all carcinogenesis. There is conclusive evidence that levels of bcl-2 are elevated in ovarian cancer patients' indication that this protein is an ovarian cancer biomarker. The overall goal of this thesis is to functionalize a substrate for specific, sensitive and cost-effective bcl-2 capture. This surface will ultimately be incorporated into an acoustic wave-based diagnostic device for worldwide point-of-care (POC) ovarian cancer detection. This research looks to assess the capture of this analyte protein on a series of bioconjugated surfaces. For the research to be diagnostically applicable, certain factors reveal themselves as more important than others. Since the surface-bound capture antibody must recognize the bcl-2 protein, it is vital to ensure upright orientation of this specific antibody with high affinity for the analyte. Furthermore once integrated with a nanosensor, the surface will sense a change in the mass on the surface, which requires that the surface is highly resistant to non-specific binding. Bioconjugation techniques were employed to initiate self-assembled monolayers (SAM) of silanes, immobilize antibodies (via amine-crosslinking or direct adsorption of protein A/G) and disperse polyethylene glycol (PEG) reagents to reduce non-specific binding on the glass substrates. 3-aminopropyltrimethoxysilane (3-APTMS) and chlorodimethyloctylsilane (ODMS) were deposited on the surface to create initial hydrophilic and hydrophobic properties on which molecular self-assembly could occur. Testing a variety of assemblies with and without the presence of silanes, amine-crosslinking and PEGylation reagents, the substrate displaying the highest efficacy of bcl-2 capture was revealed. These various surfaces were assessed through contact angle and a novel sandwich enzyme linked immunosorbent assay (ELISA) for sensitivity and specificity of bcl-2 standard capture. The consistently low background and facile assembly of the ODMS based substrate with direct adsorption of protein A/G and the PEGylation reagent, Pluronic, was deemed the best functionalized surface for non-specific recruitment of the bcl-2 protein. The substrate also consistently displayed low signal-to-noise ratio which was of extreme importance in this research to guarantee the prevention of false-positive results when detecting nascent carcinogenic behavior. Elucidation of this substrate assembly is the first step towards the long term objective of this thesis, which is to construct a cost-effective early ovarian cancer detection device which can be implemented at the point-of-care to those who need it the most. This is ultimately expected to dramatically improve health outcomes for females worldwide.

Apoptosis is a conserved program of cell death that promotes organism homeostasis in all stages of life. Two main pathways activate caspases, which are cysteinyl-aspartate proteases that execute apoptosis. The extrinsic pathway is initiated by cell surface death receptors, while the intrinsic pathway is initiated by intracellular signals that cause permeabilization of the outer mitochondrial membrane (MOMP). The Bcl-2 protein family regulates MOMP, which causes the release of several pro-apoptotic proteins (such as cytochrome c, Smac) into the cytosol. Bcl-2 proteins share homology in up to four "BH" domains and are subdivided into three subgroups. Pro-apoptotic Bax and Bak catalyze pore formation in the mitochondria, while anti-apoptotic members (Bcl-2, Mcl-1) inhibit MOMP. The third subgroup, termed BH3-only, promotes MOMP by either antagonizing Bcl-2 proteins or by directly activating Bax/Bak, and initiate apoptosis in response to various stressors, including heat shock (HS). Hyperthermia or acute HS reportedly induces apoptosis through caspase-2-mediated cleavage of BID, engaging the intrinsic pathway. However, additional evidence suggests that this pathway could represent an amplification loop. Thus we hypothesized that during HS, another BH3-only protein such as BIM, that does not require cleavage, could engage MOMP. Herein, we report that BIM mediates an alternative HS-induced apoptosis pathway. Cells lacking BIM are resistant to HS and exhibit better short and long-term survival than either Bid[superscript -/-] or Bax[superscript -/-]Bak[superscript -/-]. Moreover, caspase-2 induces apoptosis in Bim[superscript -/-] but not Bid[superscript -/-] cells, implying that caspase-2 kills exclusively through BID. Interestingly, Bim[superscript -/-] and Bax[superscript -/-]Bak[superscript -/-] cells are entirely resistant to MOMP, but the Bax[superscript -/-]Bak[superscript -/-] cells still undergo caspase-3 activation and remain partially sensitive to HS, indicating that BIM triggers caspase-3 activation upstream of mitochondria. Thus, BIM plays an important role in HS-induced apoptosis. Hyperthermia has clinical applications for the treatment of solid tumors. Unfortunately, a practical limitation is the development of thermotolerance, which confers resistance not only to subsequent HS but also to radiotherapy and chemotherapy. Therefore, a better understanding of the molecular mechanisms involved both in heat-induced apoptosis and thermotolerance could lead to new therapeutic interventions. Here we also show evidence for a putative role for the stress kinase JNK signaling pathway in the regulation of thermotolerance. / text