Browsing by Subject "cell survival"
Now showing 1 - 5 of 5
- Results Per Page
- Sort Options
Item A Systematic Screen of the Saccharomyces Cerevisiae Deletion Mutant Collection for Novel Genes Required for DNA Damage-Induced Mutagenesis(2008-07-01) Gong, Jinjun; Siede, Wolfram; Sheedlo, Harold; Reeves, RustinA Systematic Screen of the Saccharomyces Cerevisiae Deletion Mutant Collection for Novel Genes required for DNA Damage-Induced Mutagenesis. Jinjun Gong Department of Cell Biology and Genetics, University of North Texas Health Science Center, Fort Worth, TX 76107. Summary. Deoxyribonucleic acid (DNA) damage is common in a cell’s lifetime. DNA can be damaged by endogenous factors such as reactive oxygen species (ROS) or exogenous agents such as ultraviolet (UV) or industrial chemicals. DNA damage will trigger cell responses including cell cycle arrest, transcription activation, DNA repair or apoptosis. In addition to various DNA repair mechanisms including damage reversal, base excision repair, nucleotide excision repair, mismatch repair, homologous recombination and non-homologous end joining, translesion DNA synthesis is an important DNA damage tolerance pathway that can bypass the lesion on template DNA to finish the replication for cell survival but at the risk of potential mutation in the daughter cells. Accumulation of mutation may lead to cancer occurrence. Translesion DNA synthesis components are highly conserved from yeast to humans. Important players in trans-lesion synthesis pathway such as Rev1, Rev3 and Rev7 were first discovered in budding yeast. Saccharomyces cerevisiae. Homologues were found later in human cells. I used the Saccharomyces cerevisiae deletion mutant collection to do a systematic screen to search for novel genes required for DNA damage induced mutagenesis in yeast. After CAN1 forward mutation assay for the systematic screen and reverse mutation assay for further confirmation, two candidate genes SWI6 and DOA4 were detected. Deletion of SWI6 and DOA4 decreases mutagenesis of cells. At the molecular level, Swi6, a transcription cofactor, is involved in mutagenesis by regulating expression of REV7 at the mRNA and protein levels. Rev7 is a regulatory subunit of DNA polymerase zeta, which is essential for DNA damage induced mutagenesis as well as spontaneous mutagenesis. Rev7 is not UV inducible or cell cycle regulated. The regulation of Rev7 at the transcriptional level by Swi6 is essential. Future experimental approaches are planned to address the mechanism by which DOA4 is involved in mutagenesis.Item Estrogen Signaling Protects Mitochondrial Membrane Potential Integrity from Oxidative Stress in Lens Epithelial Cells(2008-05-01) Flynn, James Martin; Cammarata, Patrick R.; Wordinger, Robert J.; Dimitrijevich, S. DanFlynn, James Martin, Estrogen Signaling Protects Mitochondrial Membrane Potential Integrity from Oxidative Stress in Lens Epithelial Cells. Doctor of Philosophy, (Cell Biology and Genetics) May, 2008, 265 pages, 36 figures, bibliography, 190 titles. Loss of mitochondrial membrane potential has been determined to be one of the initiating factors in activation of apoptosis after cellular damage. Estrogen and estrogen analogues have been shown to enhance cell survival in numerous tissues through rapid pro-survival cell signaling. This study was focused on elucidating mechanisms through which estrogen protects the cells by preventing the activation of mitochondrial permeability transition pores and the subsequent loss of mitochondrial membrane potential. It is hypothesized that the anti-apoptotic mitochondrial protein BAD, once phosphorylated by estrogen activated upstream kinases, can prevent the formation of the permeability transition pre via direct interaction. To address this, lens epithelial cells were used as a model system for the examination of mitochondrial depolarization during periods of either oxidative or hyperglycemic stress. Estrogen attenuated the loss of impermeability of the mitochondrial membrane, thus maintaining the cells during acute periods of stress. It was discovered that a number of the estrogen receptor isoforms are expressed in lens epithelium, and that the wild-type estrogen receptor-β1 isoform is localized to the mitochondria in lens epithelial cultures derived from both human males and females. siRNA treatment against estrogen receptor-β determined that is a required component to elicit estrogen’s protective abilities against oxidative stress induced mitochondrial depolarization. Furthermore, administration of exogenous estrogen rapidly activated signaling pathways, particularly ERK, which were shown to have influence over the loss of mitochondrial membrane potential. Studies using both pharmacological inhibitors of MAPK signaling, as well as siRNA of ERK2 kinase demonstrate a correlation between the activation of ERK and the severity of response to oxidative stress. Investigation of downstream substrates of ERK revealed that the mitochondrial protein BAD is phosphorylated after the administration of estrogen, yet it is not required for the prevention of mitochondrial depolarization as originally hypothesized. In conclusion, these studies have confirmed a mitochondrial targeted mechanism activated by estrogen which is rapid, gender independent, estrogen receptor-β mediated signal transduction pathway. The targeting of mitochondrial function to reduce oxidative or hyperglycemic stress, thereby preventing activation of the permeability transition pore, defines a novel concept which will contribute to innovative regimens for prevention or treatment of mitochondrial pathology.Item HIF: A Key Survival Factor for Serum-Deprived Prostrate Cancer Cells(2008-05-01) Thomas, Rusha; Jamboor Vishwanatha; Harlan Jones; Raghu KrishnamoorthyThomas, Rusha, HIF: A key survival factor for serum-deprived prostate cancer cells. Doctor of Philosophy (Molecular Biology and Immunology), May 2008, 134 pages, 47 illustrations, reference list, 105 titles. The hypoxia-inducible factor (HIF) is central to hypoxic adaptation of tumors, and consists of an oxygen-labile HIF-1α and a constitutively expressed HIF-1β subunit. In specific aim 1, we report that prolonged serum deprivation is a potent inducer of HIF-1α in PC-3 and LNCaP prostate cancer (PCa) cells, despite normal oxygen conditions. In contrast, cells grown in the presence of serum did not upregulate HIF-1α protein levels. Moreover, HIF-1α protein increase during serum deprivation correlated with increased cell survival, while suppression of HIF-1α expression significantly decreased PCa cell viability. Our results further demonstrate that HIF-1α protein increase during serum deprivation is due to increased HIF-1α protein synthesis. First, there was no significant increase in HIF-1α mRNA. Secondly, cycloheximide, a protein synthesis inhibitor, prevented HIF-1α protein increase in serum-deprived PCa cells. Moreover, the expression of HIF-1α-target genes, VEGF and IGF-2, was concomitantly increased in serum-deprived PCa cells, while suppression of HIF-1α expression markedly inhibited their induction. Most interestingly, our study showed a significant decline in PCa cell survival following inhibitor of IGF-2 activity. Taken together, our study demonstrates for the first time that PCa cells counteract the stress of prolonged serum deprivation by upregulating HIF-1α protein which increases IGF-2 expression to promote cell survival. In specific aims 2 and 3, we investigated the molecular mechanism of autocrine regulation of HIF-1α, IGF-2 and cell survival in serum-deprived PC-3 and LNCaP PCa cells. We detected a time-dependent increase in Akt activation during serum deprivation, and inhibition of Akt activation attenuated the serum deprivation-mediated increase in HIF-1α and cell survival. Importantly, IGF-2 secretion significantly increased during serum deprivation, and was accompanied by increased activation of its receptor, insulin-like growth factor-I receptor (IGF-IR). Additionally, inhibition of IGF-2 activity markedly suppressed the serum deprivation-mediated increase in IGF-IR and Akt activation, HIF-1α expression, as well as its own transcription, suggesting autocrine regulation of HIF-1α expression via IGF-2. Reciprocal regulation of the IGF-2/IGF-IR system and P13K-Akt pathway was further demonstrated by findings wherein Akt activation was prevented following suppression of IGF-IR expression, and IGF-IR activation was inhibited following P13K inhibition. Lastly, HIF-1α suppression abolished the serum deprivation-mediated increase in Akt activation, and also resulted in higher IGF-IR protein levels indicating reduced IGF-IR activation. Collectively, our study demonstrates that a HIF-1α-dependent autocrine feedback loop upregulates HIF-1α, and thus promotes survival of normoxic, serum-deprived PCa cells.Item Serum Deprivation Induces Apoptosis of Retinal Ganglion Cells Utilizing Mitochondrial Signaling Pathways(2003-12-01) Charles, Irma E.; Victoria Rudick; Raghu Krishnamoorthy; Ganesh PrasnnaCharles, Irma E., Serum Deprivation Induced Apoptosis of Retinal Ganglion Cells Utilizing Mitochondrial Signaling Pathways. Master of Science (Biomedical Sciences), December 2003, 90 pp., 10 illustrations. Apoptosis is the genetically regulated death of retinal ganglion cells (RGC) in which there is a blockade of retrograde transport. This blockade results in the loss of neurotrophic growth factors that are essential for the survival of the RGCs. This study uses several different techniques to determine mechanisms underlying apoptosis in rat RGCs deprived of growth factors. An established line of transformed RGC was subjected to serum deprivation for 2-6 days and compared to RGC cells maintained in 10% FBS to study the cellular changes that occur as a result of the treatments. The results show that serum deprivation for 48 hours resulted in a 50% cell loss due to apoptosis. Apoptotic death was associated with activation of caspases 3, 8, and 9 along with increased levels of Bax and death receptors 3 & 4. These results indicate that serum deprivation results in RGC death via mitochondrial and also extrinsic pathways.Item The Role of Glycogen Synthase Kinase-3β in the Regulation of Mitochondrial Membrane Permeability(2014-12-01) Brooks, Morgan M.; Patrick R. CammarataLens epithelial cells in a fully mature lens thrive in a hypoxic environment by developing several pro-survival mechanisms that can prevent cellular dysfunction. Many of these mechanisms focus on maintaining mitochondrial membrane integrity. Loss of integrity of either the inner or outer mitochondrial membrane results in the dissipation of the mitochondrial electrochemical gradient in a process termed mitochondrial membrane permeability transition (mMPT). The project herein focuses primarily on understanding the role of glycogen synthase kinase-3β (GSK-3β) in preventing mMPT in human lens epithelial (HLE-B3) cells; and, understanding that role in relation to extracellular signal-regulated kinase 1/2 (ERK1/2), a known regulator of GSK-3β activity. These studies further define mitoprotective mechanisms of lens cells by identifying how ERK1/2 and GSK-3β can directly (through the mitochondrial transition pore) or indirectly (through the induction of apoptosis) effect mitochondrial membrane potential). Additionally, we extended the GSK-3β studies into the field of epithelial to mesenchymal transition (EMT) research. Specifically we focused on understanding how GSK-3β in conjunction with the hypoxia inducible factor (HIF) proteins can influence the persistence of EMT and the production of vascular endothelial growth factor (VEGF). Collectively, these studies demonstrate important roles in lens epithelial cell mitoprotection for GSK-3β and ERK1/2; and, demonstrate a pivotal role for HIF-1α in the persistence of EMT under hypoxic conditions. Overall, the work described herein has provided invaluable information and understanding in the field of mitoprotection research as well as EMT research.