Browsing by Subject "Apoptosis"
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Item Age-dependent alterations in spermatogenesis in itchy mice(2012-12) Dwyer, Jessica Leigh; Richburg, John H.; Mills, Edward; DiGiovanni, John; Huibregtse, Jon; Sanders, BobSpermatogenesis is an intricate process that strongly depends on the rapid turnover of short-lived proteins, both in the differentiating germ cells and in the supportive Sertoli cells. Recent evidence has demonstrated the importance of the ubiquitin-proteasome system for this turnover, with the final enzymatic E3 ligase providing the target specificity. One E3 ligase, Itch, has been well characterized in the immune system, but its role during spermatogenesis is not yet well understood. Mice lacking functional Itch protein display a late onset autoimmune disease characterized by severe inflammation, infiltration of immune cells into various organs, and most apparently chronic dermatitis, ultimately dying from pulmonary inflammation at 6 to 9 months of age. The work presented here evaluates the testes of itchy mice at two developmental time points, during the peri-pubertal period at postnatal day (PND) 28 and at adulthood, PND 56. Itchy mice are smaller in size and have lower spermatid head counts, most likely resulting from an increase in germ cell apoptosis rather than a decrease in Sertoli cell number. Litter sizes are reduced in the homozygous itchy colonies, with data suggesting a defect during fetal development and not in gamete production, although survival rates tend to be similar to that of wild type. At PND 28, itchy mice show a delay in spermatogenesis and an increase in meiotic figures, while PND 56 mice show alterations in germ cell layers, spermatid head formation, and irregular cell division. Examination of the previously identified targets of Itch revealed no significant increases in the testis, but led to discovery of immunoglobulin (IgG) deposits within the interstitial space. Changes in protein expression outside of the seminiferous epithelium suggest that cells of the immune system may be influencing proper development and functional spermatogenesis in the testis. While the previous studies using the itchy mice focused primarily on the late onset autoimmune dysfunction in these animals, increased spleen weights and changes in testicular protein are observed as early as PND 28, indicating that the loss of Itch impacts these animals much earlier during development. Taken together, these data indicate that Itch is required for functional spermatogenesis and that it may play different cellular roles depending on the developmental age of the animal. Future work is targeted at identifying the possible testis-specific targets of Itch and deciphering whether the observed phenotypes are the result of the primary loss of Itch or are a secondary effect from the overactive immune system.Item The anticancer effects of vitamin E derivative alpha-tea in human hematological malignancies(2010-12) Lu, Na, 1978-; Sanders, Bob G.; Kline, Kimberlyalpha-TEA (alpha-tocopherol ether linked acetic acid) has been shown to induce apoptosis in human prostate, ovarian and breast cancer cells in culture and in xenograft models by promoting pro-apoptotic pathways and inhibiting anti-apoptotic pathways. Studies investigated the ability of alpha-TEA to induce apoptosis in human hematological malignant cell lines Jurkat, Raji and U266, representing T cell leukemia, B cell lymphoma and multiple myeloma, respectively. The three cell lines were cultured in the presence of different concentrations of alpha-TEA for different time periods, and examined for apoptosis by annexin V – FITC analyses, DAPI staining, and western blotting for poly (ADP-ribose) polymerase cleavage. alpha-TEA induced apoptosis in all three cell lines in a dose and time dependent manner. Levels of pro-apoptotic molecules DR5, c-Jun N-terminal protein kinase (JNK), C/EBP homologous protein (CHOP), caspase 9, and caspase 3 were upregulated in alpha-TEA treated cells in comparison to vehicle controls. Caspase 8 was activated in Jurkat and U266 cells but not in Raji cells. Apoptosis and pro-death signaling mediators were blocked by ceramide inhibitor, desipramine. The anti-apoptotic nuclear factor kappa B (NF-[kappa]B) signaling pathway was down-regulated in alpha-TEA treated Raji and U266 cells. Combinations of omega-3 fatty acid docosahexaenoic (DHA) and alpha-TEA significantly enhanced apoptosis in Jurkat cells in comparison to single treatments and vehicle control. In summary, alpha-TEA induced apoptosis in the malignant hematological cell lines is via shared and distinct pathways. ASMase/ceramide-mediated JNK activation and endoplasmic reticulum (ER) stress mitochondrial dependent apoptosis are involved in alpha-TEA induced apoptosis in the three cell lines; however, the cell lines exhibit cell type-specific responses to alpha-TEA: activation of death receptor/caspase 8 pathway is involved in Jurkat cells, suppression of NF-[kappa]B signaling is involved in Raji cells, and the U266 cells share both of these pathways for the induction of apoptosis.Item Apoptosis Determinants in Drosophila melanogaster(2007-12-17) Chew, Su Kit; Abrams, JohnApoptosis is a form of programmed cell death (PCD) that is governed by a core set of genes conserved across diverse metazoan phyla. Cells dying by apoptosis exhibit a characteristic series of morphological and biochemical changes that is also conserved. This form of PCD plays pivotal roles in homeostatic regulation of cell numbers, developmental sculpting of organs, damage and infection responses; conversely, its disregulation has profound implications in diseases such as cancers, immune disorders infertility and dystrophies. Common parallels in the regulation of the core apoptosis machinery have been elucidated in human and experimental model organisms, though many fundamental questions in our understanding of its regulation remain. A conserved node in the apoptosis pathway is the apoptosome, comprising the apical caspase and its adaptor protein. To understand the functions of this node, I generated a null allele of the apical caspase Dronc in the experimental model organism Drosophila melanogaster. Dronc is required for developmentally regulated apoptosis in multiple tissues during embryogenesis and larval development. Failure of apoptosis correlated with tissue hyperplasia. Notably, the removal of Dronc eliminated the cellular apopototic response to stresses in cells. In some of the stress contexts tested, Dronc depletion partially rescued cell viability to the same levels as pan-caspase inhibition by small peptide inhibitors, suggesting that Dronc functions map specifically to caspase activation and apoptosis. These and similar observations in its adaptor protein Dark point to the apoptosome as a key node for apoptosis in Drosophila. From these observations, I sought to use the induced apoptosis cellular response as a means to identify novel components and regulators in the apoptosis pathway. I optimized a cell culture system for high-throughput cell-based screening using RNA interference (RNAi) mediated gene silencing and a synthetic antagonist of inhibitors of apoptosis proteins (IAPs). From a genome-wide Drosophila RNAi library, I identified 42 potential genes required for apoptosis, of which I characterized 13 highly validated targets for their requirements in multiple stress contexts. One of these hits, Tango7, regulates pro-Dronc protein and represents an unprecedented point of apoptosis regulation. Collectively, my studies bolster the model for the crucial requirement of the apoptosome in apoptosis and identify new regulation entry-points into the apoptosis pathway.Item Bcl-2 Function in Drosophila(2007-12-17) Galindo, Kathleen A.; Abrams, JohnBcl-2 family members are pivotal regulators of programmed cell death (PCD). In mammals, pro-apoptotic Bcl-2 family members initiate early apoptotic signals by causing the release of cytochrome c from the mitochondria, a step necessary for the initiation of the caspase cascade. Worms and flies do not show a requirement for cytochrome c during apoptosis, but both model systems express pro- and anti-apoptotic Bcl-2 family members. Drosophila encodes two Bcl-2 family members, Debcl (pro-apoptotic) and Buffy (anti-apoptotic). To understand the role of Debcl in Drosophila apoptosis, we produced an authentic null allele at the Debcl locus. Although gross development and lifespans were unaffected, we found that debcl was required for pruning cells in the developing central nervous system. debcl genetically interacted with the ced-4/Apaf-1counterpart, dark, but was not required for killing by RPR proteins. Surprisingly, in a model of caspaseindependent cell death, we found that heterologous killing by Murine Bax required debcl to exert its pro-apoptotic activity. DebclKO mutants were also significantly affected for mitochondrial density. Taken together, these findings suggest that evolutionary functions impacting mitochondrial properties represent ancient activities which preceded the evolution of these proteins as central regulators of PCD.Item Biochemical Pathways in Apoptosis(2005-05-03) Nijhawan, Deepak; Wang, XiaodongCaspases are a family of proteases that once activated execute apoptosis, a cellular suicide pathway. Activated caspases have a unique property to cleave and activate themselves. Once the first caspase is activated, it generates a chain reaction resulting in robust caspase activity and rapid death. The central question in apoptosis is to understand how the first caspase is activated. To address this question, we present an assay that recapitulates de novo caspase activation in vitro. We describe how this assay was used to purify three proteins that were sufficient to reconstitute caspase activation in vitro: Apaf-1, cytochrome c, and caspase-9. The mechanism of caspase activation in vivo, however, is complicated by these proteins subcelluar localization. In the living cell, Apaf-1 and caspase-9 are cytoplasmic whereas cytochrome c is mitochondria, however, during apoptosis, cytochrome is released to the cytoplasm. In vitro, cytochrome c induces the formation of a stable Apaf-1/caspase-9 complex and caspase-9 autoactivation suggesting that cytochrome c release from the mitochondria to the cytosol is the rate-limiting event that leads to caspase activation. This conclusion shifted the focus of our studies upstream from what initiates caspase activation to what triggers cytochrome c release. The second part of the dissertation uses a biochemical approach to identify how cytochrome c release is regulated after exposure to ultraviolet light. Ultraviolet light irradiation of HeLa cells triggers an apoptotic response mediated by mitochondria. Biochemical analysis of such a response revealed that the initial step leading to cytochrome c release is the complete disappearance of the mRNA of Mcl-1, an anti-apoptotic member of the Bcl-2 family. This event leads to the elimination of Mcl-1 protein from cells due to the short half-life of its protein. The block or delay of Mcl-1 disappearance by either proteasome inhibitors or Mcl-1 over-expression prevents subsequent steps of this apoptotic pathway including the translocation of Bax and Bcl-xL from cytosol to mitochondria and dephosphorylation of BimEL on mitochondria. These sequential events lead to the oligomerization of Bax and Bak on the mitochondria, cytochrome c release and caspase activation.Item Characterization of small molecule smac mimetric's role in inducing apoptosis in human cancer cells(2009-09-19) Yalcin-Chin, Asligul; Wang, XiaodongInhibitor of apoptosis proteins (IAPs) regulates apoptosis by inhibiting caspases. This inhibition mechanism is an escape from death used by some human cancers. Second mitochondria-derived activator of caspases (Smac), a mitochondria-released protein during apoptosis, binds to IAPs BIR domains with four amino acid residues (AVPI) and releases the inhibition caused on caspases by IAPs. With the idea of designing a Smac mimicking drug, that will induce apoptosis in cancer cells, we synthesized a small molecule Smac mimetic compound. I tested the ability of the Smac mimetic compound to induce apoptosis on several human cancer cells in combination with chemotherapeutic agents. Unexpectedly, in 25% of the cancer cells we tested, Smac mimetic treatment alone caused apoptosis. Of the cancer cells that were sensitive to Smac mimetic, MDA-MB231 human breast cancer cells and HCC44, HCC461, H2126 lung cancer cells had the highest sensitivity. In addition, a majority of the lung cancer cell lines I tested were sensitive to TNF and/or TRAIL in combination with Smac mimetic. We identified the target of Smac mimetic to be XIAP, cIAP1, and cIAP2 in both Smac mimetic induced and TNF/Smac mimetic induced apoptosis. Moreover, we were able to mimic the Smac mimetic effect by triple knockdown experiments of IAPs in TNF induced cell death. Furthermore, we identified the target of Smac mimetic to be XIAP in the TRAIL pathway. This work identifies the targets and mechanism of Smac mimetic induced cell death in cancer cells.Item Characterizaton of Mcl-1 in Regulating Different Forms of Cell Death(2007-12-17) Gao, Wenhua; Wang, XiaodongProgrammed Cell Deaths (apoptosis, autophagic cell death and necrosis) play essential roles in animal development and certain diseases. Autophagy is a cellular process that provides nutrients to starved cells by digesting cellular constituents. Defects in autophagy have been implicated in cancer and neurodegeneration, but how autophagy causes cell death is not understood. In mammals, there are two apoptotic pathways: the extrinsic one through death receptors in the cell membrane and the intrinsic one through mitochondria that release death proteins. The Bcl-2 family of proteins function upstream in the intrinsic pathway. They can be subdivided into anti-apoptotic and proapoptotic proteins. Mcl-1 has two different splice variants, Mcl-1L and Mcl-1S. Mcl-1L is an anti-apoptotic Bcl-2 family protein with a short half-life, which functions in the apical step of the intrinsic apoptotic pathway and can inhibit the release of death proteins from mitochondria. After genotoxic treatment, Mcl-1L is rapidly degraded, resulting in mitochondria damage and apoptosis. Prevention of Mcl-1L degradation with proteasome inhibitors blocks apoptosis. An Mcl-1L ubiquitin ligase was identified using biochemical purification method and named Mule, which has five recognizable domains including UBA, WWE, HECT and two ARM repeats like domains. Mule also contains a region similar to the Bcl-2 homology region 3 (BH3) that allows it to specifically interact with Mcl-1L. Depletion of Mule by RNA interference stabilizes Mcl-1L, resulting in an attenuation of apoptosis induced by DNA-damaging agents. To further understand Mcl-1 function, I generated inducible Mcl-1 knockdown stable U2OS cell lines. Depletion of Mcl-1L but not Mcl-1S causes the cells to commit autophagic cell death. The autophagic phenotype can be blocked by knocking down Beclin 1, which is a known upstream component of the autophagic pathway. Mcl-1L knockdown induced cell death is accompanied by and requires the upregulation of p53 and p21. Loss-of-function experiments confirmed the involvement of Mcl-1S in inducing autophagy. Taken together, my data showed that Mcl-1 genes function in the apical step of both apoptosis and autophagic cell death pathways.Item Colon Cancer Chemoprotection through Epigenetic Effects of a Fish Oil/Pectin Diet(2012-10-19) Cho, Young MiAccumulated genetic and epigenetic abnormalities contribute to the development of colon cancer. We have shown that a combination of fish oil (containing decosahexaenoic acid, DHA, 22:6 n-3) and pectin (fermented to butyrate by colonic microflora) is protective against colon carcinogenesis in part by regulating the expression of genes involved in apoptosis, leading to apoptosis induction. To determine how FO/P enhances apoptosis, we measured the expression of genes involved in apoptosis. We performed a pathway analysis on differentially expressed genes identified at three times during colon tumorigenesis: initiation, aberrant crypt foci (ACF) formation, and tumor stage, and compared these results with phenotypic observations at those times. At initiation, FO/P down-regulated the expression of genes involved with cell adhesion and enhanced apoptosis compared with corn oil/cellulose (CO/C). At the ACF stage, expression of genes involved in cell cycle regulation was modulated by FO/P and proliferation was reduced in FO/P rats compared with CO/C rats. FO/P increased apoptosis and the expression of genes that promote apoptosis at the tumor endpoint compared with CO/C. We next determined if changes in expression of genes involved in apoptosis by FO/P are associated with changes in promoter methylation of a key apoptosis regulator, Bcl-2. Genomic DNA was isolated from carcinogen-induced colon tumors and non-involved tissues. FO/P increased Bcl-2 promoter methylation in tumor tissues and colonocyte apoptosis relative to those observed with CO/C. A negative correlation between Bcl-2 DNA methylation and Bcl-2 mRNA levels was observed in the tumors. Additionally, we examined gene specific promoter methylation of 24 apoptosis-related genes using human colon cancer cells. Cells were treated with DHA or linoleic acid (18:2 n-6), and select cultures were also treated with butyrate. The combination of DHA and butyrate led to a significant reduction in the methylation of pro-apoptotic genes and an increase in apoptosis. These data suggest that part of the mechanisms involved in the induction of apoptosis by FO/P may be through epigenetic regulation of genes involved in apoptosis throughout colon carcinogenesis.Item Communal Cell Death and P53 Mediated Transcriptional Control in Drosophila Melanogaster(2011-08-26T17:33:49Z) Link, Nichole Lynn; Abrams, JohnApoptosis is essential for all metazoan development. The key component that functions in apoptosis, the apoptosome, is a molecular machine that initiates caspase activation and is conserved throughout the animal kingdom. Drosophila strains that are mutated for genes encoding the apoptosome show pronounced defects in programmed cell death (PCD). Using a characteristic phenotype associated with mosaic animals, we conducted a screen in Drosophila to discover new regulators or effectors of the apoptosome. Using this model, we also discovered a unique communal form of cell death where large regions of epithelial cells are eliminated within minutes. We also produced 'saturation tile' arrays by digital optical chemistry for an unbiased sampling of transcriptional activity in the Drosophila genome. We found that the scope of unannotated transcriptional activity is extensive and widespread. A dominant population of noncanonical transcripts was stress-responsive and required p53, a master regulator of conventional stress-responsive target genes in vertebrates and invertebrates. This prompted us to examine stimulus dependent activity surrounding a single p53 enhancer in our tiled region. Through genetic analyses, we showed that this enhancer coordinates stimulus dependent induction of multiple genes spanning over 300kb throughout the Reaper region. Surprisingly, this same enhancer regulated a gene positioned across the centromere at distances over 20Mb and also controlled at least one gene mapping to a different chromosome. Chromosome conformation capture analyses placed this enhancer in close proximity to these distant targets in vivo through specific DNA looping and these interactions were influenced by p53. Therefore, a single p53 enhancer is necessary and sufficient for long range, multigenic regulation in cis and in trans.Item Development and validation of microcystin biomarkers for exposure studies(2006-05) Billam, Madhavi; Wang, Jia-Sheng; Anderson, Todd; Pence, Barbara; Shen, Leslie; Smith, ErnestMicrocystins (MCs) are hepatotoxic cyanotoxins produced mainly by the cyanobacteria Microcystis spp. They are distributed in waterbodies worldwide, and the toxicity on exposure to MCs was reported worldwide in fish, animals and in humans for over a century. There are about 70 known variants of MCs to date and of them the most toxic and widely distributed MC is Microcystin-LR (MCLR). MCLR is hepatotoxic and a potent tumor promoter. Epidemiological studies in China have linked exposure to MCs with high incidence of liver cancer. Although analytical tools have been reported to detect MCs in water and in food samples, and biomarkers for biochemical alterations like inhibition of protein phosphatases (PP) have been proposed, to date, validation of these analytical methods for simultaneous measurement of MCs in environmental samples and in body fluids of exposed individuals has not yet been done. In this study, we have developed new methods and validated existing methods to detect MCLR and its biomarkers in body fluids of animals and human hepatic cells treated with different concentrations of MCLR. The methods thus validated were used to monitor the seasonal fluctuations in MCLR concentrations in two lakes of western Texas. Studies were also conducted to explore molecular level targets of MCLR in normal (THLE-2) and cancerous (HepG2) human hepatic cell lines. Effect of MCLR on cell proliferation was explored, and validated methods were used to detect alteration in PP activity in these cell lines on exposure to MCLR. Alteration in expression of apoptosis regulatory proteins like Bax, Bcl2, Bad and PP2A on exposure to MCLR was also studied by immunoblotting in these cell lines. Studies were also conducted to explore molecular level targets of MCLR on acute exposure to a single dose, and on subacute exposure to repeated doses of MCLR in F-344 rats. We observed a dose dependent alteration in expression of PP2A, Bax, Bcl2 and Bad in both acute and subchronic exposures, as quantified by western blotting and immunohistochemistry. The study also focused on alteration in levels of sphingolipids in serum on exposure to MCLR. In another experiment, the combinative toxic effect of MCLR along with the tumor initiator aflatoxin-B1 was studied in normal and hepatocarcinoma cell lines, and the mechanism involved was explored.Item Development and validation of microcystin biomarkers for exposure studies(Texas Tech University, 2006-05) Billam, Madhavi; Wang, Jia-Sheng; Pence, Barbara; Smith, Ernest; Shen, Leslie; Anderson, ToddMicrocystins (MCs) are hepatotoxic cyanotoxins produced mainly by the cyanobacteria Microcystis spp. They are distributed in waterbodies worldwide, and the toxicity on exposure to MCs was reported worldwide in fish, animals and in humans for over a century. There are about 70 known variants of MCs to date and of them the most toxic and widely distributed MC is Microcystin-LR (MCLR). MCLR is hepatotoxic and a potent tumor promoter. Epidemiological studies in China have linked exposure to MCs with high incidence of liver cancer. Although analytical tools have been reported to detect MCs in water and in food samples, and biomarkers for biochemical alterations like inhibition of protein phosphatases (PP) have been proposed, to date, validation of these analytical methods for simultaneous measurement of MCs in environmental samples and in body fluids of exposed individuals has not yet been done. In this study, we have developed new methods and validated existing methods to detect MCLR and its biomarkers in body fluids of animals and human hepatic cells treated with different concentrations of MCLR. The methods thus validated were used to monitor the seasonal fluctuations in MCLR concentrations in two lakes of western Texas. Studies were also conducted to explore molecular level targets of MCLR in normal (THLE-2) and cancerous (HepG2) human hepatic cell lines. Effect of MCLR on cell proliferation was explored, and validated methods were used to detect alteration in PP activity in these cell lines on exposure to MCLR. Alteration in expression of apoptosis regulatory proteins like Bax, Bcl2, Bad and PP2A on exposure to MCLR was also studied by immunoblotting in these cell lines. Studies were also conducted to explore molecular level targets of MCLR on acute exposure to a single dose, and on subacute exposure to repeated doses of MCLR in F-344 rats. We observed a dose dependent alteration in expression of PP2A, Bax, Bcl2 and Bad in both acute and subchronic exposures, as quantified by western blotting and immunohistochemistry. The study also focused on alteration in levels of sphingolipids in serum on exposure to MCLR. In another experiment, the combinative toxic effect of MCLR along with the tumor initiator aflatoxin-B1 was studied in normal and hepatocarcinoma cell lines, and the mechanism involved was explored.Item Development of an Informational Video Using 3D Animation to Teach the Fundamentals of the Cellular Process of Apoptosis(2003-06-01) Litton, Rebecca; Calver, LewisThe goal of this thesis was to create an animated video, with narration, that explains the fundamentals of the process of apoptosis. The objectives were to produce a narrated 3D animation of apoptosis presented in an accurate efficient way, and format it for distribution on CD or DVD. Topics discussed in the video include: the difference between necrosis and apoptosis, the physical changes occurring in the cell during apoptosis, triggers of apoptosis and the effect apoptosis has on disease processes. The creation process began by determining subject, scope and audience. After these initial decisions were made a script was written and storyboards were produced. Narration was then recorded and combined with stills of the storyboards and preliminary animation to create an animatic. All animation was created in 3D Studio Max. Editing was accomplished using Adobe Premiere. The final product was then copied to CD and to DVD. This document discusses the process of creating this video from formation of the idea to DVD creation. Results of an informal test of the video are also iscussed as well as ideas for further research.Item E2F3a functions as an oncogene and induces DNA damage response pathway mediated apoptosis(2007) Paulson, Qiwei Xia, 1974-; Johnson, David, 1963-; Bratton, Shawn B.Mutation or inactivation of RB occurs in most human tumors and results in the deregulation of several E2F family transcription factors. Among the E2F family, E2F3 has been implicated as a key regulator of cell proliferation and E2f3 gene amplification and overexpression is detected in some human tumors. To study the role of E2F3a in tumor development, we established a transgenic mouse model expressing E2F3a in a number of epithelial tissues via a keratin 5 (K5) promoter. Transgenic expression of E2F3a leads to hyperproliferation, hyperplasia and increased levels of p53-independent apoptosis in transgenic epidermis. Consistent with data from human cancers, the E2f3a transgene is found to have a weak oncogenic activity on its own and to enhance the response to a skin carcinogenesis protocol. While E2F3a induces apoptosis in the absence of p53, the inactivation of both p53 and p73, but not p73 alone, significantly impairs apoptosis induced by E2F3a. This suggests that both p53 and p73 contribute to E2F3a induced apoptosis but that their function is compensatory. Even though data suggest that E2F3a carries out its unique apoptotic activity in part through another E2F family member E2F1, unlike E2F1, the ARF tumor suppressor is required for E2F3a-induced apoptosis. While both E2F3a and E2F1 require ATM for apoptosis, E2F3a activates ATM through a distinct mechanism from E2F1. The overexpression of E2F3a results in the accumulation of DNA damage in K5 transgenic keratinocytes and normal human fibroblasts (NHFs). In response to this, the DNA damage checkpoint kinase ATM is activated, and phosphorylation of the downstream targets p53 and the histone variant H2AX are significantly increased. Additional studies show that increased Cdk activity and aberrant DNA replication contributes to DNA damage, ATM activation and apoptosis in response to deregulated E2F3a, which suggest that aberrant replication imposed by deregulated E2F3a plays an important role in the activation of the ATM DNA damage response pathway. Activation of ATM by E2F3a is not affected by loss of ARF or E2F1. Meanwhile, E2F3a-induced ARF upregulation is not affected by E2F1 loss. The above results indicate that E2F3a engages several parallel pathways involving E2F1, ARF and the ATM kinase, and these pathways cooperate to promote apoptosis.Item The effect of a three dimensional growth environment on cell death and stress protein expression(2012-05) Song, Alfred Seunghoon; Diller, K. R. (Kenneth R.); Najjar, Amer; Dunn, Andrew; Suggs, Laura; Merchant, FatimaUnderstanding the cellular response thermal stress is important for improving thermoablative treatments of cancer. Cells generally respond to thermal stress by expressing heat shock proteins, or undergoing cell death by apoptosis or necrosis. Most of our detailed knowledge regarding these cellular phenomena has been gathered in vitro in two dimensional (2D) environments. Yet, little is known about how prostate cancer cells respond to thermal stress in a more physiologically relevant three dimensional (3D) environment. Several approaches were used to investigate this question, all of which focused on controlled heating of cells in both two dimensional (2D) and 3D culture. Tools and assays were developed to investigate cellular response to thermal stress in 2D and 3D environments. A computer-controlled heating apparatus was constructed to heat cell cultures to precise temperatures and durations. Three dimensional growth environments were produced using Matrigel, a commercially available extracellular matrix (ecm) mixture. Transcriptional expression of heat shock protein 70 (HSP70) was measured using a green fluorescent protein (GFP) reporter gene under the control of an HSP promoter. Apoptosis, necrosis and HSP70 transcription was measured using flow cytometry analysis. Quantitative polymerase chain reaction (qPCR) and microscopy revealed that transmembrane targets may be involved in the mechanism of the effect which 3D culture has on the cellular response to heat shock. The results herein demonstrate that the 3D growth environment, may be protective to the cell in that the percentage of cells that undergo apoptosis or necrosis when exposed to heat shock are reduced. Furthermore, HSP70 expression is enhanced in 3D culture at a specific thermal dose and integrins and heat shock proteins may be part of the mechanism by which the ecm exerts its protective effect against thermal stress.Item Genetic Analysis of hox Transcription Factors and Cofactors in the Regulation of Programmed Cell Death in C Elegans(2009-01-14) Potts, Malia Beth; Cameron, ScottIt has been well established that blocking apoptosis can promote cancer. Throughout the animal kingdom, apoptosis is exquisitely regulated in cell-specific and context-specific ways to ensure proper development and tissue homeostasis. In many cases, transcriptional pathways carry out this regulation by mechanisms that are not completely understood. Studies of programmed cell death in the nematode Caenorhabditis elegans provided an essential foundation for understanding the more complex pathways of apoptosis in mammals. More recent work, including my thesis research, has focused on the mechanisms that decide whether individual cells of C. elegans survive or undergo programmed cell death, and has revealed a striking concordance of transcription factors that regulate cell death and that cause cancer in humans when altered by mutation. These findings suggest that mutations affecting these transcriptional pathways can provide a survival advantage to cancer cells, and thus may represent promising novel therapeutic targets.Item Genetics of Stress-Induced Responses in Drosophila(2006-08-11) Akdemir, Fatih; Abrams, John M.Apoptosis is a highly conserved process responsible for elimination of cells during normal development and after cellular damage. Apical caspases that initiate caspase cascades are stimulated upon interaction with adaptor molecules. The Drosophila adaptor protein Dark, a homolog of nematode Ced-4 and mammalian Apaf-1, regulates the apical caspase Dronc, through interactions involving respective caspase recruitment domains (CARD). Dronc is the only caspase in the fly genome with a caspase recruitment domain. Here I pursue functional characterization of dronc and dark animals especially to find out whether they are required for all programmed cell death and whether they have distinct functions. dronc mutants have extensive hyperplasia of hematopoietic tissues and adult structures lacking dronc are disrupted for fine patterning. In diverse models of metabolic injury, dronc- cells are completely insensitive to induction of cell killing. I also show the generation and functional characterization of dark null mutant animals. Using in vivo and ex vivo assays, I demonstrate a global apoptogenic requirement for dark and show that a required focus of dark- organismal lethality maps to the central nervous system. Finally I show functional similarities of dronc and dark null mutants by a diverse set of experiments. Together these findings illustrate broad requirements for Dark and Dronc in adaptive responses during stress-induced apoptosis and in normal cell death. Treatment of cells with DNA-damaging agents upregulates the transcription of many genes, many of which are not functionally characterized. In a series of independent studies I characterize an ionizing radiation (IR)-induced gene, CG17836, designated as xrp1. Xrp1 is robustly responsive to IR, and it is a nuclear protein with DNA-binding activity as inferred from domain structure. I have characterized two different loss-of-function mutants of xrp1. In a loss-of-heterozygosity (LOH) assay xrp1 mutant animals display higher genomic instability than wild types after IR challenge. Even though xrp1 is not required for apoptosis or cell cycle arrest after IR treatment in animals, surprisingly, its overexpression in cell culture prevents cell proliferation. Thus, Xrp1 might maintain genomic stability by modulating cell cycle checkpoints upon IR exposure.Item Germ cell apoptosis and death receptor response in the rodent testis after acute mono-(2-ethylhexyl) phthalate and cisplatin exposure(2002-12) Giammona, Charles John; Richburg, John H.Item Heat shock-induced apoptosis(2013-12) Mahajan, Indra Maria; Wright, Casey Wyatt; Bratton, Shawn B.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.Item Induction of apoptosis by an insect iridescent virus in boll weevil and budworm cell culture(Texas Tech University, 1999-08) Jayaraman, RajeswariThe cotton boll weevil, Anthonomus grandis, is a major pest of cotton, causing up to $100 million in damage to the American cotton farmer. Our laboratory has found that an insect virus (boll weevil pathogenic virus, BWPV) replicates efficiently in boll weevil larvae and a protein extract from purified virus particles induces apoptosis in cell culture. The phenomenon was confirmed in budworm (CF) and boll weevil (AG) cells by observing characteristic apoptotic cytopathology (blebbing) and DNA fragmentation. In addition, a number of differential sensitivity assays were also carried out to determine the dose required to induce blebbing and DNA fragmentation. The tissue culture toxicity dose assay (an equivalent of the LD50 assay) showed that 6.3 ng/ml is sufficient to induce apoptotic blebbing in CF cells, and 54.1 ng/ml of the virion extract is required for AG cells. Further, the minimum dose required to induce DNA fragmentation was found to be 2ug/ml. The above data will have significance for the mechanism of viral induction of apoptosis and for the use of viral genes in the generation of insect-resistant plants.Item The induction of apoptosis by the E2F1 transcription factor and the emergence of a role for E2F1 in the DNA double strand break response(2006) Powers, John Thomas; Tucker, Philip W.
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