Browsing by Subject "cell death"
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Item Characterization of Protein Kinase C in Cisplatin Sensitive and Resistant Human Cervical Cancer HeLa Cells(2000-12-01) Mohanty, Sanghamitra; Basu, Alakananda; Simecka, Jerry; Dimitrijevich, DanMohanty, S., Characterization of protein kinase C in cisplatin sensitive and resistant human cervical cancer HeLa cells. Master of Science (Microbiology and Immunology), December, 2000. 37 pp., 11 illustrations, bibliography, 27 titles. Signal transduction plays a crucial role in carcinogenesis. A defect in signaling, by evading cell death or promoting cell proliferation, may result in neoplastic transformation or protection of cells from the cytotoxicity of anticancer drugs. Therefore, in order to understand the complex mechanism of drug resistance, it is relevant to probe into the important signal transduction pathways. Protein kinase C, a key signal transducer, influences cisplatin sensitivity in many cell lines. We examined whether or not the PKC signal transduction pathway is affected during development of resistance to cisplatin by tumor cells. PKC activators increased cisplatin sensitivity in both parental and cisplatin-resistant cells. Western blot analysis showed a slight decrease in cPKCα and nPKCε, an evaluation in nPKCδ and no change in the abundance of PKCϚ in HeLa/CP cells compared to HeLa cells. Though TPA-induced translocation of PKC isoforms was identical in both cell lines, down regulation of PKCδ was defective in resistant cells. Therefore, a deregulation in PKCδ was associated with cisplatin resistance.Item Detection of Androgen Receptors by Flow Cytometry(2008-05-01) Dutta, Mayurika; McClain, Robert; Singh, Meharvan; Hall, StanDutta, Mayurika, ‘Detection of androgen receptors by Flow Cytometry’. Internship Practicum report, Biotechnology, May 2008, 80 pp., 1 table, 18 figures. The use of androgen therapy is expanding given the documented potential benefits like increasing bone mineral density, muscle mass and strength. Androgen therapy also has potential risks including increasing the likelihood of prostate cancer and cardiovascular disease. So, we need a way to differentiate those who are likely to be benefitted by the therapy and those that are not. Data from Dr. Meharvan Singh’s lab has shown that activation of intracellular androgen receptors triggers cell survival pathways, while activation of the membrane androgen receptor suppresses cytoprotective pathways, and thus promotes cell death. We propose to develop a diagnostic kit that measures the relative ratio of intracellular androgen receptors and membrane androgen receptors, which is predicted to gauge relative risks or benefits associated with androgen therapy.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 Hypertrophic Versus Apoptotic Response of Vascular Smooth Muscle to β1 Adrenergic Receptor Stimulation(2004-05-01) Hannon, Sherry Beth; Stephen Grant; Glenn Dillon; Robert MalletHannon, Sherry Beth., Hypertrophic vs. Apoptotic Response of Vascular Smooth Muscle to β1 Adrenergic Receptor Stimulation. Master of Science (Biotechnology), May, 2004, 64 pp., 3 tables, 16 illustrations, references, 41 titles. This project explores how β1 adrenoceptor (β1-AR) stimulation affects cellular hypertrophy and apoptosis in PAC-1, a cultured rat pulmonary artery cell line. Insights into these responses may further the current understanding of vascular remodeling. Promoter-reporter activity for the hypertrophy-specific gene smooth muscle myosin heavy chain decreased as measured by a luciferase assay when PAC-1 cells were treated with the selective β1-AR agonist dobutamine (DOB) in 0.4% fetal bovine serum (FBS) supplemented media. However, activity of a β1-gal control vector also decreased, and neither response was attenuated by pre-treatment with a β1-AR selective antagonist metoprolol. A MTS [3-(4,5-dimethlythiazol-2-yl)-5-(3-carboxymethoxyphenly)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] viability assay shows that while there is a loss of cells with decreasing amounts of serum, this effect is not exacerbated by DOB in 0.4% FBS. DNA fragmentation assays were inconclusive as to the mode of cell death occurring. However, an increase in Bax/Bcl-2 ratio suggest that apoptosis is induced with DOB treatment in 10% FBS, but that this DOB treatment in 0.4% and serum-free media does not increase this apoptotic index compared to control. Both similar and conflicting cellular responses have been documented in rat neonate cardiomyocytes as well as in murine transgenic models selectively over-expressing adrenergic receptors in the heart. Comparison of the vascular smooth muscle cell response to the cardiomyoctye response may lead to a more tailored use of adrenergic agents for treatment during different stages of cardiovascular disease.Item Involvement of S6 Kinase in Breast Cancer(2013-12-01) Sridharan, Savitha; Basu, AlakanandaSridharan, S., Involvement of S6 Kinase in Breast Cancer. Doctor of Philosophy (Cancer Biology), November 2013, 129pp, 19 illustrations, 215 references. The 40S ribosomal protein S6 Kinase (S6K) is activated downstream of the mammalian target of rapamycin (mTOR)and is believed to play and important role in protein translation. In mammalian cells S6K is represented by two highly homologous proteins, S6K1 and S6K2. Both homologs have been shown to be amplicfied and over expressed in breast cancer cells and tissues. While the regulation and functions of S6K1 have been addressed, little is known about those of S6K2 . Hence we sought to examine the causes and consequences of elevated S6K2 levels in breast cancer cells. While the depletion of S6K1 decreased breast cancer cell death, silencing of S6K2 substantially increased it in response to apoptotic and chemotherapeutic agents. We then explored the mechanism by which S6K2 mediates survival and observed that in contrast to S6K1, S6K2 depletion decreased the activation of the prosurvival protein Akt and increased the level of proapoptotic proteins p53 and bid. Following this observation, we sought to determine the pathways(s) contributing to the overexpression of S6K2 in breast cancer cells. Due to its role as a prognostic indicator in estrogen receptor (ER) – positive tumors, we studied the role of the estrogen signaling pathways in regulating S6K2 levels. Estradiol and estrogen receptor alpha (ERα) positively regulated S6K2 protein but did not affect its mRNA levels, suggesting post-transcriptional regulation. We further observed that S6K2 regulated cell survival downstream of estrogen in ER-positive breast cancer cells. These findings strongly suggest that S6K2 is critical for the survival of breast cancer cells and that targeting S6K2 in combination with chemotherapeutic agents is a novel strategy to promote breast cancer cell death.Item Molecular Mechanisms of and Potential Therapies for Oxidative Damage to the Retinal Pigment Epithelium(2007-09-01) Wang, Zhaohui; Roque, Rouel S.; Wordinger, Robert J.; Das, HridayWang, Zhaohui, Molecular Mechanisms of and Potential Therapies for Oxidative Damage to the Retinal Pigment Epithelium. Doctor of Philosophy (Biomedical Sciences), September 2007, 161 pages, 34 illustrations, bibliography, 119 titles. Age-related macular degeneration (AMD), the most common cause of irreversible vision loss in the elderly, results mainly from degeneration of the retinal pigment epithelium (RPE) and loss of photoreceptor cells. Oxidative stress has been acknowledged as a leading cause of RPE degeneration and concomitant photoreceptor cell loss, but the exact role of reactive oxygen species (ROS) in RPE cell death remains to be established. Moreover, while mitogen-activated protein kinases (MAPKs) are suggested to be involved in RPE degeneration induced by oxidative stress, the precise functions and molecular mechanisms of MAPKs in RPE degeneration remain elusive. In spite of the numerous therapeutic modalities proposed for AMD, the treatment of AMD remains unsatisfactory. Recent studies suggesting stem cells as a potential source for trophic factors in damaged murine hearts led us to investigate a possible role for stem/progenitor cell-derived factors in protecting RPE cells from oxidative damage. Furthermore, human retinal progenitor cells promote RPE cell survival by regulating p42/p44 MAPK activity. When exposed to oxidative stress produced by glucose oxidase/glucose, human RPE cells exhibited membrane blebbing and cytoskeleton remodeling in the early phase of oxidative stress. Prolonged exposure to oxidative stress induced mitochondrial membrane potential depolarization, cell death and DNA condensation, but not DNA fragmentation. Furthermore, both p38 MAPK and p42/p44 MAPK were activated by oxidative injury. P38 MAPK inhibitor, but not p38 MAPK siRNA, inhibited RPE cell death induced by oxidative stress. Overexpression of constitutively active MEK1 inhibited RPE cell death exposed to oxidative damage. In contrast, interfering p42/p44 MAPK expression accelerated oxidative-stress induced RPE cell death. To investigate the effects of human retinal progenitor cells (hRPC) on RPE cells, we isolated and expanded hRPC in vitro. The hRPCs expressed markers of neuronal and retinal progenitor cells, and were capable of differentiating into neuronal phenotype in defined medium. In the presence of 10% fetal bovine serum, hPRC suppressed RPE cell death induced by oxidative damage. Furthermore, conditioned medium of hRPC induced activation of p42/p44 MAPK, and the protective effect of hRPC and conditioned medium was suppressed by p42/p44 MAPK inhibitor. Our studies increase our understanding of the molecular mechanisms that could be employed to rescue RPE cells from degeneration and support the therapeutic potential of retinal progenitor cells. It will provide further insight into molecular mechanisms of AMD and establish a foundation for the long-term prevention and treatment of AMDItem Protein-Protein Interactions Between Poly(ADP-Ribose) Polymerase-1 and DNA Polymerase B(2003-12-01) Confer, Nils Forgard; Alvarez, Rafael; Ben S. Aar…; Wu, MingehiConfer, Nils Forgard, Protein-Protein Interactions Between Poly(ADP-ribose) Polymerase-1 and DNA Polymerase B. Doctor of Philosophy (Biomedical Sciences), December 2003, 114 Pages, 22 Figures, 1 Graph, and 80 References. The mammalian genome is continually subjected to chemical and environmental modifications that are repaired by base excision, and when excessive, may lead to apoptosis. Interestingly, the chromosomal enzyme poly(ADP-ribose) polymerase-1 (PARP-1) appears to modulate both mechanisms, either facilitating DNA repair and/or modulating cell death. In this dissertation project, experiments were performed to address the regulatory potential of PARP-1 in base excision repair (BER) and specifically on DNA polymerase B (pol B) function. Activity gels were used to measure the DNA polymerase activity of pol B following protein-(ADP-ribosyl)ation. However, the fraction of pol B molecules (ADP-ribosyl)ated was never 100% under the reaction conditions employed. In fact, similar results were observed in activity gels specific for PARP-1, even under conditions where this polymerase is the primary nuclear acceptor for poly(ADP-ribose) Here, I also describe a newly developed electrophoretic-mobility-shift-assay (EMSA) to monitor for the specific binding of pol B to a custom-made five-nucleotide gapped DNA duplex. However, while specific for pol B, this assay was inefficient to monitor the effects of covalent poly(ADP-ribosyl)ation on pol B activity. Moreover, I also observed the specific molecular association of PARP-1 is specifically proteolyzed into peptide fragments by caspases, conditions were established for the efficient proteolysis of PARP-1 by either capase-7. Experimental results indicated that caspase-3 was more efficient than caspase-7 at splitting unmodified PARP-1 into two peptide fragments. By contrast, caspase-7 appeared best suited for the proteolysis of covalently auto-poly(ADP-ribosyl)ated-(PARP-1). Interestingly, both of the caspase-generated peptide fragments of PARP-1 specifically associated with pol B as supported by co-immunoprecipitation/immune-blotting experiments. Taken together, the experimental results presented here support the hypothesis that a molecular mechanism exists that involves interaction(s) of PARP-1 with pol B that may help to facilitate the decision making process between cell survival and cell death. Thus, upon proteolytic degredation of PARP-1 into a 24-kDa amino-terminal fragment and an 89-kDa carboxy-terminus, each truncated peptide, separately, retains physical association with pol B, and inhibits DNA repair associated pol B activity to irreversibly switch the fate of cell from BER toward chromatin degradation and, eventually, programmed cell death.Item REGULATION OF CELL DEATH BY INHIBITOR OF KAPPA-B KINASE IN TRIPLE NEGATIVE BREAST CANCER CELLS(2013-04-12) Patel, DipaliPurpose: Triple-negative breast cancer (TNBC) is characterized by the lack of estrogen receptor, progesterone receptor, and HER2/neu and hence poses problems for targeted therapy. Thus there is an urgent need to identify suitable molecular targets for the treatment of TNBC. The inhibitor of kappa-B kinase-epsilon (IKK-epsilon) is a breast cancer specific oncogene that is elevated in TNBCs and plays an important role in breast cancer cell survival. Cancer cells often evade death by overcoming apoptosis or type I programmed cell death. However, the role of autophagy or self-digestion in cancer is ambiguous. While a low level of autophagy promotes cell survival under stress, excessive autophagy may lead to type II programmed cell death. The purpose of this project is to determine the mechanism by which IKK-epsilon promotes survival of breast cancer cells. Methods: Triple-negative breast cancer MDA-MB-231 cells were transfected with non-targeting control siRNA or siRNA against appropriate genes. Proteins were separated using SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Western blot analysis and enhanced chemiluminescence were used to study the overall levels of apoptosis and autophagy markers. Results: Knockdown of IKK-epsilon in MDA-MB-231 potentiated tumor necrosis factor-alpha (TNF)-induced apoptosis. IKK-epsilon knockdown also showed a decrease in p62/SQSTM1 and an increase in proteins associated with autophagy induction. Conclusions: The increase in autophagic markers in IKK-epsilon depleted cells suggests that IKK-epsilon protects MDA-MB-231 cells by promoting autophagy. Understanding the specific mechanism of survival of TNBC cells could aid in developing effective therapy.Item ROLE AND REGULATION OF S6 KINASE IN BREAST CANCER(2013-04-12) Sridharan, SavithaPurpose: The 40S ribosomal protein S6 kinase (S6K) acts downstream of the mammalian target of rapamycin (mTOR), which is an important target for cancer therapy. mTOR inhibitors are, however, of limited success, partly due to the activation of the oncogene Akt caused by persistent inhibition of p70 S6 kinase or S6K1 via a negative feedback loop. S6K exists as two homologs, S6K1 and S6K2. While both homologs are overexpressed in breast cancer, little is known about the regulation and functions of S6K2. The objective of the present study is to determine pathways promoting S6K overexpression and whether S6K homologs perform redundant or distinct functions with respect to Akt activation and breast cancer cell survival. Methods: Breast cancer cells were transfected with non-targeting siRNA or siRNA against appropriate proteins and treated with cell death stimuli such as tumor necrosis factor-alpha (TNF), TNF-related apoptosis-inducing ligand (TRAIL), doxorubicin and paclitaxel. Cell death was monitored by staining cells with Annexin V/Yo-pro and propidium iodide. The levels of various proteins were determined by Western blotting. Results: S6K levels correlated with hormone receptor status and signaling through the hormone receptor pathway increased S6K levels. Silencing of S6K1 inhibited whereas knockdown of S6K2 potentiated cell death by various apoptotic stimuli, including TNF and TRAIL. In contrast to S6K1, depletion of S6K2 decreased basal and TNF-induced Akt phosphorylation. Ectopic expression of constitutively-active Akt in MCF-7 cells restored cell survival in S6K2-depleted cells. We have previously shown that activation of Akt induces downregulation of Bid via p53. Knockdown of S6K2 caused an increase in p53 and silencing of Bid blunted the ability of S6K2 deficiency to enhance TNF-induced apoptosis. Conclusions: Our study addresses the mechanism of S6K overexpression and demonstrates for the first time that the two homologs of S6K have distinct effects on Akt activation and cell survival. Thus, targeting S6K2 rather than mTOR may be an effective therapeutic strategy to treat cancers.Item Role of Nonfeminizing Estrogen Analogues in Neuroprotection of Rat Retinal Ganglion Cells Against Glutamate-Induced Cytotoxicity(2007-05-01) Kumar, Domalapalli Maneesh; Agarwal, Neeraj; Gracy, Robert; Garner, MargaretKumar, Domalapalli Maneesh, Role of Nonfeminizing Estrogen Analogues in Neuroprotection of Rat Retinal Ganglion Cells against Glutamate-Induced Cytotoxicity, Doctor of Philosophy (Cell Biology and Genetics), May, 2007, 210 pp., 3 tables, 23 figures, bibliography, 427 titles. Retinal ganglion cell death has been determined to be the final common pathway in glaucoma. Continuous loss of retinal ganglion cells results in irreversible progressive visual field deterioration that culminates in blindness. No effective therapy is currently available to reverse retinal ganglion cell loss. Therefore, preventing the loss of retinal ganglion cells is a logical approach to maintaining vision in effected individuals. Of the methods of investigation, in vivo models of ganglion cell death provide a physiological system in which to study neuroprotective drugs and their effects, but these systems are inefficient for initial screening studies. We have addressed this by utilizing the RGC-5 clonal rat retinal ganglion cell line. Glutamate treatment of RGC-5 cells induces apoptotic death which can be attenuated by pretreatment with the anti-oxidants N-acetyl cysteine and thiourea, implicating oxidative stress as a major component of glutamate’s cytotoxicity. Also antioxidants, estrogens have been demonstrated to be potent neuroprotectants in a variety of in vitro and in vivo models of neurodegeneration. Estrogens’ antioxidant capacity has been attributed to the ability of the phenolic A ring to quench and resonance stabilize oxidative free radicals. It is also known that the estrogen A ring is responsible for binding of these hormones to estrogen receptors, producing feminizing phenotypes. The feminizing effects of estrogens narrow or preclude their use as neuroprotectants in males, and in females that may be predisposed to their deleterious effects. To address these shortcomings we screened 13, non-feminizing, non-receptor binding estrogen analogues in our glutamate-induced RGC-5 model of oxidative stress-induced cell death. The most effective of these drugs was ZYC-3. ZYC-3 was synthesized by the addition of an adamantly group to the C2 position on the A ring of estrone. This modification produced a neuroprotective compound with potency and efficacy at least equal to the prototypical estrogen, 17β-estradiol, but with no appreciable binding affinity for estrogens receptors α or β. Our preliminary findings suggest that ZYC-3 enhances glutathione synthesis and blocks mitochondrial apoptotic pathways. However, as a novel drug we are naïve to its effects on cellular physiology and as to how it affords neuroprotection. Understanding how this drug regulates cellular destructive and protective mechanisms could lead to further innovations in drug design and in methods to prevent retinal ganglion cell degeneration. In vivo studies of this drug may then form the bridge to a better clinical approach to managing ocular disorders in which ganglion cell loss is the culprit for vision loss. Although promising, evidence supporting the application of estrogen analogues in models of ocular neurodegenerative diseases are nearly non-existent. It is our objective to study the neuroprotective effects of ZYC-3 in glaucomatous models with the goal of maintaining retinal ganglion cell viability and preventing vision loss.Item Sigma-1 Receptor Signaling in the Eye(2008-03-12) Tchedre, Kissaou T.; Yorio, Thomas; Singh, Meharvan; Machu, TinaSIGMA-1 Receptor Signaling in the Eye Kissaou T. Tchedre, Department of Biomedical Sciences, University of North Texas Health Science Center Fort Worth, TX 76107. SUMMARY The sigma-1 receptor is a discovered transmembrane protein that mediates the regulation of ion channels. Sigma-1 receptor ligands have exhibited a wide variety of actions in the central nervous system including attenuation of the neuronal death associated with glutamate excitotoxicity both in vitro and in vivo. Although the sigma-1 receptor was cloned almost a decade ago, the molecular mechanism of the neuroprotective effect remains to be elucidated. In the current proposal it was hypothesized that activation of sigma-1 receptors promotes retinal ganglion cells survival by decreased calcium signaling pathways and factors linked to cell death. We showed by the ratiometric calcium imaging and patch clamp techniques that sigma-1 receptor activation could inhibit both calcium influx and intracellular calcium mobilization. The results showed that sigma-1 receptor overexpressing RGC-5 cells also had a lower glutamate-induced intracellular calcium mobilization compared to non-overexpressing RGC-5 cells. The survival assay data showed that the sigma-1 receptor agonist, (+)-SKF10047 protected RGC-5 cells showed a significant resistance to glutamate-induced apoptosis compared to the control RGC-5 cells. The sigma-1 receptor neuroprotective mechanism also included the down regulation of Bax, and caveolin-1 protein expression levels and inhibited caspase-3 activation. We also demonstrated for the first time using a co-immunoprecipitation technique, the association between L-type calcium channels and sigma-1 receptors. Thus sigma-1 receptor ligands may indirectly influence the voltage-gated calcium channels by interacting with the sigma-1 receptor associated voltage-gated calcium channel complex. In conclusion, activation of sigma-1 receptors can regulate calcium homeostasis and signaling in retinal ganglion cells. Activation of sigma-1 receptors regulate intracellular calcium levels and pro-apoptotic gene expression to promote retinal ganglion cell survival. Sigma-1 receptor ligands may be neuroprotective and targets for potential glaucoma therapeutics.Item The Role of Calcium in Light-Induced Photoreceptor Apoptosis(2002-05-01) Krueger, Darrel Scott; Collier, Robert; Agarwal, Neeraj; Wordinger, Robert J.Krueger, Darrel Scott, The Role of Calcium in Light-Induced Photoreceptor Cell Apoptosis. Doctor of Philosophy (Biomedical Sciences), May, 2002; 305 pp., 15 tables, 31 illustrations, bibliography, 421 titles. Retinal degenerative diseases such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD) result in loss of photoreceptors by apoptosis. Photo-oxidative stress accelerates the rate of photoreceptor apoptosis in models of retinal dystrophies. Thus, a better understanding of light-induced apoptosis is important for developing preventative and treatment options for persons with these blinding diseases. The goals of this dissertation were to investigate the effect of photo-oxidative stress on [Ca2+]I in rat photoreceptor cells and determine whether cell death could be prevented by altering cells’ abilities to manage [Ca2+]i. Thirty minutes of light exposure resulted in significant elevation of [Ca2+]i, determined using Fura-2 ratiometric imaging, which increased with additional light exposure. At 120 minutes, the F340/380 ratio was 3-times the beginning baseline ratio. Using multiple techniques indicative of early and late phases of apoptosis, changes consistent with apoptosis were observed, including early labeling (30 Min) with Annexin-V, activation of caspase-3 (2IIr), TUNEL labeling and Propidium Iodidc staining. TUNEL and Propidium labeling were more intense at 3Hrs light exposure, reflecting late phase changes. Apoptosis was confirmed using electron microscopy (TEM). TEM showed mitochondrial swelling, indicative of permeabilization, prior to chromatin condensation. Pharmacological agents were utilized to mediate participation of cellular calcium sources or storage sites in the maintenance of intracellular calcium homeostasis during photo-oxidative stress. Agents were separately added to the media prior to light exposure and their effects on cell viability were assessed using the Formazan assay. Mitochondria were confirmed to be the site of action affected by elevated [Ca2+]I, since prevention of calcium uptake by ruthenium red (1-100-μM) provided significant protection of cell viability in a dose-related manner. The 100- μM concentration resulted in complete maintenance of viability. BAPTA-AM also demonstrated some protection (50%) indicating that reduction of [Ca2+]I independent of source is beneficial is maintaining cell viability. Identification of mitochondrial uptake and cytosolic buffering of calcium as key viability determinants in light-induced apoptosis is a significant discovery for targeting future research for preventing or inhibiting photoreceptor cell apoptosis associated with retinal dystrophies such as RP and AMD.