Browsing by Subject "Heat shock"
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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 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 Mechanisms of benzyl alcohol tolerance in Drosophila melanogaster(2009-12) Alhasan, Yazan Mahmoud; Atkinson, Nigel (Nigel S.); Zakon, Harold H.; Gonzales, Rueben A.; Singer, Michael C.; Bergeson, Susan E.Proper neuronal function requires the preservation of appropriate neural excitability. An adaptive increase in neural excitability after exposure to agents that depress neuronal signaling blunts the sedative drug effects upon subsequent drug exposure. This adaptive response to drug exposure leads to changes in drug induced behaviors such as tolerance, withdrawal and addiction. Here I use Drosophila melanogaster to study the cellular and neuronal components which mediate behavioral tolerance to the anesthetic benzyl alcohol. I demonstrate that rapid tolerance to benzyl alcohol is a pharmacodynamic mechanism independent of drug metabolism. Furthermore, tolerance is a cell autonomous response which occurs in the absence of neural signaling. Using genetic and pharmacological manipulations I find the synapse to play an important role in the development of tolerance. In addition, the neural circuits that regulate arousal and sleep also alter benzyl alcohol sensitivity. Beyond previously described transcriptional mechanisms I find a post-translational role of the Ca2+-activated K+-channel, slowpoke in the development of tolerance. Finally, I explore a form of juvenile onset tolerance, which may have origins that differ from rapid tolerance. The implications of this study go beyond tolerance in Drosophila melanogaster to benzyl alcohol and can shed light on human drug tolerance, withdrawal and addiction.