Diana Averill-bates - Academia.edu (original) (raw)
Papers by Diana Averill-bates
Free Radical Biology and Medicine, Oct 1, 2016
The antioxidant transcription factor Nrf2 contributes to the protective effect of mild thermotole... more The antioxidant transcription factor Nrf2 contributes to the protective effect of mild thermotolerance (40°C) against heat shock-induced apoptosis, Free Radical Biology and Medicine,
Journal of Cellular Physiology, 2007
... Liliane Eustache Okoumassoun 1,2 ,; Caterina Russo 2 ,; Francine Denizeau 3, ,; Diana Averil... more ... Liliane Eustache Okoumassoun 1,2 ,; Caterina Russo 2 ,; Francine Denizeau 3, ,; Diana Averill-Bates 3 ,; Dr. Janet E. Henderson 1,2 ... siRNA-mediated down-regulation of the α′ sub-unit in HEK293 cells decreased the expression of survivin and increased apoptosis (Tapia ...
InTech eBooks, May 9, 2013
Vitamins and hormones, 2023
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research
Biotechnology and Bioengineering, Feb 3, 2016
There is increasing demand for cryopreserved cells such as liver and pancreatic cells for clinica... more There is increasing demand for cryopreserved cells such as liver and pancreatic cells for clinical applications. Cryopreservation at ultra-low temperatures requires use of cryoprotectants (e.g., dimethyl sulfoxide (DMSO)) to maintain cell integrity during freezing and thawing processes. Standard cryoprotectants are cytotoxic and more effective cryopreservation technologies are urgently needed for long-term storage of cells. As an alternative, soluble protein extracts (WPE) from winter wheat successfully replaced DMSO as a cryoprotectant for several mammalian cell types. To identify novel cryoactive proteins, the WPE was separated by chromatography and cryoactive fractions were analyzed by mass spectrometry. The wheat protein 2-Cys peroxiredoxin BAS1 (renamed TaBAS1) was identified as a potential cryoactive candidate. Recombinant proteins were prepared and found to possess dual functions as a peroxidase antioxidant and molecular chaperone, and display cryoprotective properties for hepatocytes and insulin-secreting INS832/13 cells. Following cryopreservation with TaBAS1, cells were plateable and showed high post-thaw viability, good adhesion properties, and wellmaintained cell-specific metabolic functions. The overall quality of these cell types was equivalent or improved compared to cells that were cryopreserved with DMSO. The antioxidant and chaperone functions of TaBAS1 likely explain its efficacy in reducing oxidative/ nitrosative stresses in cryopreserved cells. The plant protein TaBAS1 could be a promising molecule to include in cryostorage protocols.
Biochimica et biophysica acta. Molecular cell research, Mar 1, 2021
Hyperthermia is a promising anticancer treatment used in combination with radiotherapy and chemot... more Hyperthermia is a promising anticancer treatment used in combination with radiotherapy and chemotherapy. Temperatures above 41.5°C are cytotoxic and hyperthermia treatments can target a localized area of the body that has been invaded by a tumor. However, non-lethal temperatures (39-41°C) can increase cellular defenses, such as heat shock proteins. This adaptive survival response, thermotolerance, can protect cells against subsequent cytotoxic stress such as anticancer treatments and heat shock (>41.5°C). Autophagy is another survival process that is activated by stress. This study aims to determine whether autophagy can be activated by heat shock at 42°C, and if this response is mediated by reactive oxygen species (ROS). Autophagy was increased during shorter heating times (< 60 min) at 42°C in cells. Levels of acidic vesicular organelles (AVO) and autophagy proteins Beclin-1, LC3-II/LC-3I, Atg7 and Atg12-Atg5 were increased. Heat shock at 42°C increased levels of reactive oxygen species (ROS). Increased levels of LC3 and AVOs at 42°C were inhibited by antioxidants. Therefore, increased autophagy during heat shock at 42°C (<60 min) was mediated by ROS. Conversely, heat shock at 42°C for longer times (1-3 h) caused apoptosis and activation of caspases in the mitochondrial, death receptor and endoplasmic reticulum (ER) pathways. Thermotolerant cells, which were developed at 40°C, were resistant to activation of apoptosis at 42°C. Autophagy inhibitors 3-methyladenine and bafilomycin sensitized cells to activation of apoptosis by heat shock (42°C). Improved understanding of autophagy in cellular responses to heat shock could be useful for optimizing the efficacy of hyperthermia in the clinic.
Free Radical Biology and Medicine, Nov 1, 2022
Cryobiology, Dec 1, 2018
The vitrification of ovarian tissue has the potential to preserve the fertility. The purpose of t... more The vitrification of ovarian tissue has the potential to preserve the fertility. The purpose of the present study was to reveal the effects of cryoprotectant agent temperature on the morphological and ultrastructural features of follicle in dog ovarian tissue. The ovarian tissues were classified as control and two vitrification groups. The forty tissues were exposed to cryoprotectants at 4 C (Group I) or room temperature (RT) (Group II). To vitrification procedure, 1 mm 3 pieces of ovarian tissue were placed in an acupuncture needle. The tissues were exposed to 7.5% ethylene glycol and 7.5% dimethyl sulfoxide (Me 2 SO 4) for 10 min and then 15% ethylene glycol and 15% Me 2 SO 4 for 2 min, either at room temperature or at 4 C and stored in liquid nitrogen at least 1 week. After then the tissues were thawed and immersed into Bouins' and glutaraldehyde solution for analysis. The tissues which were vitrified at 4 C were more preserved than at the room temperature. Lots of healthy primordial follicles were observed in the Group I. In Group II, there were very few normal follicles. In the ultrastructural examination in the Group I, it was observed that the double membrane structure of the oocytes' nucleus was preserved, the distributions and structures of the organelles were normal. In Group II ultrastructural observation, the oocyte cytoplasm was damaged and the linkages between oocytes and the follicle cells were detached. The ice damage was noticed in the tissue structure in this group. Oocytes vacuoles and interstitial vacuoles were seen. The optimal agent temperature was determined as 4 C for dog vitrification equilibration.
Biochimica et biophysica acta. Molecular cell research, Dec 1, 2016
Reactive oxygen species (ROS) are short-lived and highly reactive molecules. The generation of RO... more Reactive oxygen species (ROS) are short-lived and highly reactive molecules. The generation of ROS in cells exists in equilibrium with a variety of antioxidant defences. At low to modest doses, ROS are considered to be essential for regulation of normal physiological functions involved in development such as cell cycle progression and proliferation, differentiation, migration and cell death. ROS also play an important role in the immune system, maintenance of the redox balance and have been implicated in activation of various cellular signalling pathways. Excess cellular levels of ROS cause damage to proteins, nucleic acids, lipids, membranes and organelles, which can lead to activation of cell death processes such as apoptosis. Apoptosis is a highly regulated process that is essential for the development and survival of multicellular organisms. These organisms often need to discard cells that are superfluous or potentially harmful, having accumulated mutations or become infected by pathogens. Apoptosis features a characteristic set of morphological and biochemical features whereby cells undergo a cascade of self-destruction. Thus, proper regulation of apoptosis is essential for maintaining normal cellular homeostasis. ROS play a central role in cell signalling as well as in regulation of the main pathways of apoptosis mediated by mitochondria, death receptors and the endoplasmic reticulum (ER). This review focuses on current understanding of the role of ROS in each of these three main pathways of apoptosis. The role of ROS in the complex interplay and crosstalk between these different signalling pathways remains to be further unravelled during the coming years.
Cell Stress & Chaperones, Jun 11, 2020
Cryopreservation is used for long-term storage of cells and tissues. Cryoprotectants such as dime... more Cryopreservation is used for long-term storage of cells and tissues. Cryoprotectants such as dimethyl disulfoxide (DMSO) are used to protect cells against freeze-thaw damage. Despite the use of cryoprotectants, hepatocytes are sensitive to stresses imposed by freeze and thaw processes, which cause physical damage, loss of functionality, or cell death. As an alternative, we have developed new technology using several recombinant wheat proteins as cryoprotectants: TaENO (enolase), TaBAS1 (2-Cys peroxiredoxin), and a combination of WCS120 (dehydrin) with TaIRI-2 (inhibitor of ice recrystallization). This study aims to understand the mechanisms by which these plant proteins protect rat hepatocytes against cell death incurred during cryopreservation. Our analysis revealed that for cells cryopreserved with DMSO, cell death occurred by apoptosis and necrosis. Apoptosis was detected by activation of effector caspases-3 and -7, PARP cleavage, and nuclear chromatin condensation. These apoptotic events were inhibited when hepatocytes were cryopreserved with the different plant proteins. Cryopreservation with DMSO activated apoptosis through the mitochondrial pathway: the Bax/Bcl-2 protein ratio increased, mitochondrial membrane potential decreased, and initiator caspase-9 was activated. Furthermore, the endoplasmic reticulum pathway of apoptosis was activated: levels of the chaperone Bip/GRP78 decreased, pro-apoptotic transcription factor CHOP was induced, and initiator caspase-12 was activated. Activation of the mitochondrial and endoplasmic reticulum pathways of apoptosis was attenuated when hepatocytes were cryopreserved with the different recombinant proteins. This study improves understanding of mechanisms of cryoprotection provided by these plant proteins during freezing stress. These proteins are natural products and show promising potential by decreasing cell death during cryopreservation of hepatocytes.
Cell Stress and Chaperones, 2020
Cryopreservation is used for long-term storage of cells and tissues. Cryoprotectants such as dime... more Cryopreservation is used for long-term storage of cells and tissues. Cryoprotectants such as dimethyl disulfoxide (DMSO) are used to protect cells against freeze-thaw damage. Despite the use of cryoprotectants, hepatocytes are sensitive to stresses imposed by freeze and thaw processes, which cause physical damage, loss of functionality, or cell death. As an alternative, we have developed new technology using several recombinant wheat proteins as cryoprotectants: TaENO (enolase), TaBAS1 (2-Cys peroxiredoxin), and a combination of WCS120 (dehydrin) with TaIRI-2 (inhibitor of ice recrystallization). This study aims to understand the mechanisms by which these plant proteins protect rat hepatocytes against cell death incurred during cryopreservation. Our analysis revealed that for cells cryopreserved with DMSO, cell death occurred by apoptosis and necrosis. Apoptosis was detected by activation of effector caspases-3 and-7, PARP cleavage, and nuclear chromatin condensation. These apoptotic events were inhibited when hepatocytes were cryopreserved with the different plant proteins. Cryopreservation with DMSO activated apoptosis through the mitochondrial pathway: the Bax/Bcl-2 protein ratio increased, mitochondrial membrane potential decreased, and initiator caspase-9 was activated. Furthermore, the endoplasmic reticulum pathway of apoptosis was activated: levels of the chaperone Bip/GRP78 decreased, pro-apoptotic transcription factor CHOP was induced, and initiator caspase-12 was activated. Activation of the mitochondrial and endoplasmic reticulum pathways of apoptosis was attenuated when hepatocytes were cryopreserved with the different recombinant proteins. This study improves understanding of mechanisms of cryoprotection provided by these plant proteins during freezing stress. These proteins are natural products and show promising potential by decreasing cell death during cryopreservation of hepatocytes.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2016
Various toxic compounds produce reactive oxygen species, resulting in oxidative stress that threa... more Various toxic compounds produce reactive oxygen species, resulting in oxidative stress that threatens cellular homeostasis. Yet, lower doses of stress can stimulate defence systems allowing cell survival, whereas intense stress activates cell death pathways such as apoptosis. Mild thermal stress (40°C, 3 h) induces thermotolerance, an adaptive survival response that renders cells less sensitive to subsequent toxic stress, by activating defence systems like heat shock proteins, antioxidants, anti-apoptotic and ER-stress factors. This study aims to understand how autophagy and apoptosis are regulated in response to different doses of H 2 O 2 , and whether mild thermotolerance can protect cervical carcinoma cells against apoptosis by stimulating autophagy. Autophagy was monitored through Beclin-1 and LC3 expression and acid compartment activity, whereas apoptosis was tracked by caspase activity and chromatin condensation. Exposure of HeLa and C33 A cells to H 2 O 2 for shorter times (15-30 min) transiently induced autophagy; apoptosis was activated after longer times (1-3 h). Mild thermotolerance at 40C enhanced activation of autophagy by H 2 O 2. Disruption of autophagy using bafilomycin A1 and 3methyladenine sensitized cells to apoptosis induced by H 2 O 2 , in non-thermotolerant cells and, to a lesser extent, in thermotolerant cells. Inhibition of autophagy enhanced apoptosis through the mitochondrial, death receptor and endoplasmic reticulum pathways. Autophagy was activated by lower doses of stress and protects cells against apoptosis induced by higher doses of H 2 O 2. This work improves understanding of mechanisms that might be involved in toxicity of various compounds and could eventually lead to protective strategies against deleterious effects of toxic compounds.
The drug discovery and development process requires adequate safety testing for drug toxicity bef... more The drug discovery and development process requires adequate safety testing for drug toxicity before new drugs can be administered to patients. Hepatocytes are used in vitro to screen compounds for hepatotoxicity, induction of drug-metabolizing enzymes such as cytochrome P450 isoforms and drug-drug interactions, and to establish human relevance for metabolism. Cryopreservation makes it possible to preserve a large quantity of functional hepatocytes. Techniques for cryopreservation of hepatocytes are mainly based on dimethylsulfoxide. However, analyses of metabolic capacities of cryopreserved hepatocytes are often limited by loss of functional integrity of hepatocytes after thawing. It is therefore necessary to improve techniques of cryopreservation. We have developed a new cryopreservation technology for mammalian cells based on a wheat protein extract (WPE). We determined whether the WPE can better preserve activities of major cytochrome P450 isoforms, both in suspension and monolayer cultures of hepatocytes. This was achieved by comparing basal and inducible or metabolic activities of isoforms CYP1A1, CYP1A2, CYP2C6, CYP2D2 and CYP3A in rat hepatocytes that were cryopreserved with WPE, relative to fresh cells and those cryopreserved with DMSO. We conclusively demonstrate that rat hepatocytes cryopreserved with WPE retain their metabolic competency and their ability to respond to classical CYP inducers, when compared to freshly isolated hepatocytes. These findings clearly show that WPE are an excellent cryopreservant for rat hepatocytes. They are an efficient, non-toxic, economic natural product and universal cryoprotectant that is superior to DMSO, which has limitations due to cellular toxicity. This article has not been copyedited and formatted. The final version may differ from this version.
Radiation Research, 1995
The effect of both hyperthermia and verapamil on cytotoxicity and transport of melphalan was stud... more The effect of both hyperthermia and verapamil on cytotoxicity and transport of melphalan was studied in a pleiotropic drug-resistant Chinese hamster ovary cell line (CHRC5) and in the drug-sensitive parent line (AuxB1). The CHRC5 cell line was selected for resistance to colchicine but is also cross-resistant to other drugs including melphalan. Verapamil (10 microM) increased melphalan cytotoxicity in drug-resistant cells but not in drug-sensitive cells. Hyperthermia (40 to 45 degrees C) increased melphalan cytotoxicity in both cell lines. In drug-resistant but not drug-sensitive cells, melphalan cytotoxicity was increased further when verapamil was combined with hyperthermia (40 to 45 degrees C). The increased cytotoxicity caused by verapamil in drug-resistant cells was accompanied by alterations in membrane permeability to melphalan. The cellular uptake of melphalan after 15 min increased in the presence of verapamil (7 to 30 microM) at 37 and 42 degrees C. When verapamil (10 microM) was present, the rate of efflux of melphalan from CHRC5 cells decreased by almost 40% at 37 degrees C. The rate of efflux was increased at 42 degrees C relative to 37 degrees C, but with verapamil the rate decreased to that obtained at 37 degrees C in CHRC5 cells. In drug-sensitive cells, verapamil (< or = 50 microM) did not affect either uptake or efflux of melphalan. These findings suggest that verapamil could be beneficial by increasing the effectiveness of melphalan in the elimination of multidrug-resistant cells. The combination of hyperthermia and verapamil could be especially advantageous by increasing melphalan cytotoxicity in a localized target region.
International Journal of Radiation Oncology*Biology*Physics, 2004
Multidrug resistance (MDR) remains one of the primary obstacles in cancer chemotherapy and often ... more Multidrug resistance (MDR) remains one of the primary obstacles in cancer chemotherapy and often involves overexpression of drug efflux transporters such as P-glycoprotein and multidrug resistance protein 1 (MRP1). Regional hyperthermia is undergoing clinical investigation in combination with chemotherapy or radiotherapy. This study evaluates whether hyperthermia can reverse MDR mediated by MRP1 in human cervical adenocarcinoma (HeLa) cells. Cytotoxicity of hyperthermia and/or etoposide was evaluated using sulforhodamine-B in HeLa cells overexpressing MRP1 and their drug-sensitive counterparts. Glutathione, glutathione peroxidase (GPx), and glutathione S-transferase (GST) were quantified by spectrophotometry. GST isoenzymes were quantified by immunodetection. Caspase activation was evaluated by fluorometry and chromatin condensation by fluorescence microscopy using Hoechst 33258. Necrosis was determined using propidium iodide. The major finding is that HeLa and HeLaMRP cells are both sensitive to cytotoxicity of hyperthermia (41-45 degrees C). Hyperthermia induced activation of caspase 3 and chromatin condensation. Although total levels of cell killing were similar, there was a switch from apoptotic to necrotic cell death in MDR cells. This could be explained by decreased glutathione and GPx in MDR cells. MDR cells also contained very low levels of GST and were resistant to etoposide-induced apoptosis. Hyperthermia caused a modest increase in etoposide-induced apoptosis in HeLa and HeLaMRP cells, which required appropriate heat-drug scheduling. Hyperthermia could be useful in eliminating MDR cells that overexpress MRP1.
Free Radical Biology and Medicine, 1993
Free Radical Biology and Medicine, Oct 1, 2016
The antioxidant transcription factor Nrf2 contributes to the protective effect of mild thermotole... more The antioxidant transcription factor Nrf2 contributes to the protective effect of mild thermotolerance (40°C) against heat shock-induced apoptosis, Free Radical Biology and Medicine,
Journal of Cellular Physiology, 2007
... Liliane Eustache Okoumassoun 1,2 ,; Caterina Russo 2 ,; Francine Denizeau 3, ,; Diana Averil... more ... Liliane Eustache Okoumassoun 1,2 ,; Caterina Russo 2 ,; Francine Denizeau 3, ,; Diana Averill-Bates 3 ,; Dr. Janet E. Henderson 1,2 ... siRNA-mediated down-regulation of the α′ sub-unit in HEK293 cells decreased the expression of survivin and increased apoptosis (Tapia ...
InTech eBooks, May 9, 2013
Vitamins and hormones, 2023
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research
Biotechnology and Bioengineering, Feb 3, 2016
There is increasing demand for cryopreserved cells such as liver and pancreatic cells for clinica... more There is increasing demand for cryopreserved cells such as liver and pancreatic cells for clinical applications. Cryopreservation at ultra-low temperatures requires use of cryoprotectants (e.g., dimethyl sulfoxide (DMSO)) to maintain cell integrity during freezing and thawing processes. Standard cryoprotectants are cytotoxic and more effective cryopreservation technologies are urgently needed for long-term storage of cells. As an alternative, soluble protein extracts (WPE) from winter wheat successfully replaced DMSO as a cryoprotectant for several mammalian cell types. To identify novel cryoactive proteins, the WPE was separated by chromatography and cryoactive fractions were analyzed by mass spectrometry. The wheat protein 2-Cys peroxiredoxin BAS1 (renamed TaBAS1) was identified as a potential cryoactive candidate. Recombinant proteins were prepared and found to possess dual functions as a peroxidase antioxidant and molecular chaperone, and display cryoprotective properties for hepatocytes and insulin-secreting INS832/13 cells. Following cryopreservation with TaBAS1, cells were plateable and showed high post-thaw viability, good adhesion properties, and wellmaintained cell-specific metabolic functions. The overall quality of these cell types was equivalent or improved compared to cells that were cryopreserved with DMSO. The antioxidant and chaperone functions of TaBAS1 likely explain its efficacy in reducing oxidative/ nitrosative stresses in cryopreserved cells. The plant protein TaBAS1 could be a promising molecule to include in cryostorage protocols.
Biochimica et biophysica acta. Molecular cell research, Mar 1, 2021
Hyperthermia is a promising anticancer treatment used in combination with radiotherapy and chemot... more Hyperthermia is a promising anticancer treatment used in combination with radiotherapy and chemotherapy. Temperatures above 41.5°C are cytotoxic and hyperthermia treatments can target a localized area of the body that has been invaded by a tumor. However, non-lethal temperatures (39-41°C) can increase cellular defenses, such as heat shock proteins. This adaptive survival response, thermotolerance, can protect cells against subsequent cytotoxic stress such as anticancer treatments and heat shock (>41.5°C). Autophagy is another survival process that is activated by stress. This study aims to determine whether autophagy can be activated by heat shock at 42°C, and if this response is mediated by reactive oxygen species (ROS). Autophagy was increased during shorter heating times (< 60 min) at 42°C in cells. Levels of acidic vesicular organelles (AVO) and autophagy proteins Beclin-1, LC3-II/LC-3I, Atg7 and Atg12-Atg5 were increased. Heat shock at 42°C increased levels of reactive oxygen species (ROS). Increased levels of LC3 and AVOs at 42°C were inhibited by antioxidants. Therefore, increased autophagy during heat shock at 42°C (<60 min) was mediated by ROS. Conversely, heat shock at 42°C for longer times (1-3 h) caused apoptosis and activation of caspases in the mitochondrial, death receptor and endoplasmic reticulum (ER) pathways. Thermotolerant cells, which were developed at 40°C, were resistant to activation of apoptosis at 42°C. Autophagy inhibitors 3-methyladenine and bafilomycin sensitized cells to activation of apoptosis by heat shock (42°C). Improved understanding of autophagy in cellular responses to heat shock could be useful for optimizing the efficacy of hyperthermia in the clinic.
Free Radical Biology and Medicine, Nov 1, 2022
Cryobiology, Dec 1, 2018
The vitrification of ovarian tissue has the potential to preserve the fertility. The purpose of t... more The vitrification of ovarian tissue has the potential to preserve the fertility. The purpose of the present study was to reveal the effects of cryoprotectant agent temperature on the morphological and ultrastructural features of follicle in dog ovarian tissue. The ovarian tissues were classified as control and two vitrification groups. The forty tissues were exposed to cryoprotectants at 4 C (Group I) or room temperature (RT) (Group II). To vitrification procedure, 1 mm 3 pieces of ovarian tissue were placed in an acupuncture needle. The tissues were exposed to 7.5% ethylene glycol and 7.5% dimethyl sulfoxide (Me 2 SO 4) for 10 min and then 15% ethylene glycol and 15% Me 2 SO 4 for 2 min, either at room temperature or at 4 C and stored in liquid nitrogen at least 1 week. After then the tissues were thawed and immersed into Bouins' and glutaraldehyde solution for analysis. The tissues which were vitrified at 4 C were more preserved than at the room temperature. Lots of healthy primordial follicles were observed in the Group I. In Group II, there were very few normal follicles. In the ultrastructural examination in the Group I, it was observed that the double membrane structure of the oocytes' nucleus was preserved, the distributions and structures of the organelles were normal. In Group II ultrastructural observation, the oocyte cytoplasm was damaged and the linkages between oocytes and the follicle cells were detached. The ice damage was noticed in the tissue structure in this group. Oocytes vacuoles and interstitial vacuoles were seen. The optimal agent temperature was determined as 4 C for dog vitrification equilibration.
Biochimica et biophysica acta. Molecular cell research, Dec 1, 2016
Reactive oxygen species (ROS) are short-lived and highly reactive molecules. The generation of RO... more Reactive oxygen species (ROS) are short-lived and highly reactive molecules. The generation of ROS in cells exists in equilibrium with a variety of antioxidant defences. At low to modest doses, ROS are considered to be essential for regulation of normal physiological functions involved in development such as cell cycle progression and proliferation, differentiation, migration and cell death. ROS also play an important role in the immune system, maintenance of the redox balance and have been implicated in activation of various cellular signalling pathways. Excess cellular levels of ROS cause damage to proteins, nucleic acids, lipids, membranes and organelles, which can lead to activation of cell death processes such as apoptosis. Apoptosis is a highly regulated process that is essential for the development and survival of multicellular organisms. These organisms often need to discard cells that are superfluous or potentially harmful, having accumulated mutations or become infected by pathogens. Apoptosis features a characteristic set of morphological and biochemical features whereby cells undergo a cascade of self-destruction. Thus, proper regulation of apoptosis is essential for maintaining normal cellular homeostasis. ROS play a central role in cell signalling as well as in regulation of the main pathways of apoptosis mediated by mitochondria, death receptors and the endoplasmic reticulum (ER). This review focuses on current understanding of the role of ROS in each of these three main pathways of apoptosis. The role of ROS in the complex interplay and crosstalk between these different signalling pathways remains to be further unravelled during the coming years.
Cell Stress & Chaperones, Jun 11, 2020
Cryopreservation is used for long-term storage of cells and tissues. Cryoprotectants such as dime... more Cryopreservation is used for long-term storage of cells and tissues. Cryoprotectants such as dimethyl disulfoxide (DMSO) are used to protect cells against freeze-thaw damage. Despite the use of cryoprotectants, hepatocytes are sensitive to stresses imposed by freeze and thaw processes, which cause physical damage, loss of functionality, or cell death. As an alternative, we have developed new technology using several recombinant wheat proteins as cryoprotectants: TaENO (enolase), TaBAS1 (2-Cys peroxiredoxin), and a combination of WCS120 (dehydrin) with TaIRI-2 (inhibitor of ice recrystallization). This study aims to understand the mechanisms by which these plant proteins protect rat hepatocytes against cell death incurred during cryopreservation. Our analysis revealed that for cells cryopreserved with DMSO, cell death occurred by apoptosis and necrosis. Apoptosis was detected by activation of effector caspases-3 and -7, PARP cleavage, and nuclear chromatin condensation. These apoptotic events were inhibited when hepatocytes were cryopreserved with the different plant proteins. Cryopreservation with DMSO activated apoptosis through the mitochondrial pathway: the Bax/Bcl-2 protein ratio increased, mitochondrial membrane potential decreased, and initiator caspase-9 was activated. Furthermore, the endoplasmic reticulum pathway of apoptosis was activated: levels of the chaperone Bip/GRP78 decreased, pro-apoptotic transcription factor CHOP was induced, and initiator caspase-12 was activated. Activation of the mitochondrial and endoplasmic reticulum pathways of apoptosis was attenuated when hepatocytes were cryopreserved with the different recombinant proteins. This study improves understanding of mechanisms of cryoprotection provided by these plant proteins during freezing stress. These proteins are natural products and show promising potential by decreasing cell death during cryopreservation of hepatocytes.
Cell Stress and Chaperones, 2020
Cryopreservation is used for long-term storage of cells and tissues. Cryoprotectants such as dime... more Cryopreservation is used for long-term storage of cells and tissues. Cryoprotectants such as dimethyl disulfoxide (DMSO) are used to protect cells against freeze-thaw damage. Despite the use of cryoprotectants, hepatocytes are sensitive to stresses imposed by freeze and thaw processes, which cause physical damage, loss of functionality, or cell death. As an alternative, we have developed new technology using several recombinant wheat proteins as cryoprotectants: TaENO (enolase), TaBAS1 (2-Cys peroxiredoxin), and a combination of WCS120 (dehydrin) with TaIRI-2 (inhibitor of ice recrystallization). This study aims to understand the mechanisms by which these plant proteins protect rat hepatocytes against cell death incurred during cryopreservation. Our analysis revealed that for cells cryopreserved with DMSO, cell death occurred by apoptosis and necrosis. Apoptosis was detected by activation of effector caspases-3 and-7, PARP cleavage, and nuclear chromatin condensation. These apoptotic events were inhibited when hepatocytes were cryopreserved with the different plant proteins. Cryopreservation with DMSO activated apoptosis through the mitochondrial pathway: the Bax/Bcl-2 protein ratio increased, mitochondrial membrane potential decreased, and initiator caspase-9 was activated. Furthermore, the endoplasmic reticulum pathway of apoptosis was activated: levels of the chaperone Bip/GRP78 decreased, pro-apoptotic transcription factor CHOP was induced, and initiator caspase-12 was activated. Activation of the mitochondrial and endoplasmic reticulum pathways of apoptosis was attenuated when hepatocytes were cryopreserved with the different recombinant proteins. This study improves understanding of mechanisms of cryoprotection provided by these plant proteins during freezing stress. These proteins are natural products and show promising potential by decreasing cell death during cryopreservation of hepatocytes.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2016
Various toxic compounds produce reactive oxygen species, resulting in oxidative stress that threa... more Various toxic compounds produce reactive oxygen species, resulting in oxidative stress that threatens cellular homeostasis. Yet, lower doses of stress can stimulate defence systems allowing cell survival, whereas intense stress activates cell death pathways such as apoptosis. Mild thermal stress (40°C, 3 h) induces thermotolerance, an adaptive survival response that renders cells less sensitive to subsequent toxic stress, by activating defence systems like heat shock proteins, antioxidants, anti-apoptotic and ER-stress factors. This study aims to understand how autophagy and apoptosis are regulated in response to different doses of H 2 O 2 , and whether mild thermotolerance can protect cervical carcinoma cells against apoptosis by stimulating autophagy. Autophagy was monitored through Beclin-1 and LC3 expression and acid compartment activity, whereas apoptosis was tracked by caspase activity and chromatin condensation. Exposure of HeLa and C33 A cells to H 2 O 2 for shorter times (15-30 min) transiently induced autophagy; apoptosis was activated after longer times (1-3 h). Mild thermotolerance at 40C enhanced activation of autophagy by H 2 O 2. Disruption of autophagy using bafilomycin A1 and 3methyladenine sensitized cells to apoptosis induced by H 2 O 2 , in non-thermotolerant cells and, to a lesser extent, in thermotolerant cells. Inhibition of autophagy enhanced apoptosis through the mitochondrial, death receptor and endoplasmic reticulum pathways. Autophagy was activated by lower doses of stress and protects cells against apoptosis induced by higher doses of H 2 O 2. This work improves understanding of mechanisms that might be involved in toxicity of various compounds and could eventually lead to protective strategies against deleterious effects of toxic compounds.
The drug discovery and development process requires adequate safety testing for drug toxicity bef... more The drug discovery and development process requires adequate safety testing for drug toxicity before new drugs can be administered to patients. Hepatocytes are used in vitro to screen compounds for hepatotoxicity, induction of drug-metabolizing enzymes such as cytochrome P450 isoforms and drug-drug interactions, and to establish human relevance for metabolism. Cryopreservation makes it possible to preserve a large quantity of functional hepatocytes. Techniques for cryopreservation of hepatocytes are mainly based on dimethylsulfoxide. However, analyses of metabolic capacities of cryopreserved hepatocytes are often limited by loss of functional integrity of hepatocytes after thawing. It is therefore necessary to improve techniques of cryopreservation. We have developed a new cryopreservation technology for mammalian cells based on a wheat protein extract (WPE). We determined whether the WPE can better preserve activities of major cytochrome P450 isoforms, both in suspension and monolayer cultures of hepatocytes. This was achieved by comparing basal and inducible or metabolic activities of isoforms CYP1A1, CYP1A2, CYP2C6, CYP2D2 and CYP3A in rat hepatocytes that were cryopreserved with WPE, relative to fresh cells and those cryopreserved with DMSO. We conclusively demonstrate that rat hepatocytes cryopreserved with WPE retain their metabolic competency and their ability to respond to classical CYP inducers, when compared to freshly isolated hepatocytes. These findings clearly show that WPE are an excellent cryopreservant for rat hepatocytes. They are an efficient, non-toxic, economic natural product and universal cryoprotectant that is superior to DMSO, which has limitations due to cellular toxicity. This article has not been copyedited and formatted. The final version may differ from this version.
Radiation Research, 1995
The effect of both hyperthermia and verapamil on cytotoxicity and transport of melphalan was stud... more The effect of both hyperthermia and verapamil on cytotoxicity and transport of melphalan was studied in a pleiotropic drug-resistant Chinese hamster ovary cell line (CHRC5) and in the drug-sensitive parent line (AuxB1). The CHRC5 cell line was selected for resistance to colchicine but is also cross-resistant to other drugs including melphalan. Verapamil (10 microM) increased melphalan cytotoxicity in drug-resistant cells but not in drug-sensitive cells. Hyperthermia (40 to 45 degrees C) increased melphalan cytotoxicity in both cell lines. In drug-resistant but not drug-sensitive cells, melphalan cytotoxicity was increased further when verapamil was combined with hyperthermia (40 to 45 degrees C). The increased cytotoxicity caused by verapamil in drug-resistant cells was accompanied by alterations in membrane permeability to melphalan. The cellular uptake of melphalan after 15 min increased in the presence of verapamil (7 to 30 microM) at 37 and 42 degrees C. When verapamil (10 microM) was present, the rate of efflux of melphalan from CHRC5 cells decreased by almost 40% at 37 degrees C. The rate of efflux was increased at 42 degrees C relative to 37 degrees C, but with verapamil the rate decreased to that obtained at 37 degrees C in CHRC5 cells. In drug-sensitive cells, verapamil (< or = 50 microM) did not affect either uptake or efflux of melphalan. These findings suggest that verapamil could be beneficial by increasing the effectiveness of melphalan in the elimination of multidrug-resistant cells. The combination of hyperthermia and verapamil could be especially advantageous by increasing melphalan cytotoxicity in a localized target region.
International Journal of Radiation Oncology*Biology*Physics, 2004
Multidrug resistance (MDR) remains one of the primary obstacles in cancer chemotherapy and often ... more Multidrug resistance (MDR) remains one of the primary obstacles in cancer chemotherapy and often involves overexpression of drug efflux transporters such as P-glycoprotein and multidrug resistance protein 1 (MRP1). Regional hyperthermia is undergoing clinical investigation in combination with chemotherapy or radiotherapy. This study evaluates whether hyperthermia can reverse MDR mediated by MRP1 in human cervical adenocarcinoma (HeLa) cells. Cytotoxicity of hyperthermia and/or etoposide was evaluated using sulforhodamine-B in HeLa cells overexpressing MRP1 and their drug-sensitive counterparts. Glutathione, glutathione peroxidase (GPx), and glutathione S-transferase (GST) were quantified by spectrophotometry. GST isoenzymes were quantified by immunodetection. Caspase activation was evaluated by fluorometry and chromatin condensation by fluorescence microscopy using Hoechst 33258. Necrosis was determined using propidium iodide. The major finding is that HeLa and HeLaMRP cells are both sensitive to cytotoxicity of hyperthermia (41-45 degrees C). Hyperthermia induced activation of caspase 3 and chromatin condensation. Although total levels of cell killing were similar, there was a switch from apoptotic to necrotic cell death in MDR cells. This could be explained by decreased glutathione and GPx in MDR cells. MDR cells also contained very low levels of GST and were resistant to etoposide-induced apoptosis. Hyperthermia caused a modest increase in etoposide-induced apoptosis in HeLa and HeLaMRP cells, which required appropriate heat-drug scheduling. Hyperthermia could be useful in eliminating MDR cells that overexpress MRP1.
Free Radical Biology and Medicine, 1993