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5. Signaling proteins regulate cellular freeze response
Abstracts / Cryobiology 61 (2010) 362–408 4. Adaptations and climate change in Antarctic marine animals. Lloyd S. Peck, British Antarctic Survey, High Cross, Madingley Rd., Cambridge CB3 0ET, UK Antarctic marine species live at the end of the spectrum of marine environments on Earth, with low, constant temperatures and the strongest seasonality of light and phytoplankton productivity anywhere. The marine animals there have some of the lowest physiological rates reported with slow growth and development, metabolic rates, and activity levels of any major fauna and Earth. They also have many unique adaptations including a group of fish that has no blood pigments to carry oxygen around the body, gigantism, and the loss of the previously reported as ubiquitous heat shock response. They also have possibly the poorest abilities of any major fauna to resist or respond to elevated temperature in laboratory experiments. However, parts of the Antarctic have warmed as fast or faster than anywhere on Earth, with Antarctic peninsula air temperatures rising 3 °C and sea temperatures 1 °C in 50 years. Climate models also predict that polar areas will warm more than other regions in the future. This talk will highlight current research on vulnerabilities of this group to change, and where approaches to predicting responses to change are lacking. Conflict of interest: None declared. Source of funding: None declared. doi:10.1016/j.cryobiol.2010.10.008
Joint session with the Society for Low Temperature Biology Cryosurgery – Applying cryobiology in ablative therapy 5. Signaling proteins regulate cellular freeze response. J.G. Baust * 1, A.A. Gage 2, J.M. Baust 3, 1 Institute of Biomedical Technology, State University of New York, Binghamton, NY, United States, 2 Department of Surgery, State University of New York, Buffalo, NY, United States, 3 CPSI Biotech, Inc., Owego, NY, United States Cellular responses to freezing excursions whether during the course of cryoablative or cryopreservation protocols vary with regard to structural integrity, survival and cell response. Analysis of the biophysical parameters associated with a freezethaw excursion provide helpful data on determinates of gross structural integrity and survival provided intracellular freezing or severe excursions in cell volume occur. With successful cryopreservation, neither of these determinates are anticipated and the delayed on-set cell death observed following thawing is related to the accumulation of severe oxidative stresses (ref). Similarly, the cell death observed following a cryosurgical procedure due to the intrusive biophysical events that occur during the few minutes of freezing is limited. Importantly, a significant portion of cell death is due to the activation of diverse, interrelated cell death cascades. The cryoablative process is characterized by the cumulative effects of cellular stressors that manifest through proteomic signaling in the launch of cell death cascades. These signaling paths dictate whether a specific cell will succumb to the lethal effects of freezing via apoptosis or necrosis or recover via cell survival pathway activation. Molecular analysis of cancer cell response to freezing insults has revealed complex web of protein activation including both cell death and survival signaling. This web includes the activation of caspases, the Bcl-2 family, AKT, JUN kinase, cell surface signaling proteins (TRADD, FAS, TRAIL, etc.), among others. Further, the cell responses have been found to be cancer type specific and highly influenced by the molecular pre-disposition including androgen sensitivity, integrin expression, etc. This presentation will provide an overview of the studies conducted in our laboratory focused on dissecting the proteomic response of cells to freezing injury. These studies have provided insight into cryoablative processes allowing for the development of new neo-adjunctive strategies for treating cancer. Conflict of interest: None declared. Source of funding: Studies supported by various grants from the National Institutes of Health.
363
was to investigate if a cell’s sensitivity to freeze stress is related to cell cycle phase. We hypothesized that cells are most sensitive to cryotherapy during the G2/M phase of the cell cycle, and the cells that survive freezing would primarily be in the G1/G0 phase. The mucine prostate cancer cell line PM-9 was used as a model and frozen to 15 °C in a circulating temperature controlled liquid bath. Cell viability was assessed using the metabolic activity indicator alamarBlueTM for 3 days post-treatment and cell cycle responses were determined via flow cytometry. For determination of DNA content cells were fixed in ethanol and stored at 4 °C for at least 24 h prior to staining with a propidium iodide (PI) solution containing Rnase and run on a Guava EasyCyte Plus microcapillary flow cytometry system. Data was analyzed using ModFit LT (Verity Software House). Cells were subjected to DNA analysis by flow cytometry to determine the percentage of cells in G0/G1, S, or G2/M phase. Logarithmically growing control populations exhibited approximately 43% G0/G1, 29% S, and 28% G2/M. Cells exposed to a single 10 min exposure to 15 °C were separated into lifted and adherent populations to estimate which cells were more sensitive to freeze. In the viable post-freeze population the percentage of G0/G1 cells dropped to 21%; this could be due to a loss of cells in G2/M that do not re-enter the G1 phase of the cell cycle. The changes in cell cycle distribution seen between the control population and the population remaining after freezing suggests a role for cell cycle in low temperature injury. This indicates there may be potential for adjuvants to alter the cell cycle to improve therapeutic outcome. Understanding how cell cycle kinetics can influence response to treatment will help to identify those potential adjuvants to cryotherapy that may ultimately increase treatment efficacy and patient quality of life. Conflict of interest: None declared. Source of funding: Grants R43CA1123993-01A1 and R43CA118537-01A1. doi:10.1016/j.cryobiol.2010.10.010
7. A new approach to cryosurgery training using computation tools. Yoed Rabin *, Kenji Shimada, Department of Mechanical Engineering, Carnegie Mellon University, Pittsburgh, PA, United States This talk presents a new approach to complement cryosurgery training by using a battery of computation tools. The main objectives in this effort are: (i) to develop a software package to simulate the cryosurgical procedure and (ii) to improve surgical decisions based on biothermal considerations. The simulation tools under development far exceed the traditional bioheat-transfer simulation capabilities by making the displayed results compatible with imaging means available to the clinician in the operating room, and by incorporating visualization of virtual procedures. The proposed training approach combines concepts from recently developed computerized planning methods with common clinical practices, in an effort to help the trainee select a better cryoprobe layout and its parameters (including cryoprobe type, size, and number). As part of the training system, a database is developed to enable the comparison of past performance with the current interactive training session. This presentation displays the conceptual design of the virtual training setup, reviews recent developments in computation tools for surgical planning, and presents a new computational method for creating a three-dimensional geometric model of a target organ from image datasets. This presentation uses examples from the application of prostate cryosurgery, which is the most established minimally invasive cryoprocedure. Conflict of interest: None declared. Source of funding: The project described is supported by Award Number R01CA134261 from the National Cancer Institute of the USA. Developing the computerized planning tools has been supported by Award Number R01EB003563 from the National Institute of Biomedical Imaging and Bioengineering of the USA. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health.
doi:10.1016/j.cryobiol.2010.10.009
doi:10.1016/j.cryobiol.2010.10.011
6. Investigating the role of cell cycle in a prostate cancer cryosurgical model. Kimberly L. Santucci * 1,2, Kristi K. Snyder 2,3, John M. Baust 2,3, Robert G. Van Buskirk 1,2,3, John G. Baust 2, 1 Department of Biological Sciences, Binghamton University, Binghamton, NY, United States, 2 Institute of Biomedical Technology, Binghamton University, Binghamton, NY, United States, 3 CPSI Biotech, Owego, NY, United States
8. An in vitro investigation of cardiomyocyte response to cryoablation. Kristi K. Snyder * 1,2, John M. Baust 1,2, James Cox 4, Robert G. Van Buskirk 1,2,3, John G. Baust 2,3, 1 CPSI Biotech, Owego, NY, USA, 2 Institute of Biomedical Technology, Binghamton University, Binghamton, NY, USA, 3 Dept. of Biological Sciences, Binghamton University, Binghamton, NY, USA, 4 ATS Medical, Inc., Minneapolis, MN, USA
Cell cycle progression is regulated by a complex network of signaling pathways that are amplified in cancerous cells. Stress signals that normally activate cell cycle checkpoints often fail, allowing continued proliferation, accumulation of mutations and chromosome instability. This makes tumor cells unpredictable, resulting in the development of aggressive cancers (such as prostate) which become difficult to treat. Cryotherapy is an established therapeutic strategy for the treatment of prostate cancer, but so far no studies have investigated the effect of cryo on cell cycle, or what effects cell cycle phase can have on freeze sensitivity. Therefore, the aim of this study
Atrial and ventricular tachyarrhythmias represent major cardiovascular diseases that may be treated utilizing thermal therapies. Given this there has been a palpable increase in technologies and approaches of therapeutic intervention, including cryoablation. While increased utilization of cryoablation continues, our fundamental understanding of the response of cardiac cells to the freezing process remains limited. As such we investigated the cellular and molecular response of cardiomyocytes to a range of subfreezing temperatures in an effort to provide further insight to help guide the application of cryoablation for the treatment of cardiac arrhythmias.
Joint session with the Society for Low Temperature Biology Cryosurgery – Applying cryobiology in ablative therapy 5. Signaling proteins regulate cellular freeze response. J.G. Baust * 1, A.A. Gage 2, J.M. Baust 3, 1 Institute of Biomedical Technology, State University of New York, Binghamton, NY, United States, 2 Department of Surgery, State University of New York, Buffalo, NY, United States, 3 CPSI Biotech, Inc., Owego, NY, United States Cellular responses to freezing excursions whether during the course of cryoablative or cryopreservation protocols vary with regard to structural integrity, survival and cell response. Analysis of the biophysical parameters associated with a freezethaw excursion provide helpful data on determinates of gross structural integrity and survival provided intracellular freezing or severe excursions in cell volume occur. With successful cryopreservation, neither of these determinates are anticipated and the delayed on-set cell death observed following thawing is related to the accumulation of severe oxidative stresses (ref). Similarly, the cell death observed following a cryosurgical procedure due to the intrusive biophysical events that occur during the few minutes of freezing is limited. Importantly, a significant portion of cell death is due to the activation of diverse, interrelated cell death cascades. The cryoablative process is characterized by the cumulative effects of cellular stressors that manifest through proteomic signaling in the launch of cell death cascades. These signaling paths dictate whether a specific cell will succumb to the lethal effects of freezing via apoptosis or necrosis or recover via cell survival pathway activation. Molecular analysis of cancer cell response to freezing insults has revealed complex web of protein activation including both cell death and survival signaling. This web includes the activation of caspases, the Bcl-2 family, AKT, JUN kinase, cell surface signaling proteins (TRADD, FAS, TRAIL, etc.), among others. Further, the cell responses have been found to be cancer type specific and highly influenced by the molecular pre-disposition including androgen sensitivity, integrin expression, etc. This presentation will provide an overview of the studies conducted in our laboratory focused on dissecting the proteomic response of cells to freezing injury. These studies have provided insight into cryoablative processes allowing for the development of new neo-adjunctive strategies for treating cancer. Conflict of interest: None declared. Source of funding: Studies supported by various grants from the National Institutes of Health.
363
was to investigate if a cell’s sensitivity to freeze stress is related to cell cycle phase. We hypothesized that cells are most sensitive to cryotherapy during the G2/M phase of the cell cycle, and the cells that survive freezing would primarily be in the G1/G0 phase. The mucine prostate cancer cell line PM-9 was used as a model and frozen to 15 °C in a circulating temperature controlled liquid bath. Cell viability was assessed using the metabolic activity indicator alamarBlueTM for 3 days post-treatment and cell cycle responses were determined via flow cytometry. For determination of DNA content cells were fixed in ethanol and stored at 4 °C for at least 24 h prior to staining with a propidium iodide (PI) solution containing Rnase and run on a Guava EasyCyte Plus microcapillary flow cytometry system. Data was analyzed using ModFit LT (Verity Software House). Cells were subjected to DNA analysis by flow cytometry to determine the percentage of cells in G0/G1, S, or G2/M phase. Logarithmically growing control populations exhibited approximately 43% G0/G1, 29% S, and 28% G2/M. Cells exposed to a single 10 min exposure to 15 °C were separated into lifted and adherent populations to estimate which cells were more sensitive to freeze. In the viable post-freeze population the percentage of G0/G1 cells dropped to 21%; this could be due to a loss of cells in G2/M that do not re-enter the G1 phase of the cell cycle. The changes in cell cycle distribution seen between the control population and the population remaining after freezing suggests a role for cell cycle in low temperature injury. This indicates there may be potential for adjuvants to alter the cell cycle to improve therapeutic outcome. Understanding how cell cycle kinetics can influence response to treatment will help to identify those potential adjuvants to cryotherapy that may ultimately increase treatment efficacy and patient quality of life. Conflict of interest: None declared. Source of funding: Grants R43CA1123993-01A1 and R43CA118537-01A1. doi:10.1016/j.cryobiol.2010.10.010
7. A new approach to cryosurgery training using computation tools. Yoed Rabin *, Kenji Shimada, Department of Mechanical Engineering, Carnegie Mellon University, Pittsburgh, PA, United States This talk presents a new approach to complement cryosurgery training by using a battery of computation tools. The main objectives in this effort are: (i) to develop a software package to simulate the cryosurgical procedure and (ii) to improve surgical decisions based on biothermal considerations. The simulation tools under development far exceed the traditional bioheat-transfer simulation capabilities by making the displayed results compatible with imaging means available to the clinician in the operating room, and by incorporating visualization of virtual procedures. The proposed training approach combines concepts from recently developed computerized planning methods with common clinical practices, in an effort to help the trainee select a better cryoprobe layout and its parameters (including cryoprobe type, size, and number). As part of the training system, a database is developed to enable the comparison of past performance with the current interactive training session. This presentation displays the conceptual design of the virtual training setup, reviews recent developments in computation tools for surgical planning, and presents a new computational method for creating a three-dimensional geometric model of a target organ from image datasets. This presentation uses examples from the application of prostate cryosurgery, which is the most established minimally invasive cryoprocedure. Conflict of interest: None declared. Source of funding: The project described is supported by Award Number R01CA134261 from the National Cancer Institute of the USA. Developing the computerized planning tools has been supported by Award Number R01EB003563 from the National Institute of Biomedical Imaging and Bioengineering of the USA. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health.
doi:10.1016/j.cryobiol.2010.10.009
doi:10.1016/j.cryobiol.2010.10.011
6. Investigating the role of cell cycle in a prostate cancer cryosurgical model. Kimberly L. Santucci * 1,2, Kristi K. Snyder 2,3, John M. Baust 2,3, Robert G. Van Buskirk 1,2,3, John G. Baust 2, 1 Department of Biological Sciences, Binghamton University, Binghamton, NY, United States, 2 Institute of Biomedical Technology, Binghamton University, Binghamton, NY, United States, 3 CPSI Biotech, Owego, NY, United States
8. An in vitro investigation of cardiomyocyte response to cryoablation. Kristi K. Snyder * 1,2, John M. Baust 1,2, James Cox 4, Robert G. Van Buskirk 1,2,3, John G. Baust 2,3, 1 CPSI Biotech, Owego, NY, USA, 2 Institute of Biomedical Technology, Binghamton University, Binghamton, NY, USA, 3 Dept. of Biological Sciences, Binghamton University, Binghamton, NY, USA, 4 ATS Medical, Inc., Minneapolis, MN, USA
Cell cycle progression is regulated by a complex network of signaling pathways that are amplified in cancerous cells. Stress signals that normally activate cell cycle checkpoints often fail, allowing continued proliferation, accumulation of mutations and chromosome instability. This makes tumor cells unpredictable, resulting in the development of aggressive cancers (such as prostate) which become difficult to treat. Cryotherapy is an established therapeutic strategy for the treatment of prostate cancer, but so far no studies have investigated the effect of cryo on cell cycle, or what effects cell cycle phase can have on freeze sensitivity. Therefore, the aim of this study
Atrial and ventricular tachyarrhythmias represent major cardiovascular diseases that may be treated utilizing thermal therapies. Given this there has been a palpable increase in technologies and approaches of therapeutic intervention, including cryoablation. While increased utilization of cryoablation continues, our fundamental understanding of the response of cardiac cells to the freezing process remains limited. As such we investigated the cellular and molecular response of cardiomyocytes to a range of subfreezing temperatures in an effort to provide further insight to help guide the application of cryoablation for the treatment of cardiac arrhythmias.