http://hdl.handle.net/10027/7397 Mon, 20 May 2013 12:10:38 GMT 2013-05-20T12:10:38Z http://hdl.handle.net/10027/7761 Part I. Development of a Model System for Studying Nitric Oxide in Tumors: High Nitric Oxide-Adapted Head and Neck Squamous Cell Carcinoma Cell Lines Yarmolyuk, Yaroslav; Vesper, Benjamin J.; Paradise, William A.; Elseth, Kim M.; Tarjan, Gabor; Haines III, G. K.; Radosevich, James A. The free radical nitric oxide (NO) is over-expressed in many tumors, including head and neck squamous cell carcinomas (HNSCC). However, the role NO plays in tumor pathophysiology is still not well understood. We herein report the development of an in vitro model system which can be used to probe the role of NO in the carcinogenesis of HNSCC. Five HNSCC cell lines were adapted to a high NO (HNO) environment by gradually introducing increasing concentrations of DETA-NONOate, a nitrogen-based NO donor, to cell media. The adaptation process was carried out until a sufficiently high enough donor concentration was reached which enabled the HNO cells to survive and grow, but which was lethal to the original, unadapted (“parent”) cells. The adapted HNO cells exhibited analogous morphology to the parent cells, but grew better than their corresponding parent cells in normal media, on soft agar, and in the presence of hydrogen peroxide, an oxygen-based free radical donor. These results indicate the HNO cell lines are unique and possess biologically different properties than the parent cell lines from which they originated. The HNO/parent cell lines developed herein may be used as a model system to better understand the role NO plays in HNSCC carcinogenesis. Post print version of article may differ from published version. The original publication is available at www.springerlink.com; DOI: 10.1007/s13277-010-0101-1. Tue, 01 Feb 2011 06:00:00 GMT http://hdl.handle.net/10027/7761 2011-02-01T06:00:00Z http://hdl.handle.net/10027/7651 Molecular mechanisms of antithrombin-heparin regulation of blood clotting proteinases. A paradigm for understanding proteinase regulation by serpin family protein proteinase inhibitors Olson, Steven T.; Richard, Benjamin; Izaguirre, Gonzalo; Schedin-Weiss, Sophia; Gettins, Peter G. W. Serpin family protein proteinase inhibitors regulate the activity of serine and cysteine proteinases by a novel conformational trapping mechanism that may itself be regulated by cofactors to provide a finely-tuned time and location-dependent control of proteinase activity. The serpin, antithrombin, together with its cofactors, heparin and heparan sulfate, perform a critical anticoagulant function by preventing the activation of blood clotting proteinases except when needed at the site of a vascular injury. Here, we review the detailed molecular understanding of this regulatory mechanism that has emerged from numerous X-ray crystal structures of antithrombin and its complexes with heparin and target proteinases together with mutagenesis and functional studies of heparin-antithrombin-proteinase interactions in solution. Like other serpins, antithrombin achieves specificity for its target blood clotting proteinases by presenting recognition determinants in an exposed reactive center loop as well as in exosites outside the loop. Antithrombin reactivity is repressed in the absence of its activator because of unfavorable interactions that diminish the favorable RCL and exosite interactions with proteinases. Binding of a specific heparin or heparan sulfate pentasaccharide to antithrombin induces allosteric activating changes that mitigate the unfavorable interactions and promote template bridging of the serpin and proteinase. Antithrombin has thus evolved a sophisticated means of regulating the activity of blood clotting proteinases in a time and location-dependent manner that exploits the multiple conformational states of the serpin and their differential stabilization by glycosaminoglycan cofactors. Post print version of article may differ from published version. The definitive version is available through Elsevier at DOI: 10.1016/j.biochi.2010.05.011 Mon, 01 Nov 2010 05:00:00 GMT http://hdl.handle.net/10027/7651 2010-11-01T05:00:00Z http://hdl.handle.net/10027/7641 Kinetic evidence that allosteric activation of antithrombin by heparin is mediated by two sequential conformational changes Schedin-Weiss, Sophia; Richard, Benjamin; Olson, Steven T. The serpin, antithrombin, requires allosteric activation by a sequencespecific pentasaccharide unit of heparin or heparan sulfate glycosaminoglycans to function as an anticoagulant regulator of blood clotting proteases. Surprisingly, X-ray structures have shown that the pentasaccharide produces similar inducedfit changes in the heparin binding site of native and latent antithrombin despite large differences in the heparin affinity and global conformation of these two forms. Here we present kinetic evidence for similar induced-fit mechanisms of pentasaccharide binding to native and latent antithrombins and kinetic simulations which together support a three-step mechanism of allosteric activation of native antithrombin involving two successive conformational changes. Equilibrium binding studies of pentasaccharide interactions with native and latent antithrombins and the salt dependence of these interactions suggest that each conformational change is associated with distinct spectroscopic changes and is driven by a progressively better fit of the pentasaccharide in the binding site. The observation that variant antithrombins that cannot undergo the second conformational change bind the pentasaccharide like latent antithrombin and are partially activated suggests that both conformational changes contribute to allosteric activation, in agreement with a recently proposed model of allosteric activation. Post print version of article may differ from published version. The definitive version is available through Elsevier at DOI: 10.1016/j.abb.2010.08.021 Wed, 15 Dec 2010 06:00:00 GMT http://hdl.handle.net/10027/7641 2010-12-15T06:00:00Z http://hdl.handle.net/10027/7619 Deregulation of manganese superoxide dismutase (SOD2) expression and lymph node metastasis in tongue squamous cell carcinoma Liu, Xiqiang; Wang, Anxun; Muzio, Lorenzo L.; Kolokythas, Antonia; Sheng, Shihu; Rubini, Corrado; Ye, Hui; Shi, Fei; Yu, Tianwei; Crowe, David L.; Zhou, Xiofeng Background: Lymph node metastasis is a critical event in the progression of tongue squamous cell carcinoma (TSCC). The identification of biomarkers associated with the metastatic process would provide critical prognostic information to facilitate clinical decision making. Previous studies showed that deregulation of manganese superoxide dismutase (SOD2) expression is a frequent event in TSCC and may be associated with enhanced cell invasion. The purpose of this study is to further evaluate whether the expression level of SOD2 is correlated with the metastatic status in TSCC patients. Methods: We first examined the SOD2 expression at mRNA level on 53 TSCC and 22 normal control samples based on pooled-analysis of existing microarray datasets. To confirm our observations, we examined the expression of SOD2 at protein level on an additional TSCC patient cohort (n = 100), as well as 31 premalignant dysplasias, 15 normal tongue mucosa, and 32 lymph node metastatic diseases by immunohistochemistry (IHC). Results: The SOD2 mRNA level in primary TSCC tissue is reversely correlated with lymph node metastasis in the first TSCC patient cohort. The SOD2 protein level in primary TSCC tissue is also reversely correlated with lymph node metastasis in the second TSCC patient cohort. Deregulation of SOD2 expression is a common event in TSCC and appears to be associated with disease progression. Statistical analysis revealed that the reduced SOD2 expression in primary tumor tissue is associated with lymph node metastasis in both TSCC patient cohorts examined. Conclusions: Our study suggested that the deregulation of SOD2 in TSCC has potential predictive values for lymph node metastasis, and may serve as a therapeutic target for patients at risk of metastasis. The original version is available through BioMed Central at DOI: 10.1186/1471-2407-10-365. © 2010 Liu et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Fri, 09 Jul 2010 05:00:00 GMT http://hdl.handle.net/10027/7619 2010-07-09T05:00:00Z