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ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
administration demonstrated minimal to no siRNA uptake in target organs. Importantly, our results demonstrate that siRNA can be effectively administered to various organ systems by standard or hydrodynamic IV injection in vivo thus offering a potential therapeutic option for specific gene knockdown as a novel treatment strategy for certain diseases.
94. MEDICAL INFERENCE BY NETWORK INTEGRATION OF TEMPORAL DATA USING DYNAMIC BAYESIAN ANALYSIS. R. M. Minter, Y. He, P. J. Woolf, Z. Xiang, X. Bi, D. G. Remick; University of Michigan, Ann Arbor, MI Background: In biomedical research and clinical studies, experimental data are often collected temporally, and it is important to know if an intervention or an adverse event (i.e. drug treatment or pathogenic infection) affects the distribution of data over time. Dynamic Bayesian networks (DBNs) represent a powerful method for identifying potential causal patterns in heterogeneous, fluctuating, and complex datasets, while tolerating a large degree of data variability and nonlinearity. Purpose: The present studies were undertaken to develop a generic web-based network modeling system which will allow clinical and biomedical researchers to perform dynamic Bayesian network analysis for temporal datasets. This system will support complex inference modeling, including rational decision making systems, useful for causality analysis. Methods: A complex inference modeling system was developed to support the analysis of the dynamic and heterogeneous inflammatory response present in the setting of biliary obstruction. A generic web-based tool was developed for this analysis: Medical Inference by Network Integration of Temporal Data Using Bayesian Analysis (miniTUBA), and can be viewed at www. minituba.org. In the present studies, female ICR mice underwent Sham operation, Common Bile Duct Ligation (CBDL), or no treatment at all. Mice were followed daily and 20 l of tailvein blood was obtained three times weekly. Survival and weights were measured daily, and a complete blood cell count and differential was performed using an automated system. Plasma was assayed for bilirubin, AST, and ALT levels. Results: miniTUBA was utilized to analyze the temporal dataset obtained from the studies outlined. The network shown demonstrates the highest scoring network for the data provided. miniTUBA will also demonstrate the top five scoring networks as well as the conserved edges, or those relationships which persist across the top thirty scoring networks. Though inflammatory mediators were also measured in the plasma, initial analyses were undertaken to demonstrate that miniTUBA was able to identify known relationships. As shown, procedure (CBDL, Sham, No Tx) relates to liver injury parameters, weight loss, survival, and platelet count. Additionally, the total white blood cell count relates to the neutrophil and lymphocyte counts as would be expected. Of interest, however, is that even with this simple dataset comprised only of clinical data, miniTUBA is able to identify an unknown relationship between neutrophil count and survival following CBDL. Closer examination of this relationship within miniTUBA reveals that CBDL animals with a low neutrophil count are ten times more likely to expire the following day than those mice with a normal or high neutrophil count. Conclusion: miniTUBA represents a powerful generic web-based tool which can be utilized by investigators for the analysis of complex and heterogeneous temporal datasets. This system will allow for the analysis of an infinite number of variables at any level of an organism; allowing investigators to identify clinical predictors as well as potential putative mediators or pathways which can then be targeted for perturbation or therapeutic intervention.
ONCOLOGY III: CELL SIGNALING 95. EGFR REGULATES HAS3 EXPRESSION IN COLON CANCER CELLS. R. Singh 1, Y. Zhao 2, J. Wang 2, G. Howell 2, M. Brattain 2, A. Rajput 3, K. Bullarddunn 3; 1University at Buffalo, State University of New York, Department of Surgery, Buffalo, NY, 2Roswell Park Cancer Institute, Buffalo, NY, 3University at Buffalo, State University of New York, Department of Surgery, Roswell Park Cancer Institute, Buffalo, NY Background: Hyaluronan (HA) and its biosynthetic enzymes (hyaluronan synthases; HAS1, 2 and 3) have been implicated in cancer growth and progression. We have previously shown that HAS3 is upregulated in metastatic colon cancer cells, and mediates anchorage-independent cell growth and Matrigel invasion; however the mechanism by which this enzyme is regulated remains poorly understood. Epidermal Growth Factor (EGFR) is a tyrosine kinase receptor that activates the PI3-kinase (PI3K) pathway and appears to be critical to cancer cell survival, proliferation and migration. HCT116 cells are highly tumorigenic human colon cancer cells that express constitutively active EGFR. This cell line also possesses both mutant PI3K (gain of function) and wild-type PI3K. Manipulation of EGFR activity alters HCT116 cell growth and invasion. HCT116 cells also synthesize and retain pericellular HA. Based upon our previous observation that HAS3 mediates cellular growth and invasion, we hypothesized that EGFR and/or PI3K activity would alter expression of HAS3 in HCT116 cells. Materials and Methods: To test this hypothesis, we investigated HAS expression in cell lines derived from HCT116. To investigate the role of EGFR activation, we used HCT116b cells (isoparental clones of HCT116) that do not have constitutively active EGFR and are less tumorigenic. We also assessed HAS expression in HCT116 cells treated with the EGFR inhibitor Tarceva (erlotinib). Finally, HCT116 cells were manipulated by homologous recombination techniques to delete one of the two PI3K alleles, generating a PI3K mutant/- (gain of function; active) and PI3K wild type/-. HAS isozyme expression was first assessed using semi-quantitative RT-PCR; band intensities were normalized to GAPDH. Results were then confirmed using quantitative PCR. Experiments were repeated in triplicate. Data were analyzed with Students’t-test. Results: None of the cell lines expressed HAS1. HAS2 was detected in all four cell lines, but expression did not differ. In contrast, HCT116 cells were found to express high levels of HAS3, and its expression correlated with EGFR and PI3K activity. HCT116b cells expressed less HAS3 than HCT116 cells. PI3K wild-type/- cells expressed less HAS3 than the PI3K mutant/- cells. Treatment with Tarceva also decreased HAS3 expression, confirming that EGFR activity was responsible for upregulated HAS3 expression in HCT116 cells. Conclusions: EGFR activation increases HAS3 expression in HCT116 colon cancer cells, and EGFR inhibition decreases expression. In addition, PI3K activation is as-
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
administration demonstrated minimal to no siRNA uptake in target organs. Importantly, our results demonstrate that siRNA can be effectively administered to various organ systems by standard or hydrodynamic IV injection in vivo thus offering a potential therapeutic option for specific gene knockdown as a novel treatment strategy for certain diseases.
94. MEDICAL INFERENCE BY NETWORK INTEGRATION OF TEMPORAL DATA USING DYNAMIC BAYESIAN ANALYSIS. R. M. Minter, Y. He, P. J. Woolf, Z. Xiang, X. Bi, D. G. Remick; University of Michigan, Ann Arbor, MI Background: In biomedical research and clinical studies, experimental data are often collected temporally, and it is important to know if an intervention or an adverse event (i.e. drug treatment or pathogenic infection) affects the distribution of data over time. Dynamic Bayesian networks (DBNs) represent a powerful method for identifying potential causal patterns in heterogeneous, fluctuating, and complex datasets, while tolerating a large degree of data variability and nonlinearity. Purpose: The present studies were undertaken to develop a generic web-based network modeling system which will allow clinical and biomedical researchers to perform dynamic Bayesian network analysis for temporal datasets. This system will support complex inference modeling, including rational decision making systems, useful for causality analysis. Methods: A complex inference modeling system was developed to support the analysis of the dynamic and heterogeneous inflammatory response present in the setting of biliary obstruction. A generic web-based tool was developed for this analysis: Medical Inference by Network Integration of Temporal Data Using Bayesian Analysis (miniTUBA), and can be viewed at www. minituba.org. In the present studies, female ICR mice underwent Sham operation, Common Bile Duct Ligation (CBDL), or no treatment at all. Mice were followed daily and 20 l of tailvein blood was obtained three times weekly. Survival and weights were measured daily, and a complete blood cell count and differential was performed using an automated system. Plasma was assayed for bilirubin, AST, and ALT levels. Results: miniTUBA was utilized to analyze the temporal dataset obtained from the studies outlined. The network shown demonstrates the highest scoring network for the data provided. miniTUBA will also demonstrate the top five scoring networks as well as the conserved edges, or those relationships which persist across the top thirty scoring networks. Though inflammatory mediators were also measured in the plasma, initial analyses were undertaken to demonstrate that miniTUBA was able to identify known relationships. As shown, procedure (CBDL, Sham, No Tx) relates to liver injury parameters, weight loss, survival, and platelet count. Additionally, the total white blood cell count relates to the neutrophil and lymphocyte counts as would be expected. Of interest, however, is that even with this simple dataset comprised only of clinical data, miniTUBA is able to identify an unknown relationship between neutrophil count and survival following CBDL. Closer examination of this relationship within miniTUBA reveals that CBDL animals with a low neutrophil count are ten times more likely to expire the following day than those mice with a normal or high neutrophil count. Conclusion: miniTUBA represents a powerful generic web-based tool which can be utilized by investigators for the analysis of complex and heterogeneous temporal datasets. This system will allow for the analysis of an infinite number of variables at any level of an organism; allowing investigators to identify clinical predictors as well as potential putative mediators or pathways which can then be targeted for perturbation or therapeutic intervention.
ONCOLOGY III: CELL SIGNALING 95. EGFR REGULATES HAS3 EXPRESSION IN COLON CANCER CELLS. R. Singh 1, Y. Zhao 2, J. Wang 2, G. Howell 2, M. Brattain 2, A. Rajput 3, K. Bullarddunn 3; 1University at Buffalo, State University of New York, Department of Surgery, Buffalo, NY, 2Roswell Park Cancer Institute, Buffalo, NY, 3University at Buffalo, State University of New York, Department of Surgery, Roswell Park Cancer Institute, Buffalo, NY Background: Hyaluronan (HA) and its biosynthetic enzymes (hyaluronan synthases; HAS1, 2 and 3) have been implicated in cancer growth and progression. We have previously shown that HAS3 is upregulated in metastatic colon cancer cells, and mediates anchorage-independent cell growth and Matrigel invasion; however the mechanism by which this enzyme is regulated remains poorly understood. Epidermal Growth Factor (EGFR) is a tyrosine kinase receptor that activates the PI3-kinase (PI3K) pathway and appears to be critical to cancer cell survival, proliferation and migration. HCT116 cells are highly tumorigenic human colon cancer cells that express constitutively active EGFR. This cell line also possesses both mutant PI3K (gain of function) and wild-type PI3K. Manipulation of EGFR activity alters HCT116 cell growth and invasion. HCT116 cells also synthesize and retain pericellular HA. Based upon our previous observation that HAS3 mediates cellular growth and invasion, we hypothesized that EGFR and/or PI3K activity would alter expression of HAS3 in HCT116 cells. Materials and Methods: To test this hypothesis, we investigated HAS expression in cell lines derived from HCT116. To investigate the role of EGFR activation, we used HCT116b cells (isoparental clones of HCT116) that do not have constitutively active EGFR and are less tumorigenic. We also assessed HAS expression in HCT116 cells treated with the EGFR inhibitor Tarceva (erlotinib). Finally, HCT116 cells were manipulated by homologous recombination techniques to delete one of the two PI3K alleles, generating a PI3K mutant/- (gain of function; active) and PI3K wild type/-. HAS isozyme expression was first assessed using semi-quantitative RT-PCR; band intensities were normalized to GAPDH. Results were then confirmed using quantitative PCR. Experiments were repeated in triplicate. Data were analyzed with Students’t-test. Results: None of the cell lines expressed HAS1. HAS2 was detected in all four cell lines, but expression did not differ. In contrast, HCT116 cells were found to express high levels of HAS3, and its expression correlated with EGFR and PI3K activity. HCT116b cells expressed less HAS3 than HCT116 cells. PI3K wild-type/- cells expressed less HAS3 than the PI3K mutant/- cells. Treatment with Tarceva also decreased HAS3 expression, confirming that EGFR activity was responsible for upregulated HAS3 expression in HCT116 cells. Conclusions: EGFR activation increases HAS3 expression in HCT116 colon cancer cells, and EGFR inhibition decreases expression. In addition, PI3K activation is as-