In this project research is conducted to characterize and develop new animal models of human disease and to develop the means to better characterize a models relevance for a human condition. The project aims include the evaluation of research technologies and markers. Progress was made in developing cancer diagnostics and in research resources useful in developing and characterizing new models of human cancer. Methods and reagents were developed and technologies were applied for use in validating animal models of human cancer. This research project included developing capabilities in molecular diagnostics for cancer models, developing methods for automated morphmetric image analysis of cancer specimens for quantitative pathology, investigating the role of S100 in ovarian cancer and cell cycle regulation in nondividing cells undergoing Ras oncogene activation. Research advances in ovarian cancer biomarker development are yielding discovery of serum proteins that have the potential to serve as tumor biomarkers. Applying mass spectrometry-based proteomics, we sought to elucidate an unanswered biomarker research question regarding ability to determine tumor burden detectable by an ovarian cancer biomarker protein emanating directly from the tumor cells. Since aggressive serous epithelial ovarian cancers account for most mortality, a xenograft model using human SKOV-3 serous ovarian cancer cells was established to model progression to disseminated carcinomatosis. Using a method for low molecular weight protein enrichment, followed by liquid chromatography and mass spectrometry analysis, a human-specific peptide sequence of an S100 was identified in sera from mice with advanced-stage experimental ovarian carcinoma. This S100 expression was documented in cancer xenografts as well as from ovarian cancer patient tissues. Longitudinal study revealed that serum S100 concentration is directly related to tumor burden predictions from an inverse regression calibration analysis of data obtained from a detergent-supplemented antigen capture immunoassay and whole-animal bioluminescent optical imaging. The result from the animal model was confirmed in human clinical material as this S100 family member was found to be significantly elevated in the sera from women with advanced stage ovarian cancer compared to those with early stage disease. This S100 is expressed in ovarian and other cancer tissues, but has not been documented previously in ovarian cancer disease sera. This S100 family member is found in serum in concentrations that correlate with experimental tumor burden and with clinical disease stage. The data signify that S100 may prove useful in detecting and/or monitoring ovarian cancer, when used in concert with other biomarkers. In additional research efforts, MPU validated a mouse model of oncogenic Ras expression in adult cardiac myocytes using heart tissue sections to work out protocols for processing limited tissue sizes for mass spectrometry proteomic studies. In contrast to the role oncogenic Ras plays in typical cytoproliferative responses, Ras signaling in the myocardium in response to a variety of ionotropic and chronotropic stimuli, may lead to myocardial hypertrophy. Cardiac Ras signaling can also be manifested as pathogenic myocardial hypertrophy and subsequent heart failure. A two-transgene tetracycline (tet-off) system was used to regulate expression of Ras (H-Ras-G12V) in M/R mice to examine whether Ras-induced pathogenic myocardial hypertrophy could resolve after removal of Ras signaling. Ras activation at weaning for 2-weeks caused hypertrophy, while activation for 4-8 weeks led to cardiomyopathy and heart failure. Discontinuing Ras transgene expression after early cardiomyopathy onset led to improved survival and cardiomyopathy lesion scores, with reduced heart/body weight ratios, compared to mice with continuous Ras activation, demonstrating the reversibility of early pathogenic hypertrophy. This system is anticipated to yield new information on important protein signaling during healing of the cardiac myocyte with pre-malignant injury and hypertrophy secondary to Ras oncogene. The significant materials, equipment or methods in this project include use of recombinant DNA technology, in vitro cell culture, DNA sequence analysis, immunodiagnostics, molecular imaging, morphometrics, computer assisted image analysis, optical imaging, molecular pathology, and veterinary medical diagnosis.
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