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Browsing by Author "Quackenbush, Sandra L., advisor"

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    Characterization of walleye dermal sarcoma virus Orf B during tumor development
    (Colorado State University. Libraries, 2008) Daniels, Candelaria Christina, author; Quackenbush, Sandra L., advisor
    Walleye dermal sarcoma virus is a complex retrovirus associated with walleye dermal sarcomas (WDS). These sarcomas develop and regress on a seasonal basis, providing a unique model to study mechanisms of tumor development and regression in vertebrates. WDS is experimentally transmissible to walleye with cell-free, regressing tumor homogenates. During the fall, low levels of spliced accessory gene transcripts, A and B, are present in developing tumors suggesting that their encoded proteins, rv-cyclin and Orf B, may play a role in oncogenesis. Infectious virus and high levels of full-length viral RNA and spliced accessory and env transcripts are expressed during tumor regression, the following spring. The three accessory proteins Orf A (rv-cyclin), Orf B, and Orf C function in tumor development and regression. In explanted tumor and mammalian cells stably expressing the 35kDa Orf B protein, Orf B is localized at the cell periphery in structures similar to focal adhesions and along actin stress fibers. Results from these studies demonstrate Orf B interacts directly or in a complex with several cellular proteins important in signal transduction pathways: receptor for activated C kinase (RACK1), protein kinase C alpha (PKCα), Src, phosphatidylinositol-3-kinase (PI3K), and protein phosphatase 2A (PP2A). The cellular proteins BAD, 90kDa ribosomal S6 kinase (p90RSK), PKCα, and protein kinase B (AKT), which are important in controlling apoptosis and/or proliferation, are activated in Orf B-expressing cells. Orf B protects cells from staurosporine-induced apoptosis and induces cell proliferation of Orf B-expressing cells under serum-deprived conditions suggesting a mechanism of action for tumor development. Expression of Orf B induces transformation of NIH3T3 cells in vitro and a PI3K and mTOR inhibitor prevented transformation, providing the first evidence that Orf B induces a transformed phenotype. The regulation of cell signaling pathways is one way in which viruses induce oncogenesis. Orf B ensures the establishment of dermal sarcoma by activating signal transduction pathways that control cell survival and proliferation such as PKC and Akt.
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    Sinus tumors of Rocky Mountain bighorn sheep: investigation of an infectious etiology
    (Colorado State University. Libraries, 2013) Fox, Karen A., author; Quackenbush, Sandra L., advisor; Miller, Michael W., committee member; Wootton, Sarah K., committee member; Huyvaert, Kathryn P., committee member
    Rocky Mountain bighorn sheep are an icon in Colorado. As our state animal, bighorn sheep are a well-recognized symbol of the wildlife, wildlands, and wilderness-centric people that Colorado is famous for. Efforts to manage and conserve this species are a priority in Colorado and throughout western North America. As part of those efforts a great deal of research has been conducted to understand bighorn sheep respiratory disease, the leading infectious cause of death in these animals. In the process of investigating respiratory disease in bighorn sheep in Colorado, we discovered a surprisingly high occurrence of sinus tumors within the upper respiratory tracts of many animals. This disease had not been described previously and became the focus of work for this dissertation. Here, I have compiled our findings regarding the characterization of bighorn sheep sinus tumors and the results of our efforts to identify an infectious etiology for this disease. Through the examination of naturally-occurring cases, we identified characteristic histologic and gross features of bighorn sheep sinus tumors to define this disease. We also analyzed factors associated with sinus tumors at a population level. The results of this study suggest that bighorn sheep sinus tumors are an infectious disease, maintained within specific geographic areas corresponding to distinct populations of animals. Our results also suggest a role for bighorn sheep sinus tumors in predisposing animals to secondary infections by bacterial agents that can cause pneumonia. To specifically test the hypothesis that bighorn sheep sinus tumors are a transmissible disease, we experimentally inoculated bighorn sheep and domestic sheep lambs with a cell-free filtrate derived from a naturally-occurring bighorn sheep sinus tumor and its associated exudates. Within 18 months post-inoculation we demonstrated transmission of the disease to both bighorn sheep and domestic sheep species, supporting our hypothesis that bighorn sheep sinus tumors represent an infectious process. This experiment also provided an opportunity to examine tumors early in development, further characterize the cells comprising the tumors, and suggest mechanisms for pathogenesis. With evidence that bighorn sheep sinus tumors are caused by an infectious agent, we also attempted to identify a specific etiology for this disease. We primarily used PCR methods with degenerate PCR primers to evaluate samples from bighorn sheep sinus lining tissues for the presence of herpesviruses and retroviruses, which are well-known causes of infectious tumors. We successfully identified the presence of herpesviral and (likely endogenous) retroviral sequences in our samples, but we were unable to find an association between these viruses and the occurrence of sinus tumors. Based on similarities between bighorn sheep sinus tumors and oncogenic retroviral diseases of domestic sheep and goats, we specifically screened our samples for the presence of Jaagsiekte sheep retrovirus (JSRV), and enzootic nasal tumor viruses (ENTV-1 and ENTV-2). We successfully identified ENTV-2-specific sequences from some of our samples, but an association between this virus and bighorn sheep sinus tumors was not clear. We found an association between ENTV-2 and early tumor cases, but not well-defined tumors. While our PCR data alone did not definitively identify ENTV-2 as the cause of bighorn sheep sinus tumors, our histologic, histochemical, and immunohistochemical results have helped us to develop a hypothesis for the pathogenesis of bighorn sheep sinus tumors, and provided additional support for the hypothesis that this disease is caused by ENTV-2. Our working hypothesis for the pathogenesis of bighorn sheep sinus tumors is that epithelial cells of the sinus lining are infected by ENTV-2, but that uninfected periosteal pluripotent cells are stimulated to replicate, resulting in predominantly stromal tumors. This hypothesis is based on histologic observations, histochemical stains used to differentiate cell types, and IHC results specifically identifying the presence of ENTV antigen within surface epithelial cells of experimentally-induced tumors, but not within the predominating stromal cells of the tumors. These results help to explain why detection of the virus is uncommon in well-developed stromal tumors, but more easily detected in early tumor cases with less stromal proliferation. Additional research will help to further elucidate the pathogenesis of bighorn sheep sinus tumors, and the potential role that tumors may play in predisposing bighorn sheep to fatal respiratory disease. The definitive identification of an etiologic agent for bighorn sheep sinus tumors, and the development of an antemortem diagnostic assay will greatly enhance efforts to understand and manage this disease.
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    The role of retroviral cyclin in the development of walleye dermal sarcoma
    (Colorado State University. Libraries, 2015) Birkenheuer, Claire, author; Rovnak, Joel, advisor; Quackenbush, Sandra L., advisor; Nyborg, Jennifer, committee member; VandeWoude, Susan, committee member; Wilusz, Jeff, committee member
    The retroviral cyclin (RV-cyclin) is an accessory protein encoded by walleye dermal sarcoma virus (WDSV). This virus causes the formation of walleye dermal sarcoma, and requires the tumor tissue to replicate. RV-cyclin is one of only two proteins expressed by the virus during tumor development before production of the WDSV virion, and the mechanism by which RV-cyclin causes tumor formation was explored and is presented here. RV-cyclin interacts with TAF9 and CDK8, which are cellular proteins that regulate RNA Pol II transcription. RV-cyclin's influence on transcription was explored by analyzing transcript levels of CCND1, CDKN2D, FOS, EGR1, and JUN. All of these genes are important oncogenes in human cancers, and were hypothesized to contribute to development of walleye dermal sarcoma (WDS). Quantitative reverse transcription PCR analysis of these genes in HeLa and HCT116 cell lines expressing wt or mutant RV-cyclin, or over-expressing cyclin C was carried out. These analyses showed that wt RV-cyclin enhances expression of all genes tested and that the interactions with TAF9 and with CDK8 contribute to this enhancement. Western blot analysis of phosphorylated Elk1 and SRF, two transcription factors that initiate RNA Pol II transcription, revealed that RV-cyclin's mechanism of gene activation is downstream of transcription factor phosphorylation. Messenger RNA (mRNA) decay assays demonstrated that RV-cyclin does not alter the rate of mRNA decay, framing RV-cyclin's mechanism of activation to the level of RNA Pol II transcription. Nuclear run-on and chromatin immunoprecipitation analysis of EGR1 with an RNA Pol II antibody support a role for RV-cyclin in transcription elongation and re-initiation. ChIP analysis of the mutant RV-cyclin cells and cells over-expressing cyclin C demonstrated that RV-cyclin requires both a functional CDK8 and TAF9 interaction for efficient activation and extension of the EGR1 serum response. CDK8 ChIPs demonstrated that the RV-cyclin enhances CDK8 occupancy at the EGR1 gene locus, and HA-ChIPs show that CDK8 and RV-cyclin occupancy correlate with each other on the locus. At this point, it was hypothesized that RV-cyclin functions to enhance CDK8 kinase activity in addition to bringing more CDK8 to select oncogenic loci in vivo. For this reason kinase assays using recombinant, baculovirus-produced CDK8/RV-cyclin or CDK8/cyclin C constructs were attempted. CDK8 bound to RV-cyclin was able to autophosphorylate to similar levels as CDK8 bound to cyclin C. Other CDK8 substrates remain to be tested. RV-cyclin's function in transcription requires activation of the MAPK pathway, suggesting RV-cyclin needs an outside signal like serum stimulation for efficient WDS formation. This signal likely comes from the presence of the Orf b protein, the other WDSV accessory protein expressed during tumor development. The protein Orf b causes phosphorylation and activation of the p90RSK complex. This event likely causes aberrant phosphorylation of SRF and Elk-1 giving RV-cyclin the activated transcription factors required for its function on specific proto-oncogenes. Taken together, the Orf b protein and RV-cyclin illustrate a previously un-described, trans-acting mechanism of retroviral-induced oncogenesis.