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Browsing by Author "Weil, Michael, committee member"

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    Biomarkers of disease progression and chemotherapeutic resistance in canine osteosarcoma
    (Colorado State University. Libraries, 2011) O'Donoghue, Liza E., author; Duval, Dawn L., advisor; Thamm, Douglas H., committee member; Bouma, Gerrit J., committee member; Weil, Michael, committee member
    Osteosarcoma is the most common primary bone malignancy in both humans and dogs. Over 10,000 canine patients develop this highly aggressive cancer annually and many succumb to metastatic disease in less than a year. In recent years, canine osteosarcoma has been increasingly recognized as an excellent model for the disease in humans, especially with regard to the molecular biology of the disease. Thus, research targeted at canine osteosarcoma benefits not only dogs but the field of human oncology as well. Research into the genetic and molecular derangements of osteosarcoma in both species has identified a number of oncogenes and tumor suppressor genes that may contribute to tumorigenesis. Additionally, some mediators of invasion and metastasis have been recognized (e.g. Ezrin, matrix metallopeptidases). Despite this, only a limited number of studies have been performed that examine the molecular genetics of osteosarcoma in the context of patient outcome. Thus, with the aim of identifying new target genes and pathways that contribute to disease progression and chemoresistance in osteosarcoma, we first performed transcriptomic and genomic analyses of primary tumors from dogs that had experienced good or poor outcomes following definitive treatment for osteosarcoma. These broad survey experiments yielded a selection of targets for future investigation. To further focus in on the genes that were most deranged from "normal" expression patterns, we compared gene expression patterns from tumors to those of normal bone. This study provided valuable perspective on genes that were identified in the outcome-based experiments, allowing selection of four promising gene targets to pursue. We next set out to validate in vitro models of canine osteosarcoma so that mechanistic studies could be pursued. Assays to test species and short tandem repeat identity were adapted to cell lines in use in our facility and presumed osteosarcoma cell lines were verified to be bone-derived via PCR testing of a bone-specific marker. Additionally, four anti-human antibodies were validated for use in canine samples. Two genes whose expression progressively altered with increased tumor aggressiveness where chosen for further study: insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) and n-Myc downstream regulated gene 2 (NDRG2). IGF2BP1 has been identified as an oncofetal protein and its mRNA was strongly overexpressed in patients with the worst outcome while it was virtually undetectable in normal bone. We identified one possible mechanism for dysregulation of this gene in OSA and we also discovered that knock down of this gene in a canine osteosarcoma cell line inhibited cell invasion. NDRG2 has been dubbed a tumor suppressor in a number of different tumor types yet had not been previously investigated in osteosarcoma. We found NDRG2 mRNA to be underexpressed in all tumors relative to normal bone; patients with poor outcomes had the lowest expression levels. Multiple isoforms of the gene were found to be expressed in canine samples: these were cloned and transfected into a low-NDRG2-expressing cell line. Exogenous expression of NDRG2 in this in vitro system enhanced sensitivity to doxorubicin, one of the drugs most commonly used to treat osteosarcoma. Additionally, three possible mechanisms of dysregulation of this gene were identified. The studies presented herein progress from fact-finding surveys to in-depth functional examination of two genes that likely contribute to osteosarcoma invasion and chemoresistance. Furthermore, additional genes identified in our survey experiments offer promise for future studies into molecular mechanisms of osteosarcoma metastases and chemotherapeutic resistance. Finally, these studies have laid the groundwork for the development of gene-expression-based prognostic screens for dogs with osteosarcoma.
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    Clinical and molecular characterization of canine small cell B-cell lymphocytosis disorders
    (Colorado State University. Libraries, 2020) Rout, Emily, author; Avery, Anne, advisor; Avery, Paul, committee member; Zabel, Mark, committee member; Weil, Michael, committee member
    To view the abstract, please see the full text of the document.
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    Global analysis reveals differential regulation of mRNA decay in human induced pluripotent stem cells
    (Colorado State University. Libraries, 2013) Neff, Ashley T., author; Wilusz, Jeffrey, advisor; Wilusz, Carol J., advisor; Thamm, Douglas H., committee member; Weil, Michael, committee member
    Induced Pluripotent Stem (iPS) cells are able to proliferate indefinitely while maintaining the capacity for unlimited differentiation and these properties are reflected by global changes in gene expression required for reprogramming of differentiated cells. Although the rate of transcription is an important regulator of steady-state mRNA levels, mRNA decay also plays a significant role in modulating the expression of cell-specific genes. The contribution of regulated mRNA decay towards establishing and maintaining pluripotency is largely unknown. To address this, we sought to determine global mRNA decay rates in iPS cells and the genetically-matched fibroblasts (HFFs) they were derived from. Using a microarray based approach, we determined half-lives for 5,481 mRNAs in both cell lines and identified three classes of mRNAs whose decay is differentially regulated in iPS cells compared to HFFs. We found that replication-dependent histone mRNAs are more abundant and more stable in iPS cells, resulting in increased histone protein abundances. This up-regulation of histone expression may facilitate the unique chromatin dynamics of pluripotent cells. A large set of C2H2 ZNF mRNAs are also stabilized in iPS cells compared to HFFs, possibly through reduced expression of miRNAs that target their coding regions. As many of these mRNAs encode transcriptional repressors, stabilization of these transcripts may support the overall increased expression of C2H2 ZNF transcription factors in early embryogenesis. Finally, we found that mRNAs containing C-rich elements in their 3'UTR are destabilized in iPS cells compared to HFFs and many of these mRNAs encode factors important for development. Interestingly, we also identified the Poly(C)-Binding Protein (PCBP) family as differentially regulated in iPS cells and investigated their possible involvement in regulation of the mRNAs in our dataset identified as destabilized in iPS cells and having C-rich 3'UTR elements. Thus, we identified several interesting classes of mRNAs whose decay is differentially regulated in iPS cells compared to HFFs and our results highlight the importance of post-transcriptional control in stem cell gene expression. Coordinated control of mRNA decay is evident in pluripotency and characterization of the mechanisms involved would further contribute to our limited understanding of pluripotent gene expression and possibly identify additional targets for reprogramming.
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    Modeling human immunodeficiency virus-1 (HIV-1) infection in the male reproductive tract (MRT) using humanized mice
    (Colorado State University. Libraries, 2016) Fisher, Suhrim, author; Akkina, Ramesh, advisor; Aboellail, Tawfik, committee member; Dean, Gregg, committee member; Kendall, Lon, committee member; Weil, Michael, committee member
    Thirty-five million people are currently living with human immunodeficiency virus (HIV) globally. While 9.7 million infected people are receiving antiretroviral therapy, 2.3 million people are newly infected every year. Transmission via semen is one of the most prevalent methods of HIV-1 transmission, accounting for up to 80% of new infections every year. However, the source of infected leukocytes and the method of virus replication in semen and/or in the male reproductive tract (MRT) is not well described. It has been shown that infected germinal cells such as spermatogonia and spermatocytes as well as infected leukocytes are present in the MRT of HIV infected human patients and simian immunodeficiency virus (SIV) infected macaque models. Studies have also shown that the seminal viral strain differs from the serum viral strains in HIV infected human patients. Furthermore, HIV-1 continues to persist in the semen and in the MRT of infected male patients on highly active antiretroviral therapy (HAART) despite undetectable serum viral load. To investigate the spread and replication of HIV-1 in the MRT in human patients, development of an animal model is essential. Use of humanized mice allows researchers to study the transmission, pathogenesis and drug interactions of HIV-1 in the human immune system in vivo. The human-hematopoietic stem cell (hu-HSC) mouse model created by transplantation of HSC into Rag2−/−γc−/− mice, has shown excellent susceptibility to HIV-1 and ability to sustain high viremia for months. However, so far no studies have been conducted to demonstrate whether or not HIV-1 infiltrates and replicates in the semen or MRT of infected humanized mice. The presence of HIV-1 in the semen or MRT of infected humanized mice could play a vital role in using humanized mice for describing the pathogenesis mechanism of the virus in semen and drug development in reducing seminal HIV-1 viral load in human patients. The aim of this study is to model HIV-1 infection in the MRT using humanized mice to 1) evaluate the severity of disease using histologic scoring of MRT and morphometric analysis of the testis and epididymis in viremic and negative control mice, 2) characterize leukocytes in the testis and epididymis of viremic and negative control mice via immunohistochemistry, 3) assess the presence of HIV-1 viral RNA in the MRT of viremic mice via in situ hybridization (HIV-1 mRNA), and 4) assess seminal viral loads from vaginal plugs of viremic mice via qRT-PCR. We showed that both viremic (16/25; 64%) and negative control (9/28; 32%) mice developed histopathologic lesions in their testis and epididymis parallel to the lesions found in the testes and epididymis of chronically infected HIV-1 and AIDS patients. When broken down by two age groups, 5-7 month old and 8-12 month old, the viremic mice from both age groups showed significantly higher frequency of testicular oligospermia/azoospermia (p<0.05) compared to the age-matched negative control groups. Five to seven month old viremic mouse group showed significantly higher frequency of testicular germinal cell degeneration (p<0.05) compared to the age-matched negative control group. The histopathological lesion scores were significantly higher in both 5-7 month old and 8-12 month old viremic mouse group compared to that of age-matched negative control mouse groups (p<0.05), suggesting the lesions in viremic mice are related to HIV-1 infection. Morphometric analysis of the testicular and epididymal epithelium however, did not show any statistical differences on the levels of germinal cell degeneration between the viremic (n=8) and negative control (n=4) group. We were able to identify the presence of CD4 positive cells in viremic (4/22; 22%) and negative control (7/17; 41%) mice in the testicular and epididymal interstitium. The majority of the CD4 cells were associated with histopathologic lesions within primary reproductive organs. RNA in situ hybridization showed successful infiltration of HIV-1 infected cells in the MRT of viremic mice (9/11; 82%), most often found in penis or prepuce (6/11; 55%), and less often in seminal vesicles (4/11; 9%), testes (3/11; 27%), epididymides (1/11; 9%) and prostate glands (1/11; 9%), suggesting that HIV-1 indeed can infect and infiltrate the MRT via CD4 positive cells. Vaginal plugs failed to show detectable HIV-1 viral loads using qRT-PCR. Our study offers first glance at HIV-1 infection in the MRT of viremic humanized mice and provides further evidence that leukocytes play an important role in HIV-1 infection in the MRT.
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    Utilization of a canine cancer cell line (FACC) panel in comparative and translational studies of gene expression and drug sensitivity
    (Colorado State University. Libraries, 2015) Fowles, Jared S., author; Gustafson, Daniel, advisor; Duval, Dawn, committee member; Hess, Ann, committee member; Thamm, Douglas, committee member; Weil, Michael, committee member
    Canine cancer is the leading cause of death in adult dogs. The use of the canine cancer model in translational research is growing in popularity due to the many biologic and genetic similarities it shares with human cancers. Cancer cell tissue culture has long been an established tool for expanding our understanding of cancer processes and for development of novel cancer treatments. With the high rate of genomic advancements in cancer research over the last decade human cancer cell line panels that combine pharmacologic and genomic information have proven very helpful in elucidating the complex relationships between gene expression and drug response in cancer. We have assembled a panel of canine cancer cell lines at the Flint Animal Cancer Center (FACC) at Colorado State University to be utilized in a similar fashion as a tool to advance canine cancer research. The purpose of these studies is to describe the characteristics of the FACC panel with the available genomic and drug sensitivity data we have generated, and to show its utility in comparative and translational oncology by focusing specifically on canine melanoma and osteosarcoma. We were able to confirm our panel of cell lines as being of canine origin and determined their genetic fingerprint through PCR and microsatellite analyses, creating a point of reference for validation in future studies and collaborations. Gene expression microarray analysis allowed for further molecular characterization of the panel, showing that similar tumor types tended to cluster together based on general as well as cancer specific gene expression patterns. In vitro studies that measure phenotypic differences in the panel can be coupled with genomic data, resulting in the identification of potential gene targets worthy of further exploration. We also showed that human and canine cancer cells are similarly sensitive to common chemotherapy. Next we utilized the FACC panel in a comparative analysis to determine if signaling pathways important in human melanoma were also activated and sensitive to targeted inhibition in canine melanoma. We were able to show that despite apparent differences in the mechanism of pathway activation, human and canine melanoma tumors and cell lines shared constitutive signaling of the MAPK and PI3K/AKT pathways, and responded similarly to targeted inhibition. These data suggest that studies involving pathway-targeted inhibition in either canine or human melanoma could potentially be directly translatable to each other. Evidence of genetic similarities between human and canine cancers led us to ask whether or not non-pathway focused gene expression models for predicting drug sensitivity could be developed in an interspecies manner. We were able to show that models built on canine datasets using human derived gene signatures successfully predicted response to chemotherapy in canine osteosarcoma patients. When compared to a large historical cohort, dogs that received the treatment our models predicted them to be sensitive to lived significantly longer disease-free. Taken together, these studies show that human and canine cancers share strong molecular similarities that can be used advantageously to develop better treatment strategies in both species.