137 research outputs found
Development of pituitary tumor-transforming gene (PTTG) transgenic mice for the treatment of ovarian cancer.
This dissertation is a hypothesis-driven research oriented study to determine the role of the pituitary-tumor transforming gene (PTTG) in ovarian cancer, specifically if it is involved in neoplastic transformation leading to tumorigenesis through oncogene activation and the involvement of tumor-suppressor gene, p53. Furthermore, generation of a useful ovarian cancer mouse model provides a platform technology to screen for ovarian cancer diagnosis and treatment options. This dissertation is divided into four chapters covering the etiology of ovarian cancer and a novel treatment strategy for ovarian cancer. The first chapter reviews the related literature encompassing the etiology of ovarian cancer, mouse models of ovarian cancer, the biological function of PTTG, the role of PTTG in cancer and diabetes, and mouse models using PTTG as a transgene. The second chapter studies the role of PTTG in tumorigenesis in vivo through the generation ofa PTTG transgenic (TgPTTG) mouse model observed at various ages, ranging from 4 to 10 months. The third chapter is a preliminary study investigating the signaling mechanisms affected by chemotherapy agent doxorubicin in combination with withaferin A in vitro and in vivo for the treatment of ovarian cancer. The fourth chapter is a discussion of the utility of ovarian cancer mouse models and the consequences of the lack of a working model
Studies in Cellular Immunology
Antisera were prepared against pigeon thymus and bursa lymphocytes and against saline extracts of thymus and bursa. All unabsorbed sera were screened for differential reactivity against thymus and bursa-derived tissue by several techniques. Selected sera were absorbed with thymus and bursa tissue to demonstrate specificity. One serum with specificity against thymus extracts was identified by immunodiffusion. The thymus specificity was absent from extracts of pigeon bursa, brain, liver and breast muscle, but present in spleen extracts. This thymus specificity does not appear analogous to lymphocyte specificities identified in other species. Shared tissue specificities and a possible quantitative antigenic difference among the tissue extracts were also demonstrated by immunodiffusion and absorption analysis. In lymphocytotoxicity tests, fresh rabbit normal serum is highly toxic for pigeon thymus and bursa cells. This toxicity, in general, resembles the natural antibody present in rabbit and guinea pig sera against heterologous thymus cells. A rabbit anti-pigeon gamma globulin serum was rendered specific for bursa cells by absorption with thymus cells. Some standard antilymphocyte sera were shown to contain an antibody fraction specific for thymus cells. Some or all of these reagents may be useful for distinguishing cooperating cell populations in a variety of immune responses.
An antigen was demonstrated on red cells from all pigeon squabs less than four days old. The antigen appears not to be secondarily adsorbed to the red cells from the fluids of the egg or the embryo. In vitro, masking of the antigen by components of adult serum does not occur under the conditions tested. Although the squab antigen behaves similarly to a known fetal red cell antigen in doves, it is probably qualitatively different from that antigen and from the known chick red cell antigen. The squab antigen is not detectable on lymphocytes from the bursa or the thymus.
Virgin female CBA/J mice were obtained after a variety of treatments and observed for primary tumors until either tumor onset or death. Included were mice which were: (1) immunosuppressed as adults by injection of anti-thymocyte serum (ATS); (2) injected with normal rabbit serum; (3) immunized with an irrelevant antigen or (4) untreated. Data were collected on tumor histology, incidence and time of onset for all groups. No tumors appeared during the period of ATS-immunosuppression or for several months following treatment. The most frequently observed subsequent tumor was the typical mammary tumor. Although the first tumors appeared in ATS-treated mice, the mean age at tumor onset was not significantly affected by ATS-immunosuppression. No unusual tumors and no lymphomas were observed. Tumor incidences among groups of mice purchased at different times were different, but unrelated to ATS-immunosuppression. The failure of ATS-immunosuppression to affect growth is consistent with the fact that cellular immunity to mammary tumors is often specifically compromised.</p
Expression of Activated PIK3CA in Ovarian Surface Epithelium Results in Hyperplasia but Not Tumor Formation
activation is one of the early genetic events in ovarian cancer. However, its role in malignant transformation of ovarian surface epithelium (OSE) is largely unclear..
Tumour Antigens and Herpes Simplex Virus Oncogenesis
Herpes simplex virus (HSV) has been implicated in the aetiology of human cervical cancer, but despite intensive investigation the oncogenic properties of HSV are still not well understood. Studies reported here, explore one possible molecular mechanism by which this virus may cause oncogenic transformation; namely, the induction of cellular polypeptides which play a role in oncogenesis. A group of cellular polypeptides specific to cells showing the immortalized phenotype were previously detected in a range of cell lines, including the Bn5T cell line which is derived from rat embryo fibroblasts transformed by a fragment of the HSV type 2 (HSV-2) genome. These polypeptides which will be referred to as Bn5T:TBS polypeptides, are not detectable in control rat embryo fibroblasts. Bn5T:TBS polypeptides are immunoprecipitated by tumour bearing serum (TBS) and studies presented in this thesis confirm that they are also immunoprecipitated by the monoclonal antibody TG7A. The TG7A monoclonal was raised against affinity purified DNA binding proteins from HSV-2 infected cells and recognizes cellular polypeptides induced on infection by the virus. This suggests that Bn5T:TBS polypeptides are related to polypeptides induced in HSV-2 infection. Another feature of Bn5T:TBS polypeptides is that they all show oncofoetal expression, suggesting that they have a physiological role in cell proliferation and differentiation. Members of the Bn5T:TBS set have always been found to be either co-expressed or not expressed at all, in a range of different cell types and culture conditions. This suggests that these polypeptides may have a common precursor or be induced by the same factor(s) and it also suggests that they may have a common function. Three of the six members of the Bn5T:TBS set give similar peptide maps, suggesting that they possess similar or shared domains. In addition, pulse labelling and pulse-chase labelling experiments suggest that at least two members of the set result from post-translational processing events. Immunological and peptide mapping studies indicate that a Bn5T:TBS polypeptide of 97kDa molecular weight, is related to the 90kDa heat shock protein (HSP90) and that it may share a domain with an HSV-2-induced form of HSP90. The TG7A monoclonal recognizes a highly conserved epitope present on a bacterial heat-shock protein, an HSV-2-induced 90kDa polypeptide (also recognized by a monoclonal against HSP90) and a 97kDa transformed cell polypeptide belonging to the Bn5T:TBS set. These findings suggest that an HSV-2-induced form of the HSP90 may play a role in oncogenic transformation by the virus. The synthesis of Bn5T:TBS polypeptides has not been detected in the rabbit reticulocyte lysate in vitro translation system and possible reasons for this have been investigated. Further, data from an amino acid sequencing study suggests that the 97kDa polypeptide in the Bn5T:TBS set has a domain with homology to one form of bovine casein; likely explanations are discussed in detail
VEGFR3 and Notch signaling in angiogenesis
Blood and lymphatic vessels form extensive networks throughout the body, which function in order to deliver oxygen and nutrients to the tissues, to remove extravasated fluid and to absorb dietary lipids. The formation of new blood and lymphatic vessels (termed angiogenesis and lymphangiogenesis) is critical during embryonic development and in the adult, and is regulated by multiple signaling pathways. Vascular Endothelial Growth Factors (VEGFs) and their receptors (VEGFRs), as well as the Notch signaling system, are key governors of blood and lymphatic endothelial cell fate, and regulate angiogenesis and lymphangiogenesis in health and disease. Despite the numerous recent advances in the field of vascular biology, many steps in the complex processes of angiogenesis and lymphangiogenesis remain unclear. In this study we investigated the role of VEGFR3 signaling in blood endothelial cells, tip cell specification, as well as the interplay of the receptor with the VEGFR2 and the Notch signaling pathways during angiogenesis.
VEGFR3 is a tyrosine kinase receptor that is mainly expressed in lymphatic endothelial cells in the adult. We observed VEGFR3 expression in sprouts that guide the blood vascular endothelium in angiogenic conditions. VEGFR3 blockade with a monoclonal antibody displayed synergistic properties with simultaneous VEGFR2 targeting in reducing angiogenesis and inhibiting tumor growth. Furthermore we found that Notch signaling suppresses VEGFR3 expression in endothelial cells, identifying VEGFR3 as a novel tip cell marker, which is normally repressed by Notch activation.
In the next step we employed a combination of genetic and in vitro models to show that loss of VEGFR3 results in a hypervascular phenotype, accompanied by loss of Notch signaling. VEGFR3 could be stimulated by VEGFC and activate Notch in blood endothelial cells. Our results point towards a mechanism where VEGFC produced by macrophages at the vascular front acts via VEGFR3 to activate Notch and turn tip into stalk cells; thus promoting the formation of stable vascular loops. Furthermore we identified the transcription factor FOXC2 as the downstream target of the VEGFC/VEGFR3/Notch signaling cascade. These data reinforce the idea that VEGFR3 has two distinct signaling modalities, one ligand-dependent and one ligand-independent, and that different perturbations in VEGFR3 expression and function result in diverse vascular phenotypes.
Subsequently we investigated the interplay of VEGFR2 and VEGFR3 in postnatal angiogenesis and lymphangiogenesis, using a genetic approach of conditional mutagenesis. Various combinations of genetic ablation of VEGFRs and pharmacological inhibition of Notch showed that VEGFR2 is irreplaceable during sprouting angiogenesis, also in endothelial cells with low Notch signaling, and that it acts upstream of VEGFR3 expression in angiogenic settings. On the other hand VEGFR3 suppressed VEGFR2 expression in a negative feedback loop. Finally we employed for the first time lymphatic endothelial specific deletion of VEGFRs in postnatal conditions and found no significant role for VEGFR2 in lymphatic vessel growth and remodeling, while VEGFR3 signaling was indispensable.
Our results unravel previously unknown roles for VEGFR3 in sprouting angiogenesis and provide new insight into the signaling cross-talk of the receptor with other important regulators of blood vessel development. Increasing our understanding of the molecular mechanisms underlying this process is crucial in order to comprehend the pathophysiology of angiogenesis-related diseases, characterized by excessive or insufficient blood vessel growth, and promote the emergence of novel angiogenic therapies.Not availabl
INCREASED BMP SIGNALING DECREASES EPITHELIAL OVARIAN CANCER TUMOUR GROWTH ON THE CHICK CAM BY INHIBITING ANGIOGENESIS
Current limitations regarding the treatment of metastatic epithelial ovarian cancer (EOC) are attributed to our poor overall understanding of its progression due to the limited number of appropriate model systems. To this end, I have characterized EOC tumour growth and angiogenesis using the innovative chick chorioallantoic membrane (CAM) model system. Bone morphogenetic protein (BMP) signaling has been implicated in multiple processes of EOC metastasis, although its role in tumour angiogenesis has never been assessed. I found an inverse correlation between the level of BMP signaling in mouse EOC cells and their abilities to induce angiogenesis both in vitro using HUVEC tube formation assays and in vivo using a chick CAM angiogenesis collagen onplant assay. These results support the further implementation of the chick CAM as an important tool to study EOC metastasis. Additionally, the putative anti-angiogenic role of BMP4 signaling from my work highlights potential future implications for this pathway in prognostics and treatment of EOC
Development and Characterization of a Recombinant Vesicular Stomatitis Virus (rVSV) for the Treatment of Glioblastoma
Background: Over the past 30 years, little has changed in the treatment modalities and prognosis of patients suffering from Glioblastoma multiforme (GBM), the most common and by far the most devastating adult primary malignant brain tumor. Conventional therapies provide only a marginal increase in survival of GBM patients, post-diagnosis. Therefore, more novel means of treating GBM are needed to increase long-term survival and quality of life for those affected. Replication competent oncolytic viruses (OVs) have recently emerged as a possible option for treatment of high-grade gliomas. Particularly, recombinant vesicular stomatitis virus (rVSV), an enveloped, negative strand RNA virus, has shown promising results in preclinical studies. Tumor selectivity of VSV is thought to be associated with tumor specific defects in the interferon (IFN) pathway. However, largely due to insufficient attention on the role the immune system plays in efficacy of treatment, potential OVs have been obstructed from moving through the clinical trial pipeline past Phase I/II studies. rNCP12.1 is a novel recombinant VSV vector possessing specific mutations in the matrix protein. Thes mutations have been shown to promote viral attenuation in normal cells while maintaining cytotoxicity in a number of tumor cell lines. We aim to characterize and further develop this novel agent for the treatment of GBM. Methods: In order to determine differences between rNCP12.1 and wtVSV and to determine specificity of rNCP12.1 for tumor over normal cells, cell rounding assays, one-step growth curves, and cytotoxicity assays were performed in normal glial and tumor glial cell lines. To understand the basis of this selectivity and whether it correlated with the antiviral responses of IFN, expression levels of IFN and IFN stimulated genes (ISGs) were quantified, production of active IFN was measured, and the ability of cells to inhibit viral infection in response to exogenous IFN was determined. In vivo experiments were designed and carried out to test for oncolytic activity of rNCP12.1 in immunocompetent animal models of intracranial glioma. A single injection of rNCP12.1 was administered into previously implanted F98-GFP tumors. Tumor load and parameters of morbidity were assessed at 15 days following tumor implantation and long term at the time of euthanasia. Viral induced immune responses were assessed by the IFN bioassay and detection of circulating anti-VSV antibodies were achieved by Western blot analysis and a neutralizing antibody assay. Experimental methods of virus administration for treatment of glioma were further tested including multiple injections, injections using different VSV serotypes, continuous infusion of virus using implantable osmotic pumps, and pre-infected autologous carrier cells. These methods were designed to enhance anti-tumor effect by managing the negative effects of the tumor microenvironment and of a functional immune system on viral therapy. Results and Conclusion: rNCP12.1 was shown to be an attenuated strain of VSV that has clear differences in its growth and induction of the IFN response pathway in normal cells. It has a preference for growth in tumor cells as determined by viral titers, cell rounding, and cell viability post infection. This preference varied based on cellular expression of a particular IFN phenotype. The importance of this molecular versus histological cell profile was evident even in the performance of rNCP12.1 on human glioma cell lines that differ in their expression of IFN. In vivo evaluation of rNCP12.1 against a highly IFN resistant rat glioma cell line, F98, demonstrated its ability to decrease tumor size while eliciting a peripheral response to virus that protects normal tissue but also shortens its therapeutic window and the ability to sustain reduction of tumor over time. Several experimental methods in delivery of virus proved to be beneficial, including administering an additional dose of virus using a different serotype to bypass antiviral neutralizing responses and by shielding virus from the immune system through the use of tumor carrier cells. As an additional benefit, the latter was shown to have a unique pattern in eliciting tumor specific antibodies that was different from those increased by therapy with virus alone. This method also increased recovery of virus from brain tissue even after 20 days post treatment.
Our data supports the capability of rVSV vectors as treatment for GBM. Specifically rNCP12.1 therapy increased survival while decreasing tumor load, depending on method of administration. When given alone, virus is ultimately immunogenic and prompts anti-viral as well as anti-tumor immune responses. However, when shielded from the immune system, antiviral responses are minimal while anti-tumor responses are sustained. To this end, therapy cannot be fully addressed without addressing the effects the immune system has on therapy and on the host. Future studies, should include not only evaluation of tumor load, morbidity, and side effects of viral therapy but also immune responses especially those that are likely to enhance therapy past the acute stages of disease
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