Cancer remains to be one of the leading causes of death worldwide. Current treatment methods are not effective, and so newer therapies are needed, particularly in applied virology, where aspects of viral growth and replication are targeted to tumour lysis (termed viral oncolysis). Reoviruses are naturally occurring oncolytic viruses. Since most cancers are epithelial in origin, but also include fibroblastic properties due to the epithelial-mesenchymal transitions within tumours, my investigations mainly focused on the biology of Reoviruses in both fibroblast and epithelial cells. We studied the genetic basis for Protein Kinase R sensitivity and its role in lung tropism, as well as the study of tumour oncolysis by Reoviruses and their reassortants. We also investigated the growth and protein production of Reovirus and four oncolytic reassortants and their interferon susceptibilities in L929 fibroblast cells, CT26 mouse colon tumour cells, interferon-deficient Vero cells and interferon producing CV-1 cells. We found that PKR protected the bronchiolar epithelium from infection from Reovirus. In PKR knockout mice lungs, the ability to infect the bronchial epithelium was attributed to the T3D σ1s non-structural protein, encoded by the S1 gene segment. T1L infection of PKR+/+ Balb-c mice enhanced disease, whereas T3D growth and replication was limited by PKR. Studies in viral oncolysis showed that treatments of CT26 tumour-bearing mice with high viral doses of Reovirus or oncolytic reassortants resulted in death from enhanced virus toxicity. However, treatments at lower and multiple doses significantly increased survival rates of mice. Growth of parental Reoviruses in mouse colon tumour CT26 cells showed that there is an enhanced effect of the virus infection leading to the 18 hour time point, where the virus induced the breakdown of host factors. There was also a major increase in T3D viral proteins at this time point, where we also observed the decrease in host proteins such as actin, eIF2α, interferon regulatory factors and PKR. We proposed that the Reovirus μ2 protein may be involved in this process, which induces the degradation of host inhibitors to result in enhanced viral gene and protein expression. Our results also showed that parental and oncolytic reassortant viruses behaved differently with respect to growth and IFN response in different cell lines. We demonstrated a complicated interplay between Reovirus gene products, resulting in a unique interaction involving other viral and cell proteins to achieve a certain trait, in the context of the cell type.

Cancer remains to be one of the leading causes of death worldwide. Current treatment methods are not effective, and so newer therapies are needed, particularly in applied virology, where aspects of viral growth and replication are targeted to tumour lysis (termed viral oncolysis). Reoviruses are naturally occurring oncolytic viruses. Since most cancers are epithelial in origin, but also include fibroblastic properties due to the epithelial-mesenchymal transitions within tumours, my investigations mainly focused on the biology of Reoviruses in both fibroblast and epithelial cells. We studied the genetic basis for Protein Kinase R sensitivity and its role in lung tropism, as well as the study of tumour oncolysis by Reoviruses and their reassortants. We also investigated the growth and protein production of Reovirus and four oncolytic reassortants and their interferon susceptibilities in L929 fibroblast cells, CT26 mouse colon tumour cells, interferon-deficient Vero cells and interferon producing CV-1 cells. We found that PKR protected the bronchiolar epithelium from infection from Reovirus. In PKR knockout mice lungs, the ability to infect the bronchial epithelium was attributed to the T3D σ1s non-structural protein, encoded by the S1 gene segment. T1L infection of PKR+/+ Balb-c mice enhanced disease, whereas T3D growth and replication was limited by PKR. Studies in viral oncolysis showed that treatments of CT26 tumour-bearing mice with high viral doses of Reovirus or oncolytic reassortants resulted in death from enhanced virus toxicity. However, treatments at lower and multiple doses significantly increased survival rates of mice. Growth of parental Reoviruses in mouse colon tumour CT26 cells showed that there is an enhanced effect of the virus infection leading to the 18 hour time point, where the virus induced the breakdown of host factors. There was also a major increase in T3D viral proteins at this time point, where we also observed the decrease in host proteins such as actin, eIF2α, interferon regulatory factors and PKR. We proposed that the Reovirus μ2 protein may be involved in this process, which induces the degradation of host inhibitors to result in enhanced viral gene and protein expression. Our results also showed that parental and oncolytic reassortant viruses behaved differently with respect to growth and IFN response in different cell lines. We demonstrated a complicated interplay between Reovirus gene products, resulting in a unique interaction involving other viral and cell proteins to achieve a certain trait, in the context of the cell type.

Mammalian reovirus is a naturally benign virus that preferentially replicates in cancer cells (reovirus oncolysis) and has been tested as a potential cancer therapy in vitro, in vivo and in clinical trials for treating cancers from a wide range of origins. Reovirus-induced apoptosis has been shown to be important for reovirus oncolysis. The tumor suppressor protein p53 plays vital roles in mediating host apoptosis but its effect on reovirus oncolysis has not been fully understood and hence investigated. Data here show that p53 does not affect reovirus replication or reovirus-induced apoptosis in human cancer cells. However, significant enhancement of the reovirus-mediated apoptosis is induced by addition of p53 accumulators/activators such as a MDM2 antagonist Nutlin-3a or sub-lethal concentrations of chemotherapy drugs. This enhanced cell death is p53-dependent, requires NF-kappaB activation and p53 target genes p21 and bax. Furthermore, a combination of reovirus and p53 accumulators/activators directly results in significantly higher level of reovirus dissemination and spread. One of the hurdles for current chemotherapy in patients is the side effects caused by high concentrations of cytotoxic drugs. Hence, a therapeutic regime using the combination of reovirus and sub-lethal (lower concentrations of) chemotherapeutics that induce p53 activation/accumulation potentially can both enhance tumor regression and reduce the side effects in patients. Ras mutation, one of the most prevalent mutations in human cancer, has been implicated to determine the susceptibility of cancer cells to reovirus infection. However, the underlying mechanism of reovirus preferential replication needs to be further delineated. Quantitative analysis was used to compare individual steps of reovirus replication between non-transformed and Ras-transformed NIH 3T3 cells. Contrary to previous reports, reoviral protein synthesis is shown to be comparable between non-Ras and Ras-transformed cells. Meanwhile, although reovirus binding and internalization is not affected by Ras-transformation, reovirus uncoating is enhanced in Ras-transformed cells. Ras-transformation also enables reovirus to better spread to neighboring cells through apoptosis. Furthermore, reovirus infection of Ras effector mutant cells that activate specific Ras effector pathways indicate that sub-Ras pathways play different roles in enhancing reovirus preferential replication in Ras-transformed cells and therefore provide additional targets for cancer therapy.

Cancer remains to be one of the leading causes of death worldwide. Current treatment methods are not effective, and so newer therapies are needed, particularly in applied virology, where aspects of viral growth and replication are targeted to tumour lysis (termed viral oncolysis). Reoviruses are naturally occurring oncolytic viruses. Since most cancers are epithelial in origin, but also include fibroblastic properties due to the epithelial-mesenchymal transitions within tumours, my investigations mainly focused on the biology of Reoviruses in both fibroblast and epithelial cells. We studied the genetic basis for Protein Kinase R sensitivity and its role in lung tropism, as well as the study of tumour oncolysis by Reoviruses and their reassortants. We also investigated the growth and protein production of Reovirus and four oncolytic reassortants and their interferon susceptibilities in L929 fibroblast cells, CT26 mouse colon tumour cells, interferon-deficient Vero cells and interferon producing CV-1 cells. We found that PKR protected the bronchiolar epithelium from infection from Reovirus. In PKR knockout mice lungs, the ability to infect the bronchial epithelium was attributed to the T3D σ1s non-structural protein, encoded by the S1 gene segment. T1L infection of PKR+/+ Balb-c mice enhanced disease, whereas T3D growth and replication was limited by PKR. Studies in viral oncolysis showed that treatments of CT26 tumour-bearing mice with high viral doses of Reovirus or oncolytic reassortants resulted in death from enhanced virus toxicity. However, treatments at lower and multiple doses significantly increased survival rates of mice. Growth of parental Reoviruses in mouse colon tumour CT26 cells showed that there is an enhanced effect of the virus infection leading to the 18 hour time point, where the virus induced the breakdown of host factors. There was also a major increase in T3D viral proteins at this time point, where we also observed the decrease in host proteins such as actin, eIF2α, interferon regulatory factors and PKR. We proposed that the Reovirus μ2 protein may be involved in this process, which induces the degradation of host inhibitors to result in enhanced viral gene and protein expression. Our results also showed that parental and oncolytic reassortant viruses behaved differently with respect to growth and IFN response in different cell lines. We demonstrated a complicated interplay between Reovirus gene products, resulting in a unique interaction involving other viral and cell proteins to achieve a certain trait, in the context of the cell type.

Cancer remains to be one of the leading causes of death worldwide. Current treatment methods are not effective, and so newer therapies are needed, particularly in applied virology, where aspects of viral growth and replication are targeted to tumour lysis (termed viral oncolysis). Reoviruses are naturally occurring oncolytic viruses. Since most cancers are epithelial in origin, but also include fibroblastic properties due to the epithelial-mesenchymal transitions within tumours, my investigations mainly focused on the biology of Reoviruses in both fibroblast and epithelial cells. We studied the genetic basis for Protein Kinase R sensitivity and its role in lung tropism, as well as the study of tumour oncolysis by Reoviruses and their reassortants. We also investigated the growth and protein production of Reovirus and four oncolytic reassortants and their interferon susceptibilities in L929 fibroblast cells, CT26 mouse colon tumour cells, interferon-deficient Vero cells and interferon producing CV-1 cells. We found that PKR protected the bronchiolar epithelium from infection from Reovirus. In PKR knockout mice lungs, the ability to infect the bronchial epithelium was attributed to the T3D σ1s non-structural protein, encoded by the S1 gene segment. T1L infection of PKR+/+ Balb-c mice enhanced disease, whereas T3D growth and replication was limited by PKR. Studies in viral oncolysis showed that treatments of CT26 tumour-bearing mice with high viral doses of Reovirus or oncolytic reassortants resulted in death from enhanced virus toxicity. However, treatments at lower and multiple doses significantly increased survival rates of mice. Growth of parental Reoviruses in mouse colon tumour CT26 cells showed that there is an enhanced effect of the virus infection leading to the 18 hour time point, where the virus induced the breakdown of host factors. There was also a major increase in T3D viral proteins at this time point, where we also observed the decrease in host proteins such as actin, eIF2α, interferon regulatory factors and PKR. We proposed that the Reovirus μ2 protein may be involved in this process, which induces the degradation of host inhibitors to result in enhanced viral gene and protein expression. Our results also showed that parental and oncolytic reassortant viruses behaved differently with respect to growth and IFN response in different cell lines. We demonstrated a complicated interplay between Reovirus gene products, resulting in a unique interaction involving other viral and cell proteins to achieve a certain trait, in the context of the cell type.

<p> Oncolytic viruses are able to selectively replicate in tumour cells and are an attractive new avenue of cancer therapy that lacks the toxic side effects of current treatment modalities. HSV-1 mutants lacking ICPO are promising oncolytic vectors, however, the mechanisms behind viral oncolysis remain unclear. Since PML contributes to the repression of HSV-1 and also is downregulated in various types of cancer, but particularly in prostate cancer, PML has been implicated as a factor influencing the permissiveness of tumour cells to I CPO-null HSV-1 oncolysis. By screening a series of immortalized patient matched normal and tumour prostate epithelial cells for sensitivity to ICPO-null HSV-1 oncolysis and evaluating the levels of PML in each cell line, we were unable to establish a link between PML status and permissiveness to ICPO-null HSV-1 oncolytic vectors. Also, since a large proportion of the population possesses pre-existing immunity to HSV -1, which may hinder systemic administration of HSV-1 vectors, we sought to determine if BHV-1 could be an alternative oncolytic herpesvirus. BHV-1 was cytotoxic to various human immortalized and transformed cell lines in vitro, but was generally more restricted from normal human cells, suggesting that BHV -1 may have potential as an oncolytic virus. However, the sensitivity of human cells to BHV -1 infection did not correlate with type I IFN signaling, as has been demonstrated for other oncolytic viruses. Furthermore, neutralizing antibodies against HSV-1 were unable to cross-react with BHV -1 in vitro suggesting that pre-existing immunity to HSV -1 in humans may not hinder BHV -1 infection. It is hoped that these results will contribute to the understanding of viral mediated oncolysis and also provide some evidence that BHV-1 may be a new alternative oncolytic herpesvirus, however, in vivo studies are necessary to evaluate the oncolytic efficacy of BHV -1. </p> / Thesis / Master of Science (MSc)

Auf Adenoviren (Ads) basierende Vektoren wurden als ein gezielter Anti-Krebs-Wirkstoff entwickelt, der erfolgversprechende Resultate in prä-klinischen Studien erzielen konnte. Solche onkolytischen Ads sind zwar in klinischen Studien generell als sicher eingestuft worden, konnten jedoch die therapeutischen Erwartungen nicht erfüllen. In dieser Doktorarbeit konnte, unter Verwendung der Genexpressionsprofile von Ovarialkarzinom-Zellen, der epitheliale Phänotyp als Hindernis für allgemein verwendete onkolytische Ads, die auf den Coxsackie- und Adenovirusrezeptor (CAR) oder CD46 ausgerichtet sind, identifiziert werden. Der Zugang zu den Virus-Rezeptoren war zwingend an Zelldepolarisation und den Verlust der epithelialen Zonulae occludens und adherens gekoppelt, was Merkmale der Epithelial-zu-Mesenchymal-Transition (EMT) darstellt. Bedeutsam ist in diesem Zusammenhang, dass Tumore in situ als auch Xenograft-Tumore zum größten Teil aus Epithelzellen oder epithelial/mesenchymalen (E/M) Hybrid-Zellen bestehen. Diese E/M Hybrid-Zellen sind die einzigen Zellen, welche an Zellkulturbedingungen adaptieren, wo sie durch EMT während weiterem Passagieren in Mesenchymzellen differenzieren. Bemerkenswert ist hierbei die Tatsache, dass nur Mesenchymzellen und E/M Hybrid-Zellen, die sich im EMT-Prozess befanden, sensitiv zu viraler Onkolyse waren. In Versuchen, die festgestellte Resistenz zu überwinden, wurde herausgefunden, dass bisher nur wenig erforschte Adenovirus-Serotypen (Ad3, Ad7, Ad11 und Ad14), welche einen anderen Rezeptor als CAR oder CD46 auf Zellen benutzen, besser geeignet sind, um polarisierte Epithelzellgewebe zu infizieren. Diese Ads induzierten EMT-ähnliche Prozesse in Ovarialkarzinom-Kulturen mit epithelialem Phänotyp, was zu deren effizienter Onkolyse führte. Die vorliegende Arbeit trägt somit zur Aufklärung der Diskrepanz zwischen der Virustherapie-Effizienz in vivo und in vitro bei und bietet Anhaltspunkte für die Konstruktion von zukünftigen onkolytischen Ads. / Vectors based on adenoviruses have been designed as targeted anti-cancer therapeutics that showed promising results in pre-clinical applications. In clinical trials, these oncolytic adenoviruses have generally been proved safe in patients, but have fallen short of their expected therapeutic value. In this thesis the susceptibility of primary ovarian cancer cells to oncolytic adenoviruses was studied in order to identify cellular mechanisms that confer resistance to virotherapy. Using gene expression profiling of cancer cells either resistant or susceptible to viral oncolysis, it was discovered that the epithelial phenotype of ovarian cancer represents a barrier to infection by commonly used oncolytic adenoviruses targeted to coxsackie- and adenovirus receptor (CAR) or CD46. Accessibility to viral receptors was critically linked to depolarization and the loss of tight and adherens junctions, both hallmarks of epithelial-mesenchymal transition (EMT). Importantly, tumors in situ as well as xenograft tumors derived from primary ovarian cancer cells mostly contained epithelial cells and cells that are in an epithelial/mesenchymal (E/M) hybrid stage. These E/M cells are the only xenograft-derived cells that can be cultured and with passaging undergo EMT to differentiate into mesenchymal cells. Notably, only mesenchymal cells and E/M cells in the process of EMT were susceptible to viral oncolysis. In attempts to overcome the observed resistance, it was found that thus far little explored adenovirus serotypes (Ad3, Ad7, Ad11, and Ad14), which use cellular receptor(s) other than CAR and CD46, have superior oncolytic abilities on polarized epithelial tissue. This study therefore contributes to the clarification of observed discrepancies between virotherapy performances in vitro and in vivo and gives a rationale for the construction of future oncolytic adenoviruses.

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