| Adenoviruses have proven to be particularly helpful in unraveling the intricacies of cell growth regulation and apoptosis, greatly increasing our understanding of oncogenesis. At the onset of this work, adenoviruses were thought to induce apoptosis via a p53-dependent cell death pathway exclusively, and we set out to develop an assay system to study the ability of viral proteins to inhibit p53-induced apoptosis. We started using the p53-null human Saos-2 osteogenic carcinoma cells when we found that p53 expression led to rapid cell death. The morphology of these cells by electron microscopy confirmed that death was by apoptosis and this death was significantly reduced with Bcl-2 overexpression. We then demonstrated that the E1B-55K protein of human adenovirus type 5 (Ad5), which was known to bind and repress p53 transcriptional activity, blocks cell death following p53 expression. These results directly show that E1B-55K is able to inhibit p53-induced apoptosis. This ability is important for adenovirus replication because alone, the 289-residue (289R) and 243R E1A products of Ad5 induce p53-dependent apoptosis which would inhibit progeny production.;During the course of this work, we also found that the 289R E1A protein is able to induce p53-independent apoptosis, and the remainder of this thesis work is focused on that question. Preliminary results suggested that p53-independent cell death required expression of additional Ad5 early gene products. We found that E1B-19kDa and Bcl-2 inhibited this p53-independent killing. Neither early regions E2 or E3 were necessary for cell death, but analysis of a series of E1A mutants indicated that mutations disrupting transactivation of E4 gene expression abolished killing. Furthermore, Saos-2 cells infected with an E1B +/E4- viral mutant remained viable for considerably longer times than those infected with wt Ad5. In addition, an E1-/E4- adenovirus vector was unable to induce cell killing in E1A-expressing cell lines, indicating that an E4 product is essential for E1A-induced p53-independent apoptosis.;To identify which E4 products are involved, studies were conducted in p53-deficient human Saos-2 cells infected with various Ad5 E4 mutants. An E4orf6-deficient mutant was defective in cell killing whereas another which expressed only E4orf6 and E4orf4 killed like wt virus, suggesting that E4orf6 may be responsible for cytotoxicity; however, a mutant expressing only E4orf4 induced high levels of cell death, indicating that this E4 product may also be cytotoxic. To define the E4 cell death-inducing functions more precisely, cDNAs encoding individual E4 products were introduced into cells by DNA transfection together with a cDNA encoding firefly luciferase. Enzymatic activity was high in all cases except with E4orf4 where levels were less than 20% of controls. In addition, drug selection of cells following transfection with a puromycin-containing retroviral vector encoding individual E4 products yielded a high number of cell colonies except when E4orf4 was expressed. These data demonstrated that E4orf4 is the only E4 product capable of independent cell killing. Death was due to apoptosis, as evidenced by the morphology of DAPI stained cell nuclei. This identification of a p53-independent apoptosis pathway has offered considerable insight into how the virus may ultimately be killing the host cell. Additionally, it has directed us to another cellular apoptosis pathway which will contribute to our understanding of the life and death balance in the cell. |
