Ceramide regulation by E4orf4 gene during adenoviral infection -

Abstract

Cancer remains the leading cause of global mortality. Advanced and metastatic cancer often remains incurable by current treatment options underscoring the need for the development of novel approaches. Therapies based on viruses are promising in the field of cancer, including adenovirus type 5 (Ad5). Advantages of adenovirus use include its ability to infect both dividing and arrested cells, and the ease of obtaining high concentrations. E4orf4, an early adenoviral gene, is a candidate oncolytic viral gene as it triggers p53- and caspase-independent apoptosis selectively in cancer cells. Yet a deeper understanding of its mechanism of action is necessary to elucidate its function before its clinical application. The potency of E4orf4’s lytic activity precludes its stable transfection in cells as they would quickly succumb. For this purpose, we used a T-Rex tetracycline inducible system. The E4orf4 gene was introduced into a vector pcDNA4-T0, where its expression is repressed by another vector pcDNA6-TR. In theory, this system would allow us to tightly control the expression of E4orf4 in “stably” transfected A549 cell line and subsequently be able to study its effects on apoptosis and ceramide. Real-time PCR and western blot analysis were used to confirm the expression of E4orf4 in these cells in response to tetracycline treatment. Analyses of viability by Trypan Blue, cell cycle distribution, expression levels of apoptotic-related proteins and ceramide levels were measured. Surprisingly, we observed that the expression of E4orf4 in A549 cells did not have any effect on the cell growth, apoptosis, or ceramide levels as compared to the A549-Ø (empty vector) cells. Moreover, E4orf4 increased the number of cells in the S phase along with an elevation of cyclin A levels in A549-E4orf4 cells. In an effort to understand these unexpected findings, we examined ceramide levels and the expression of several antiapoptotic and proapoptotic proteins of the BCL2 family in our system. We discovered that th