DNA replication of first-generation adenovirus vectors in tumor cells

A major role of the early gene 1A and 1B products (E1A and E1B) in adenovir us infection is to create a cellular environment appropriate for viral DNA replication. This is, in part, achieved by inactivation of tumor suppressor gene products such as pRb or p53, The functions of these same cellular pro teins are also frequently lost in tumor cells. Therefore, we hypothesized t hat tumor cell lines with deregulated p53 and/or pRb pathways might support replication of E1A/E1B-deleted, first-generation adenovirus vectors (AdE1( -)). Here, we analyzed the impact of virus uptake, cell cycling, and the st atus of cell cycle regulators on AdE1(-) DNA synthesis. Cellular internaliz ation of AdE1(-) vectors varied significantly among different tumor cell li nes, whereas nuclear import of incoming viral DNA appeared to be less varia ble, Replication assays performed under equalized infection conditions demo nstrated that all analyzed tumor cell lines supported AdE1(-) synthesis to varying degrees. There was no obvious correlation between the efficiency of viral DNA replication and the status of p53, pRb, and p16, However, the am ount of virus attached and internalized changed with the cell cycle, affect ing the intracellular concentration of viral DNA and thereby the replicatio n efficacy. Furthermore, infection with AdE1(-) vectors caused a partial G( 2)/M arrest or delay in cell cycle progression, which became more pronounce d in consecutive cell cycles. Correspondingly, vector DNA replication was f ound to be enhanced in cells artificially arrested in G(2)/M, Our findings suggest that cell cycling and thus passing through G(2)/M supports AdE1(-) DNA replication in the absence of E1A/E1B, This has potential implications for the use of first-generation adenovirus vectors in tumor gene therapy.