Adenoviral-mediated gene transfer of the apoptotic gene E2F-1 has been shown to induce apoptosis in a variety of tumor cells and acts in an additive or cooperative fashion with several specific chemotherapeutic agents to induce tumor cell death. The apoptotic function of E2F-1 is dependent on its ability to bind DNA; cyclin A kinase activity has been shown to negatively regulate the DNA-binding capacity of E2F-1. In the present study, we sought to determine whether cyclin A kinase activity is involved in mediating the interaction between E2F-1 and chemotherapeutic agents in colon cancer cells. Therefore, human colon adenocarcinoma (SW620) cells were treated with an adenovirus expressing E2F-1 (Ad-E2F-1, multiplicity of infection 20). Immediately following infection, a panel of conventional chemotherapeutic agents with varying modes of cytotoxic action were administered at LD(25 )doses. Three days following treatment, viability and growth inhibition were determined by trypan blue exclusion assay. Apoptosis was confirmed using cellular morphology, poly (ADP-ribose) polymerase cleavage, and flow-cytometric analysis. E2F-1 overexpression and cyclin A protein expression were monitored by immunoblot, and cyclin A kinase activity was determined by kinase assay. Vincristine (VIN), camptothecin (CPT), and actinomycin D were found to have a cooperative (>38% over the additive single therapy values) effect on E2F-1-mediated apoptosis. Etoposide, cisplatin (CIS), and 5-fluorouracil (5-FU) showed the least cooperation (<or=11.5% over the additive single therapy values) with E2F-1. Ad-E2F-1 treatment alone results in 3.4-fold increase of cyclin A kinase activity compared to Ad-LacZ control (p < 0.05); when combined with chemotherapeutic agents, cyclin A kinase activity was inhibited significantly by VIN, actinomycin D, and etoposide (p < 0.005), but not with CPT, CIS, and 5-FU (p > 0.1) compared to Ad-E2F-1 treatment alone. Combination of Ad-LacZ/5-FU and Ad-LacZ/actinomycin D significantly inhibited cyclin A kinase activity compared to Ad-LacZ treatment alone (p < 0.005). No other Ad-LacZ/drug combinations significantly affected cyclin A kinase activity (p > 0.05). In conclusion, combinations of E2F-1 adenovirus and VIN, CPT, or actinomycin D at LD(25 )had significant cooperative effects on colon cancer apoptotic cell death in vitro. Although inhibition of cyclin A kinase activity was observed in most Ad-E2F-1/drug combination treatments compared to Ad-E2F-1 treatment alone, there was no consistent correlation between degree of inhibition of cyclin A kinase activity and the cooperative effect. Nonetheless, inhibition of cyclin A kinase activity may be an important mechanism by which the chemogene therapy effects involving E2F-1 are modulated.
Copyright 2002 S. Karger AG, Basel