Inhibition of Cyclin A Kinase Activity in E2F-1 Chemogene Therapy of Colon Cancer

Abstract

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 LD₂₅doses. 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 (≤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 LD₂₅had 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.