Differential cytotoxic pathways of topoisomerase I and II anticancer agents after overexpression of the E2F-1/DP-1 transcription factor complex

The transcription factor complex E2F-1/DP-1 regulates the G(1)-to-S-phase t ransition and has been associated with sensitivity to the S-phase-specific anticancer agents camptothecin and etoposide, which poison DNA topoisomeras e I and II, respectively, To investigate the relationship between E2F-1 and drug sensitivity in detail, we established human osteosarcoma U-2OS-TA cel ls expressing full-length E2F-1/DP-1 under the control of a tetracycline-re sponsive promoter, designated UE1DP-1 cells. Topoisomerase I levels and act ivity as well as the number of camptothecin-induced DNA single- and double- strand breaks were unchanged in UE1DP-1/tc- cells with >10-fold E2F-1/DP-1 overexpression. However, UE1DP-1/tc- cells were hypersensitive to camptothe cin in both a clonogenic assay and four different apoptotic assays, This in dicates that camptothecin-induced toxicity in this model is due to the acti vation of an E2F-1/DP-1-induced post-DNA. damage pathway rather than an inc rease in the number of replication forks caused by the S-phase initiation. In contrast, topoisomerase II alpha levels (but not topoisomerase II beta l evels), together with topoisomerase II alpha promoter activity, increased 2 -3-fold in UE1DP-1/tc- cells, Furthermore, the number of etoposide-induced DNA single- and double-strand breaks increased in UE1DP-1/tc- cells togethe r with a rise in clonogenic sensitivity to etoposide, but an equal apoptoti c sensitivity to etoposide, The increase in topoisomerase II alpha promoter activity in UE1DP-1/tc- cells was shown to be due to S-phase initiation pe r se because it was blocked by ectopic expression of dominant negative cycl in-dependent kinase 2. In conclusion, overexpression of E2F-1/DP-1 in U-2OS -TA cells is sufficient to increase clonogenic sensitivity to both topoisom erase I- and II-targeted anticancer drugs. However? the mechanism by which this occurs appears to be qualitatively different. The UE1DP-1 cell model m ay be used to elucidate post-DNA damage mechanisms of cell death induced by topoisomerase I-directed anticancer agents.