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Differential Cytotoxic Pathways of Topoisomerase I and II Anticancer Agents after Overexpression of the E2F-1/DP-1 Transcription Factor Complex¹

Laboratory [K. Ho., J. F., M. S.] and Finsen [K. Ho., J. F., P. B. J.] Centres, Rigshospitalet, DK-2100 Copenhagen, Denmark, and the European Institute of Oncology, 20141 Milan, Italy [B. O. P., K. He.]

The transcription factor complex E2F-1/DP-1 regulates the G₁-to-S-phase transition and has been associated with sensitivity to the S-phase-specific anticancer agents camptothecin and etoposide, which poison DNA topoisomerase I and II, respectively. To investigate the relationship between E2F-1 and drug sensitivity in detail, we established human osteosarcoma U-2OS-TA cells expressing full-length E2F-1/DP-1 under the control of a tetracycline-responsive promoter, designated UE1DP-1 cells. Topoisomerase I levels and activity 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 camptothecin in both a clonogenic assay and four different apoptotic assays. This indicates that camptothecin-induced toxicity in this model is due to the activation of an E2F-1/DP-1-induced post-DNA damage pathway rather than an increase in the number of replication forks caused by the S-phase initiation. In contrast, topoisomerase II levels (but not topoisomerase IIß levels), together with topoisomerase II 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 together with a rise in clonogenic sensitivity to etoposide, but an equal apoptotic sensitivity to etoposide. The increase in topoisomerase II promoter activity in UE1DP-1/tc- cells was shown to be due to S-phase initiation per se because it was blocked by ectopic expression of dominant negative cyclin-dependent kinase 2. In conclusion, overexpression of E2F-1/DP-1 in U-2OS-TA cells is sufficient to increase clonogenic sensitivity to both topoisomerase I- and II-targeted anticancer drugs. However, the mechanism by which this occurs appears to be qualitatively different. The UE1DP-1 cell model may be used to elucidate post-DNA damage mechanisms of cell death induced by topoisomerase I-directed anticancer agents.

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