Figure 1.
Etoposide at low concentrations induces RPA foci in S phase cells only.
(A) U2OS cells were treated with or without 10µM etoposide for 2 hours and then fixed for staining with antibodies against RPA. DNA was stained with Hoechst. (B) U2OS cells were pre-incubated with EdU for 15 minutes before the addition of 10µM etoposide. After two further hours of incubation, cells were fixed and stained for RPA, EdU, and CenpF. DNA was stained with Hoechst. RPA foci were slightly reduced in intensity by the EdU staining protocol but remained discrete and clearly visible. Arrows indicate the two nuclei to be shown in enlarged format in (C). (C) Enlarged pictures of the nuclei indicated by the arrows in (B). (D) Percentages of RPA foci positive cells in each cell cycle stage were quantified and plotted.
Figure 2.
RPA focus induction by low concentration etoposide is dependent on replication but not on transcription.
U2OS cells were pre-treated with aphidicolin (A) or DRB (B) for 30 minutes and then with 10µM etoposide for 2 hours. (EdU was added 15 minutes before etoposide). They were fixed, and stained for RPA, EdU, and CenpF. DNA was stained with Hoechst. (C) Enlarged picture of the nucleus indicated by the arrow in (B). (D) The percentages of RPA foci positive cells under each condition were quantified and plotted.
Figure 3.
Etoposide at high concentrations induces RPA foci in S and G2 phase cells.
(A) U2OS cells were pre-treated with EdU for 15 minutes and then with 250µM etoposide for 2 hours. They were fixed and stained for RPA, EdU, and CenpF. DNA was stained with Hoechst. (B) Enlarged picture of the nucleus indicated by the arrow in (A). (C) Percentages of RPA foci positive cells in each cell cycle stage were quantified and plotted.
Figure 4.
RPA focus induction by high concentration etoposide is dependent on replication and transcription.
Cells were pre-treated with Aph (A), DRB (B), or both Aph and DRB (C) for 30 minutes and then with 250µM etoposide for 2 hours. (EdU was added 15 minutes before etoposide). They were then fixed, and stained for RPA, EdU, and CenpF. DNA was stained with Hoechst. The nuclei indicated by the arrows are also shown in enlarged format in the center panels. (D) The percentages of RPA foci positive cells under each condition were quantified and plotted.
Figure 5.
RPA focus induction by the replication-dependent mechanism does not require the 26S proteosome-mediated degradation of Top2cc.
(A) MG132 was added 30 minutes before etoposide (and 15 minutes before EdU). Cells were fixed and stained for RPA, EdU, and CenpF. DNA was stained with Hoechst. (B) Enlarged picture of the nucleus as indicated by the arrow in (A). (C) Percentages of RPA foci positive cells under each condition were quantified and plotted.
Figure 6.
RPA focus induction by the transcription-dependent mechanism is dependent on the 26S proteosome-mediated degradation of Top2cc.
Cells were pre-treated with MG132 (A), MG132 and Aph (B) or MG132 and DRB (C). They were then treated with 250µM etoposide for 2 hours, fixed, and stained for RPA, EdU, and CenpF. DNA was stained with Hoechst. The nuclei indicated by arrows are also shown in enlarged format in the center panels. (D) Percentages of RPA foci positive cells under each condition were quantified and plotted.
Figure 7.
Top2α is the major isoform mediating the replication-dependent DSB induction mechanism.
Cells were treated with two rounds of control siRNAs or Top2α and Top2β siRNAs for 72 hours. They were then treated with 10µM etoposide for 2 hours, fixed, and stained for RPA, EdU, and CenpF. DNA was stained with Hoechst. (A)-(D): siRNA treated cells stained for RPA, EdU, CenpF, and DNA. The nuclei indicated by arrows are also shown in enlarged format in the center panels. (E). Western blot analysis of Top2α and Top2β levels in siRNA treated cells. Different amounts of U2OS cell lysates were used as quantification standards.
Figure 8.
Top2α and Top2β can both mediate the transcription-dependent DSB induction mechanism.
Cells were treated with two rounds of control siRNAs or Top2α and Top2β siRNAs for 72 hours. They were then treated with 250µM etoposide for 2 hours, fixed, and stained for RPA, EdU, and CenpF. DNA was stained with Hoechst. The nuclei indicated by arrows are also shown in enlarged format in the center panels.
Figure 9.
Model for DSB Induction by Etoposide in Cells.
At low concentrations of etoposide, one subunit of Top2 is occupied by the drug to form a ss-Top2cc. Upon collision with the replication fork, ss-Top2cc is converted into a DSB, either directly by replication run-off (depicted) or indirectly by nucleolytic processing of collapsed replication forks (not depicted). At high concentrations of etoposide, both subunits of Top2 are occupied by the drug, resulting in a ds-Top2cc. It can be converted into a DSB by collision with the replication machinery or by degradation, which is stimulated by collision with the transcription machinery. Degradation might also occur independently of transcription.