A Role for VCP/p97 in the Processing of Drug-Stabilized TOP2-DNA Covalent Complexes

Abstract

DNA topoisomerase II (TOP2) poisons induce protein-DNA crosslinks termed TOP2-DNA covalent complexes, in which TOP2 remains covalently bound to each end of an enzyme-induced double-strand DNA break (DSB) via a 5'-phosphotyrosyl bond. Repair of the enzyme-induced DSB first requires the removal of the TOP2 protein adduct, which, among other mechanisms, can be accomplished through the proteasomal degradation of TOP2. VCP/p97 is a AAA ATPase that utilizes energy from ATP hydrolysis to unfold protein substrates, which can facilitate proteasomal degradation by extracting target proteins from certain cellular structures (such as chromatin) and/or by aiding their translocation into the proteolytic core of the proteasome. In this study, we show that inhibition of VCP/p97 leads to the prolonged accumulation of etoposide-induced TOP2A and TOP2B complexes in a manner that is epistatic with the proteasomal pathway. VCP/p97 inhibition also reduces the etoposide-induced phosphorylation of histone H2A.X, indicative of fewer DSBs. This suggests that VCP/p97 is required for the proteasomal degradation of TOP2-DNA covalent complexes and is thus likely to be an important mediator of DSB repair after treatment with a TOP2 poison. SIGNIFICANCE STATEMENT: TOP2 poisons are chemotherapeutic agents used in the treatment of a range of cancers. A better understanding of how TOP2 poison-induced DNA damage is repaired could improve therapy with TOP2 poisons by increasing TOP2 poison cytotoxicity and reducing genotoxicity. The results presented herein suggest that repair of TOP2-DNA covalent complexes involves the protein segregase VCP/p97.

Figure 1. Effect of the VCP/p97 inhibitor NMS-873 on the processing of etoposide-induced TOP2-DNA complexes to DSBs.

(A) The TARDIS reversal assay was used to measure levels of TOP2-DNA complexes in K562 cells treated with 100μM etoposide (VP-16) alone or in combination with 5μM NMS-873 (or MG132 for comparison). After 2 hours, etoposide was removed from the culture medium and cells were incubated for up to 2 hours in etoposide-free medium containing DMSO, MG132 or NMS-873. Cells were collected at 0, 0.5, 1 and 2 hours after etoposide removal. Averages are normalised to a 2-hour 100μM etoposide control, and statistical analysis performed by two-way ANOVA. and Bonferroni post hoc test (B) K562 cells were treated with 100μM etoposide (VP-16) alone or in combination with 5μM NMS-873 for 2 hours. Cells were processed as per the TARDIS assay and probed with anti-ubiquitin antibody (clone FK2). All values were normalised to a 100μM VP-16 control, and statistical comparisons made by unpaired t-test. (C) K562 cells were treated with 10μM etoposide (VP-16) alone or in combination with 5μM NMS-873 for up to 4 hours, and protein-free DSBs were measured by yH2AX assay. Statistical significance was determined by two-way ANOVA and Bonferroni post hoc test. Averages were normalised to a 1-hour 10μM etoposide positive control. For each graph bars represent mean values +/- SD, individual values (medians of fluorescence value per cell from individual replicates) are shown as blue-lined circles.

Figure 2. siRNA knockdown of VCP/p97 and the effect on TOP2-DNA complex processing to DSBs.

(A) VCP/p97 siRNA knockdown from triplicate experiments was tested by western blot. Each replicate represents the cells used in yH2AX and TARDIS experiments shown in Figures B and C. (B) Cells treated with VCP/p97 siRNA or control siRNA (CON) were exposed continuously to 10μM etoposide for up to 4 hours, and levels of protein-free DSBs were measured using the yH2AX assay. (C) VCP/p97 siRNA or control (CON) siRNA cells were treated with 100μM etoposide for 2 hours followed by 2 hours incubation in etoposide-free media. The TARDIS assay was used to measure levels of TOP2A- and TOP2B- DNA complexes at 0, 0.5, 1 and 2 hours after etoposide removal. Statistical significance was determined by two-way ANOVA and Bonferroni post hoc test.