Evidence for the Reversibility of Cellular DNA Lesion Induced by Mammalian Topoisomerase I1 Poisons*

Like many intercalative antitumor drugs, the non- intercalative antitumor drug epipodophyllotoxin VM-26 (teniposide) induces topoisomerase 11-linked DNA breaks as revealed by cell lysis with a strong protein denaturant such as sodium dodecyl sulfate or alkali. We show that the majority of topoisomerase 11-linked DNA breaks reflect the formation of reversible topoi- somerase 11-DNA cleavable complexes in drug-treated cells by demonstrating the reversibility of this unusual type of DNA damage at elevated temperatures (e&. 65 “C).

passing activity of topoisomerase I1 (10). Exposure of this otherwise reversible enzyme-DNA-drug-cleavable complex to a strong protein denaturant such as SDS or alkali leads to the formation of topoisomerase 11-linked single-and doublestrand DNA breaks (7)(8)(9). Detailed characterization of the cleaved product induced by protein denaturants has shown that a topoisomerase I1 polypeptide is covalently linked to the 5'-phosphoryl end of the broken DNA strand via a tyrosyl phosphate bond (10). The 3'-hydroxyl end, in the case of a double-strand break, is 4-base recessed (10). These studies suggest that the major cellular DNA lesion induced by topoisomerase I1 poisons in mammalian cells may be the reversible topoisomerase 11-DNA cleavable complexes, and topoisomerase 11-linked DNA breaks may be produced by SDS or alkali treatment during cell lysis. We have obtained additional evidence in the present studies that the cellular DNA lesion induced by topoisomerase I1 poisons is indeed reversible, consistent with the cleavable complex hypothesis.

EXPERIMENTAL PROCEDURES
Materials-DNA topoisomerase I1 from HeLa cells was purified to homogeneity by slight modification of the published procedures (11). pBR322 DNA was purified by phenol deproteinization of cleared lysates followed by CsCl/ethidium isopycnic centrifugation and gel filtration on an A-50m column. VM-26 (teniposide) was a gift from Bristol-Myers Co. Media and fetal bovine serum for cell culture work were purchased from GIBCO. Rabbit antisera against purified calf thymus DNA topoisomerase I and I1 were prepared as described previously (11,12).
Immunoblot Analysis of Topoisomerase Contents-Immunoblot analysis using rabbit antisera against human topoisomerase I and I1 was done with minor modifications as described previously (12,13). Potassium-SDS Co-precipitation Assay-Potassium-SDS co-precipitation assays, both in the purified system and in drug-treated cells, were done as described previously (6,10).

RESULTS AND DISCUSSION
Exposure of topoisomerase I-DNA cleavable complexes to an elevated temperature (e.g. 65 " C ) has been shown to abolish subsequent DNA cleavage upon addition of a strong protein 9713 denaturant (12). This apparent reversal of DNA cleavage at an elevated temperature, which has also been observed for ColEl relaxation complex (14), is another unusual property of the reversible topoisomerase I-DNA cleavable complex. To test whether topoisomerase 11-DNA cleavable complexes induced by topoisomerase I1 poisons exhibit a similar property toward elevated temperatures, the mammalian topoisomerase I1 poison, VM-26 (teniposide), was selected for the present study. In the presence of purified HeLa topoisomerase 11, VM-26 induced large amounts of cleavable complexes, which upon addition of SDS led to fragmentation of end-labeled pBR322 DNA (Fig. 1, lune P ) . Brief exposure of the cleavable complexes to 65 "C prior to the addition of SDS resulted in time-dependent reduction in subsequent DNA cleavage upon the addition of SDS (Fig. 1, lunes Q-T). Within 3 min, the majority of smaller DNA fragments had been converted into larger DNA molecules (Fig. 1, compare lunes P and R ) . The residual DNA fragments, although still evident in the gel, were considerably less than those produced before heat treatment (Fig. 1, compare lunes P and R ) . This effect was also studied by a potassium-SDS co-precipitation assay using the same 32P end-labeled pBR322 DNA (Fig. 2B). The amounts of protein-linked DNA in the precipitate were similarly reduced following heating the reaction mixture of 65 "C ( Fig.  2B). These results suggest that the reversibility of the VM-26-induced topoisomerase 11-DNA cleavable complexes can be demonstrated by incubation of the complex at an elevated temperature.
The possibility that VM-26-induced DNA lesion in cultured mammalian cells could be reversed by a brief heat treatment at an elevated temperature was tested using MELC. Previous studies have shown that topoisomerase 11-linked DNA fragments can be selectively precipitated from lysates of VM-26treated cells using the potassium-SDS co-precipitation assay (6). As shown in Fig. 2A, the amounts of protein-linked DNA in lysates of VM-26-treated MELC were rapidly reduced following exposure of drug-treated cells to 65 "C ( Fig. 2A). A different type of assay which monitors the unbound cellular topoisomerase I1 in cell lysates was also performed to test the effect of elevated temperatures on the possible formation of topoisomerase 11-DNA cleavable complexes in VM-26-treated cells (Fig. 3A). As shown previously, the majority of cellular topoisomerase I1 molecules which are trapped covalently onto chromosomal DNA in VM-26-treated cells cannot enter a 7.5% SDS-polyacrylamide gel and therefore cannot be de- tected by immunoblot analysis using topoisomerase I1 antisera. Reversal of DNA cleavage in drug-treated cells at an elevated temperature is expected to release covalently trapped topoisomerase I1 molecules from chromosomal DNA and to result in the recovery of unbound topoisomerase I1 which can be detected by immunoblot analysis. Fig. 3A shows the results of such an assay. The recovery of unbound topoisomerase I1 is dependent on the temperature. There appears to be a sharp transition temperature around 52 "C above which the majority (70%) of trapped topoisomerase I1 molecules were recovered (Fig. 3C). The trapping and recovery of topoisomerase I1 molecules in drug-treated cells were specific as the cellular level of topoisomerase I was unaffected by VM-26 and elevated temperatures (Fig. 3B). The rate of recovery of unbound topoisomerase I1 in VM-26-treated cells at 65 "C was also measured by the same method (Fig. 4A). Within 1 min at 65 "C, recovery is already complete (Fig. 4C). Again, the cellular topoisomerase I level did not change under such conditions (Fig. 4B).
SV40 virus-infected cells have been successfully used as an experimental system for studying the DNA lesion induced by topoisomerase I1 poisons (9). Both linearized (from 111) and nicked (form 11) SV40 DNA, which have been shown previously to represent topoisomerase 11-linked DNA breaks, were produced in VM-26-treated cells (Fig. 5, lane B). Brief heating (to 65 "C) of the SV40 virus-infected cells in the presence of VM-26 rapidly abolished both linearized and nicked SV40 DNA in cell lysates (Fig. 5, lunes B-F).
Together, our results provide strong evidence supporting the hypothesis that the unusual cellular DNA lesion induced by topoisomerase I1 poisons is the formation of reversible topoisomerase 11-DNA cleavable complexes. However, the mechanism of reversal of DNA cleavage at elevated temper-