DNA topoisomerase I and II (top1 and top2) are primary targets for cancer chemotherapy and can be inhibited by physiological and carcinogenic DNA modifications. Topoisomerase poisons act by stabilizing enzyme-linked DNA breaks which can be detected as protein-associated DNA breaks in drug-treated cells (cleavage complexes).We had previously reported that various DNA modifications including mismatches, abasic sites, oxidative base damage (8-oxoguanine) and carcinogenic adducts (ethenoadenine and benzo[a]pyrene guanine adducts), and demonstrated for the first time the induction of top1-DNA adducts in cells exposed to the carcinogenic isomer of benzo[a]pyrene diol epoxide. This observation suggests the possible involvement of top1 in the mutations observed after carcinogenic exposure. It also demonstrates how minor groove alkylation can trap top1, which is relevant for drug development as we found that the minor groove guanine N2 alkylator ecteinascidin 743 is a top1 poison.We have also found that top1 cleavage complexes can be observed after treatment with nucleoside analogs (cytosine arabinoside, gemcitabine), which suggests that top1-mediated DNA damage contributes to the antiproliferative activity of cytosine arabinoside and gemcitabine.We have developed a ligation-mediated PCR (LM-PCR) assay to analyze replication-mediated DNA double-strand breaks induced by top1 cleavage complexes in human colon carcinoma HT29 cells at the nucleotide level. We found that conversion of top1 cleavage complexes into replication- mediated DNA double-strand breaks is only detectable on the leading strand for DNA synthesis, which suggests an asymmetry in the way that top1 cleavage complexes are metabolized on the two arms of a replication fork. Extension by Taq DNA polymerase was not required for ligation to the LM-PCR primer, indicating that the 3' DNA ends are extended by DNA polymerase in vivo closely to the 5' ends of the topoisomerase I cleavage complexes. These findings suggest that the replication-mediated DNA double-strand breaks generated at top1 cleavage sites are produced by replication run-off. We also found that the 5' ends of these DNA double-strand breaks are phosphorylated in vivo, which suggests that a DNA 5' kinase activity acts on the double-strand ends generated by replication run-off. The replication-mediated DNA double-strand breaks were rapidly reversible after cessation of the top1 cleavage complexes, suggesting the existence of efficient repair pathways for removal of top1-DNA covalent adducts in ribosomal DNA.We have pursued our studies with ecteinascidin 743, which is, at the present time in Phase II clinical trials with responses in sarcomas. Ecteinascidin 743 (Et743, NSC 648766) differs from other anticancer drugs in clinical use because it forms covalent adducts at specific guanines in the minor groove of DNA. To further elucidate the mechanism of action of Et743, we have generated an Et743-resistant cell line (HCT116/ER5). The HCT116/ER5 cells exhibit enhanced microsatellite instability and aneuploidy. Complete lack of expression of the DNA repair gene XPG was found in HCT116/ER5 cells as a result of chromosome 13q DNA copy-number loss, due to an unbalanced t(13; 14) translocation, and loss of heterozygosity at 13q33.3, due to a frameshift mutation of XPG at codon 240 resulting in a stop codon at position 243. Transfection of XP-G cDNA restored the sensitivity of the HCT116/ER5 cells to Et743 demonstrating that proteins involved in the nucleotide excision repair pathway such as the endonuclease XPG are essential for the antiproliferative activity Et743. Et743 defines a novel class of anticancer drugs in which enhanced antiproliferative activity parallels enhanced cellular DNA-repair capability.

Agency
National Institute of Health (NIH)
Institute
Division of Basic Sciences - NCI (NCI)
Type
Intramural Research (Z01)
Project #
1Z01BC006150-19
Application #
6433070
Study Section
(LMP)
Project Start
Project End
Budget Start
Budget End
Support Year
19
Fiscal Year
2000
Total Cost
Indirect Cost
Name
Basic Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code
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