Batracylin (NSC 320846), a Dual Inhibitor of DNA Topoisomerases I and II Induces Histone γ-H2AX as a Biomarker of DNA Damage

Abstract

Batracylin (8-aminoisoindolo [1,2-b]quinazolin-10(12H)-one; NSC320846) is an investigational clinical anticancer agent. Previous animal studies showed activity against solid tumors and Adriamycin-resistant leukemia. We initially sought to test the proposed Top2-mediated DNA cleavage activity of batracylin and identify potential biomarkers for activity. COMPARE analysis in the NCI-60 cell lines showed batracylin activity to be most closely related to the class of Top2 inhibitors. The 50% growth inhibition (GI 50 ) value for batracylin in HT29 colon carcinoma cells was 10 Mmol/L. DNA-protein cross-links, consistent with Top2 targeting, were measured by alkaline elution. DNA single-strand breaks were also detected and found to be protein associated. However, only a weak induction of DNA double-strand breaks was observed. Because batracylin induced almost exclusively DNA single-strand breaks, we tested batracylin as a Top1 inhibitor. Batracylin exhibited both Top1-and Top2A/B-mediated DNA cleavage in vitro and in cells. The phosphorylation of histone (;-H2AX) was tested to measure the extent of DNA damage. Kinetics of ;-H2AX ''foci'' showed early activation with low Mmol/L concentrations, thus presenting a useful early biomarker of DNA damage. The halflife of ;-H2AX signal reversal after drug removal was consistent with reversal of DNA-protein cross-links. The persistence of the DNA-protein complexes induced by batracylin was markedly longer than by etoposide or camptothecin. The phosphorylated DNA damage-responsive kinase, ataxia telangiectasia mutated, was also found activated at sites of ;-H2AX. The cell cycle checkpoint kinase, Chk2, was only weakly phosphorylated. Thus, batracylin is a dual Top1 and Top2 inhibitor and ;-H2AX could be considered a biomarker in the ongoing clinical trials. [Cancer Res 2007;67(20):9971-9]

Potentiation of Bleomycin in Jurkat Cells by Fungal Pycnidione

“…37) Several topoisomerase inhibitors have been reported; NSC 320846 and F11782, inhibitors of topoisomerases I and II, induced cell death 33,38) and H2A.X phosphorylation, 33) and R16, which was an inhibitor of topoisomerase II 39) and an inducer of genotoxic damage similar to bleomycin, 40) reduced Chk1 levels. 41) As demonstrated, pycnidione also induced cell death (Fig.…”

“…32,33) Therefore, phospho-histone H2A.X (Ser139) levels were analyzed to investigate whether bleomycin-induced DNA damage occurs in the presence of pycnidione (Fig. 4A).…”

The Iron Chelator Dp44mT Causes DNA Damage and Selective Inhibition of Topoisomerase IIα in Breast Cancer Cells

“…Cell survival was determined using a colony formation assay after the indicated treatments as reported previously (27). Colonies were counted manually.…”

Compound-Specific Toxicities Detected in CFU-GM, Rat Kidney NRK Cells, Rat Bladder RBLAK Cells, and Rat Liver Slices following Batracylin or N-Acetyl Batracylin Exposure

“…Mechanistically, BAT was confirmed to inhibit Topo II [3] and these studies evaluated BAT and its analogs for efficacy against the HL-60 and potency for Topo II inhibition. Studies examining BAT efficacy (including the use of -H2AX as a marker) and acetylation (with the use of human HPC) determined tumor growth inhibition and HPC toxicity occurs in the low M range [4,5,26]. With data (in vitro and in vivo) indicating the biotransformation of BAT to NAB leads to greater toxicity, the level of toxicity exhibited in preclinical animal species tested reflects this (rats > mice ≫ dogs) and that oral administration of NAB resulted in almost nil bioavailability [8][9][10] the question of how humans would fare after BAT exposure is perhaps dependent on the acetylation capability of the individual.…”

“…It has demonstrated activity against solid tumors and adriamycin-resistant leukemia [1,2] and has been evaluated preclinically in multiple species. Studies have confirmed BAT induces ATP-independent topoisomerase II (Topo II) inhibition [3,4] and subsequent DNA strand breaks that can be detected using the -H2AX marker [5].…”