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

¹ Laboratory of Molecular Pharmacology and ² Developmental Therapeutics Program, National Cancer Institute, Department of Health and Human Services, Bethesda, Maryland

Requests for reprints: Yves Pommier, National Cancer Institute, Department of Health and Human Services, NIH, 37 Convent Drive, 37-5068, Bethesda, MD 20892. Phone: 301-496-5944; Fax: 301-402-0752; E-mail: pommier{at}nih.gov.

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 (GI₅₀) value for batracylin in HT29 colon carcinoma cells was 10 µmol/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 Top2/ß-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 µmol/L concentrations, thus presenting a useful early biomarker of DNA damage. The half-life 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]

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