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CHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY

Cytokeratin-RNA Cross-Linking Mediated by the Antitumor Aminoflavone, 5-Amino-2,3-fluorophenyl-6,8-difluoro-7-methyl-4H-1-benzopyran-4-one

Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (L.-h.M., Z.-h.M., Y.P.); and Laboratory of Proteomics and Analytical Technologies, Advanced Technology Program, SAIC-Frederick Inc., National Cancer Institute-Frederick, Frederick, Maryland (Z.M., T.D.V.)

Aminoflavone (AF) is an anticancer drug in early clinical trials, and its antiproliferative activity involves the induction of DNA-protein cross-links. To identify the proteins cross-linked to nucleic acids, cesium chloride (CsCl) gradient centrifugation was used to isolate proteins tightly bound to nucleic acids in AF-treated human breast carcinoma MCF-7 cells. The identified proteins included structural proteins (several cytokeratins), transcription regulators, and stress response proteins. The identification of the cytokeratins was validated using direct immunoblotting of the high-density CsCl (nucleic acid) fractions isolated from AF-treated cells. Ribonuclease A pretreatment caused the cytokeratin signal in the heaviest CsCl fractions to disappear, suggesting that AF mediates RNA-cytokeratin cross-links. Additional experiments using radiolabeled AF showed that AF formed adducts with total RNA and mRNA with similar affinity to that of DNA. Moreover, 18S RNA was selectively pulled down using an anti-cytokeratin antibody after AF treatment. Consistent with the formation of these adducts, we found that AF inhibits RNA and protein synthesis in a dose- and time-dependent manner. This study provides evidence for the formation of AF-mediated cytokeratin-RNA cross-links and the presence of cytokeratin-RNA complexes. Thus, in addition to its anticancer activity, AF might be a useful molecular probe to study the potential role of cytokeratins in the subcellular localization and metabolism of RNA.

Address correspondence to: Dr. Yves Pommier, Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 37 Convent Drive, Bldg. 37, Room 5068, Bethesda, MD 20892-4255. E-mail: pommier{at}nih.gov