Cytokeratin-RNA Crosslinking Mediated by the Antitumor Aminoflavone, NSC 686288
Journal of Pharmacology and Experimental Therapeutics
Aminoflavone (AF) is an anticancer drug in early clinical trials, and its antiproliferative activity involves the induction of DNA-protein crosslinks. To identify the proteins crosslinked to nucleic acids, 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 crosslinks. Additional experiments using radiolabeled-AF showed that AF formed adducts with total RNA and mRNA with similar affinity as for 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 dose- and time-dependent manners. This study provides evidence for the formation of AF-mediated cytokeratin-RNA crosslinks 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.
Cytokeratin, RNA, crosslinking, antitumor, aminoflavone
Meng, Lingua; Miao, Ze-hong; Veenstra, Timothy D.; and Pommier, Yves, "Cytokeratin-RNA Crosslinking Mediated by the Antitumor Aminoflavone, NSC 686288" (2008). Pharmaceutical Sciences Faculty Publications. 326.