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
Available at: http://works.bepress.com/timothy-veenstra/150/