Photoswitching FRET Studies of Doxorubicin-Chromatin Interactions

Abstract

The molecular basis for the action of anti-cancer drugs is of interest for further drug development as well as for better understanding of adverse side effects. Doxorubicin is a DNA intercalating anti-cancer drug thought to work by inhibition of topoisomerase II leading to double-strand breaks and subsequent cancer cell death. It is also proposed to display a novel mechanism of action which results in eviction of some core histones from chromatin and leads to deregulation of the cancer cell transcriptome. Uncommon amongst anti-cancer drugs, many anthracyclines such as doxorubicin have absorption spectra in the visible range which allows Förster Resonance Energy Transfer (FRET) studies of their interactions with chromatin. Since Fluorescence Lifetime Imaging Microscopy (FLIM) FRET studies do not require an acceptor to be fluorescent to measure energy transfer, previous works concentrated on doxorubicin-chromatin interactions within intact tumors by imaging the fluorescence lifetimes of EGFP labeled histone H1 or H2B. Our previously developed photoswitching FRET (psFRET) method mimics the advantage of FLIM by also not requiring a fluorescent acceptor. This property allows us to monitor energy transfer between doxorubicin and the core histone proteins tagged with the photoswitchable fluorescent protein, Dronpa. By tagging the N or C termini of the core histones, the Dronpa protein can be placed at multiple different positions on the periphery of a nucleosome and thus allow a survey of FRET efficiencies from numerous points. Here, we present psFRET studies in our efforts to better define binding sites for doxorubicin as well as its potential for histone eviction from chromatin.