Cellular uptake and interaction with purified membranes of rebeccamycin derivatives

Rebeccamycin is an antitumor antibiotic possessing a DNA-intercalating indo locarbazole chromophore linked to a glycosyl residue. The carbohydrate moie ty of rebeccamycin and related synthetic analogues, such as the potent anti tumor drug NB-506 (6-N-formylamino-12,13-dihydro-1,11-dihydroxy-13-(beta-D- glucopyranosyl)5H-indolo [2,3-a]pyrrolo-[3,4-c]carbazole-5,7-(6H)-dione), i s key element for both DNA-binding and inhibition of DNA topoisomerase I. I n this study, we have investigated the cellular uptake of rebeccamycin deri vatives and their interaction with purified membranes. The transport of rad iolabeled [H-3]dechlorinated rebeccamycin was studied using the human leuke mia HL60 and melanoma B16 cell lines as well as two murine leukemia cell li nes sensitive (P388) or resistant (P388CPT5) to camptothecin. In all cases, the uptake is rapid but limited to about 6% of the drug molecules. In HL60 cells, the uptake entered a steady-state phase of intracellular accumulati on of about 0.26 +/- 0.05 pmol/10(6) cells, which persisted to at least 90 min, The efflux of exchangeable radiolabeled molecules was relatively weak. Fluorescence studies were performed to compare the interaction of a rebecc amycin derivative and its aglycone with membranes purified from HL60 cells. The glycosylated drug molecules bound to the cell membranes can be extract ed upon washing with buffer or by adding an excess of DNA. In contrast, the indolocarbazole drug lacking the carbohydrate domain remains tightly bound to the membranes with very little or no exchange upon the addition of DNA, The membrane transport and binding properties of indolocarbazole drugs rel ated to rebeccamycin are reminiscent to those of other DNA-intercalating an titumor agents. The uptake most likely occurs via a passive diffusion throu gh the plasma membranes and the glycosyl residue of the drug plays an essen tial role for the translocation of the drug from the membranes to the inter nal cell components, such as DNA. (C) 2000 Elsevier Science B.V. All rights reserved.