Synthesis and Evaluation of Fluoromycin: A Novel Fluorescence-labeled Derivative of Talisomycin S_10b

Abstract

We have synthesized fluoromycin (FLM), a novel fluorescein-labeled derivative of talisomycin S_10b (TLM S_10b), and used it to evaluate cellular drug accumulation and distribution in bleomycin (BLM)-sensitive and-resistant cell lines. The fluorescence intensity of FLM was 300- to 400-fold greater than that of BLM A₂, TLM S_10b, or the lipophilic BLM analogue, liblomycin. FLM possessed an antiproliferative potency similar to liblomycin in BLM-sensitive human A-253 squamous carcinoma cells but was less potent than BLM A₂ or TLM S_10b. C-10E cells, a clone of A-253 cells with 40- to 50-fold resistance to BLM A₂ and TLM S_10b, were 50-fold resistant to FLM. A partially revertant cell population (C-10E ND) regained sensitivity to BLM A₂, TLM S_10b, and FLM. FLM like BLM cleaved pGEM-3Z plasmid DNA in vitro in a concentration-dependent manner. Flow cytometric analysis of FLM content in C-10E and C-10E ND cell lines showed 4-fold and 2-fold lower fluorescence intensity, respectively, compared with A-253 cells. Similar results were seen by fluorescence spectrophotometry with cell extracts. Fluorescence microscopy indicated heterogeneous distribution among A-253 cells with at least 50% of the cells exhibiting marked nuclear fluorescence localization. In contrast, C-10E cells displayed lower cellular fluorescence and predominantly cytoplasmic localization. C-10E ND cells exhibited a mixed population of nuclear and cytoplasmic vesicular localization with fluorescence levels that were intermediate between A-253 and C-10E cells. Thus, BLM-resistant cells have reduced levels of FLM and appear to have a lower nuclear:cytoplasmic ratio of FLM. FLM may be useful in studying the intracellular fate of BLM-like drugs as well as providing a tool to detect and isolate BLM-resistant cells.