Quaternary Benzo[c]phenanthridine Alkaloids - Novel Cell Permeant and Red Fluorescing DNA Probes

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Year of publication 2007
Type Article in Periodical
Magazine / Source Cytometry Part A
MU Faculty or unit

Faculty of Medicine

Web http://www3.interscience.wiley.com/journal/33945/home
Field Morphological specializations and cytology
Keywords benzophenanthridine alkaloids; cell-permeant DNA dye; chelirubine; flow cytometry; fluorescence microscopy; DNA content; cell cycle; macarpine; multicolor analysis; sanguinarine
Description Background: Quaternary benzo[c]phenanthridine alkaloids (QBAs) are naturally occurring compounds isolated from plants of Fumariaceae, Papaveraceae, Ranunculaceae and Rutaceae families. In addition to wide biological activities, they are attractive for their fluorescence. We observed interesting fluorescent characteristics of QBAs - macarpine (MA), sanguirubine (SR), chelirubine (CHR), sanguilutine (SL), chelilutine (CHL), sanguinarine (SA) and chelerythrine (CHE) upon interacting with living cells. Methods: Water stock solutions of the alkaloids (10-100 mg/ml) were added to intact cells and upon brief incubation the alkaloids stain the cells. Human cell lines HL60 (human promyelocytic leukemia), HeLa (human cervix adenocarcinoma) and LEP (human lung fibroblasts) and piglet blood were used in experiments. Blood cells were stained with MA in a combination with FITC-conjugated anti CD45 surface marker antibody. Cells were analysed by fluorescence microscopy and by flow cytometry. Results: All tested alkaloids immediately enter living cells and MA, CHR and SA bound DNA. MA showed the best DNA staining properties. Fluorescence microscopy of MA, CHR and SA stained cells reveals nuclear architecture and clearly defines chromosomes and apoptotic fragments in living cells. Moreover MA can rapidly report the cellular DNA content of living cells at a resolution level adequate for cell cycle analysis. QBAs were excitable by common argon lasers (488 nm) emitting at the range 575 - 755 nm (i.e. fluorescence detectors FL2-5). Spectral characteristics of MA allow simultaneous surface immunophenotyping. Conclusions: It was proven that MA, CHR and SA stain nucleic acids in living cells. They can be used as supravital fluorescent DNA probes both in fluorescence microscopy and flow cytometry including multiparameter analysis of peripheral blood and bone marrow. MA binds DNA stochiometrically and can report the DNA content.
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