Caffeine dissociates complexes between DNA and intercalating dyes: application for bleaching fluorochrome-stained cells for their subsequent restaining and analysis by laser scanning cytometry.
BACKGROUND
Removal of the nucleic acid-bound fluorochrome is desirable when stained cells have to be reanalyzed using other fluorochromes. It is also often desirable to remove DNA-bound antitumor drugs from drug-treated cells, to improve cell staining. We have previously observed that in aqueous solutions, the methylxanthine caffeine (CFN) decreases interactions between planar aromatic molecules such as intercalating dyes or antitumor drugs and nucleic acids. The aim of this study was to explore whether this property of CFN can be utilized to remove the DNA-bound intercalating dyes propidium iodide (PI) or 7-aminoactinomycin D (7-AAD) from the cells and whether the bleached cells can be restained and reanalyzed.


METHODS
HL-60 cells were fixed in 70% ethanol and their DNA was stained with PI or 7-AAD. The cells were then rinsed with a 0.05 M solution of CFN in phosphate-buffered saline (PBS) or with PBS alone. The decrease in intensity of cell fluorescence during rinsing was measured by laser scanning cytometry (LSC) to obtain the bleaching kinetics of individual cells. The bleached cells were then restained with PI, 7-AAD, or the protein-specific fluorochrome sulforhodamine 101(S101). Their fluorescence was measured again by LSC. In addition, free DNA was subjected to gel electrophoresis, DNA bands in the gels were stained with ethidium bromide (EB), and the gels were rinsed with a solution of CFN or PBS to bleach the DNA band's fluorescence.


RESULTS
Rinsing the PI or 7-AAD-stained cells with solutions of CFN led to nearly complete removal of PI and a more than 75% decrease in 7-AAD fluorescence after 10 min. The rinse with PBS decreased the PI cell fluorescence intensity by less than 30% and the 7-AAD fluorescence by about 50%. The differences in kinetics of PI or 7-AAD removal by CFN from G2/M versus G1 cells suggest that these intercalators bind more strongly to DNA in chromatin of G2/M than G1 cells. The CFN-bleached cells were then successfully stained with S101 and again with PI or 7-AAD. The bivariate analysis of the LSC merged files of the cells sequentially stained with PI and S101 revealed typical DNA/protein distributions. The fluorescence of EB-stained DNA bands in gels was also nearly completely removed by rinsing gels in 0.05 M CFN; PBS alone had a distinctly lesser effect.


CONCLUSIONS
Solutions of CFN can dissociate the DNA-bound PI, 7-AAD, EB, and possibly other intercalating fluorochromes. The bleached cells can be restained and reanalyzed by LSC. This approach can also be used to remove such fluorochromes from nucleic acids immobilized in gels and perhaps in other solid matrices. Analysis of the kinetics of fluorochrome removal from cells can possibly be used to study their binding affinities to nucleic acids in situ.
Bedner
E
E
Brander Cancer Research Institute, New York Medical College, Valhalla, New York, USA.
Du
L
L
Traganos
F
F
Darzynkiewicz
Z
Z
D002110:Caffeine / Q000494:pharmacology*; D004247:DNA / Q000032:analysis* / Q000378:metabolism*; D003609:Dactinomycin / Q000031:analogs & derivatives / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D005434:Flow Cytometry; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism*; D018922:HL-60 Cells; D006801:Humans; D007364:Intercalating Agents / Q000378:metabolism*; D007700:Kinetics; D007834:Lasers; D008956:Models, Chemical; D011419:Propidium / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*
D005456:Fluorescent Dyes; D007364:Intercalating Agents; D003609:Dactinomycin; D011419:Propidium; D002110:Caffeine; C025942:7-aminoactinomycin D; D004247:DNA