Spatial Distribution Analysis of AT- and GC-rich Regions in Nuclei Using Corrected Fluorescence Resonance Energy Transfer

Abstract

(JRL) S U M M A R Y We employed microscopic intensity-based fluorescence resonance energy transfer (FRET) images with correction by donor and acceptor concentrations to obtain unbiased maps of spatial distribution of the AT-and GC-rich DNA regions in nuclei. FRET images of 137 bovine aortic endothelial cells stained by the AT-specific donor Hoechst 33258 and the GC-specific acceptor 7-aminoactinomycin D were acquired and corrected for the donor and acceptor concentrations by the Gordon's method based on the three fluorescence filter sets. The corrected FRET images were quantitatively analyzed by texture analysis to correlate the spatial distribution of the AT-and GC-rich DNA regions with different phases of the cell cycle. Both visual observation and quantitative texture analysis revealed an increased number and size of the low FRET efficiency centers for cells in the G 2 /Mphases, compared to the G 1 -phase cells. We have detected cell cycle-dependent changes of the spatial organization and separation of the AT-and GC-rich DNA regions. Using the corrected FRET (cFRET) technique, we were able to detect early DNA separation stages in late interphase nuclei. C ytological diagnosis of human tumors has recently become a more important clinical examination to differentiate between malignant and benign lesions. Chromatin patterns of tumor cell nuclei are among the most important information sources for cytological diagnosis. Previously we have reported a medical diagnosis based on the investigation of the chromatin structure using fluorescent DNA staining (Ashihara et al. 1986;Murata et al. 1988Murata et al. ,1990. A similar method using fluorescent DNA staining has been reported for DNA structure and cell cycle analysis, and the medical diagnosis (Latt 1974;Rousselle et al. 1999). The majority of these microscopic fluorescence studies have used the steady-state fluorescence approach with a single probe. The structural and topological changes of the DNA were interpreted in terms of changes in fluorescence intensity, which is known to be related to local DNA concentration (Bruno et al. 1991;Urata et al. 1991;Colomb and Martin 1992;Santisteban and Brugal 1995). A few reports have focused on the DNA structure in interphase nuclei, where additional information content of the fluorescence resonance energy transfer (FRET) by steady-state fluorescence measurement was exploited (Bottiroli et al. 1989;Prosperi et al. 1994;Szollosi et al. 2002).FRET is a non-radiative transfer of the excited-state energy from the initially excited donor to an excitable acceptor (Forster 1948;Stryer and Haugland 1967;Stryer 1978;Jovin and Arndt-Jovin 1989;Clegg 1992;Lakowicz 1999;Murata et al. 2000b). Because the efficiency of FRET depends on the sixth power of the distance between the donor and the acceptor, the microscopic FRET is an excellent ruler for quantification of distances on the molecular scale in cells (Jovin and Arndt-Jovin 1989).Recently we have applied fluorescence lifetime imaging microscopy (FLIM) for FRET measurements be-

Expression of aquaporin 3 (AQP3) in normal and neoplastic lung tissues

“…The method was basically as described previously [21]. Briefly, rabbit anti-AQP3 antibody (1:500 dilution) was used as a primary antibody.…”

Fluorescence lifetime imaging in biosciences: technologies and applications

“…The applications of FLIM in the biosciences are manifold and reach from monitoring cellular parameters (Ca 2+ 55-57 and Na + concentrations 58 , pH [59][60][61][62][63][64] , pO 2 and ROS concentrations [65][66][67] , pCO 2 68 ) and the distances between proteins on the nm-scale by means of FRET 5,35,42,[46][47][48][68][69][70][71][72][73][74][75][76][77][78][79][80][81] to observing central cellular processes in real time, e.g. interactions between proteins in living cells 48,[82][83][84][85][86] , photophysics of complexes involved in plant photosynthesis 87 or the redox metabolism 38,50,[88][89][90] .…”

Application of microscopic Förster resonance energy transfer to cytological diagnosis of the thyroid tumors

“…The donor-acceptor pair exhibited a good spectral match for the FRET experiments. 19,26 Ho and 7-AAD were obtained from the Molecular Probes ͑Eugene, OR͒. Other experimental details are described elsewhere.…”

“…The three filter sets were: ͑1͒ The donor filter set ͑D͒ consisting of the 360-370 nm excitation filter, the 400 nm dichroic mirror, and the 420-460 nm emission filter; ͑2͒ the acceptor filter set ͑A͒ consisting of the 530-550 nm excitation filter, the 590 nm dichroic mirror, and the 590 nm long-pass emission filter; ͑3͒ the FRET filter set ͑F͒ consisting of the 360-370 nm excitation filter, the 400 nm dichroic mirror, and the 590 nm long pass emission filter. 26,29 We acquired three fluorescence intensity images with the three filter sets in order to calculate the corrected energy transfer signal represented as the ϪI cFRET image of each cell…”

“…Therefore measurements of the distance change between segments of DNA molecules related to the structural and topological changes of the DNA are possible. 19,20,25,26 In this paper, we present a novel medical application of our previous microscopic FRET work to the clinical cytological diagnosis of thyroid tumor cells. 19,20,24,26 We describe in more detail difference in the DNA organization between the papillary carcinoma and the follicular lesions of the thyroid by means of the microscopic FRET measurement between the AT-specific dye Hoechst 33258 ͑Ho͒ and the GC-specific dye 7-aminoactinomycin D ͑7-AAD͒.…”