Journal of Histochemistry and Cytochemistry, Vol. 51, 951-958, July 2003, Copyright © 2003, The Histochemical Society, Inc.


ARTICLE

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

Shin-ichi Murataa, Petr Hermanb, Kunio Mochizukia, Tadao Nakazawaa, Tetsuo Kondoa, Nobuki Nakamuraa, Joseph R. Lakowiczc, and Ryohei Katoha
a Department of Pathology, University of Yamanashi Medical University School of Medicine, Yamanishi, Japan
b Institute of Physics, Charles University, Prague, Czech Republic
c Center for Fluorescence Spectroscopy, Department of Biochemistry and Molecular Biology, University of Maryland at Baltimore School of Medicine, Baltimore, Maryland

Correspondence to: Shin-ichi Murata, Dept. of Pathology, U. of Yamanashi School of Medicine, 1110 Shimokato, Tamaho-cho Nakakoma-gun, Yamanashi, Japan, 409-3898. E-mail: smurata@res.yamanashi-med.ac.jp


  Summary
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Materials and Methods
Results
Discussion
Literature Cited

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 G2/M-phases, compared to the G1-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. (J Histochem Cytochem 51:951–958, 2003)

Key Words: corrected FRET, fluorescence resonance energy, transfer, texture analysis, DNA, Hoechst 33258, 7-aminoactinomycin D, cell cycle


  Introduction
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Introduction
Materials and Methods
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CYTOLOGICAL 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. 1988 , Murata 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 ; Murata 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 between the AT-specific dye Hoechst 33258 (Ho) and the GC-specific dye 7-aminoactinomycin D (7-AAD) bound in cell nuclei (Murata et al. 2000a , Murata et al. 2001b ). We have studied the spatial relation between regions of the AT- and GC-rich DNA (Murata et al. 2000a , Murata et al. 2001a , Murata et al. 2001b ). The fluorescence lifetime measurements of the donor in the presence of the acceptor revealed a higher FRET efficiency in the AT-rich DNA regions compared to the GC-rich regions (Murata et al. 2000a ). We concluded that the spatial heterogeneity of the FRET efficiency was caused by FRET between closely spaced DNA segments in the three-dimensionally condensed regions of DNA rather than to a linear energy transfer between donors and acceptors along a single helix.

The efficiency of FRET in the donor–acceptor system with multiple acceptors is known to depend on the acceptor concentration. Although sensitive to FRET, donor fluorescence lifetimes are independent of the local donor concentration (Lakowicz et al. 1992 ; Murata et al. 2000a , Murata et al. 2000b ). The same is not true for the acceptor concentration, which affects the FRET efficiency and consequently the donor lifetimes. The same FRET efficiency can be caused by the transfer from a donor to a single acceptor located within a certain distance or to a number of acceptors located at a greater distance. Therefore, a correction for the local acceptor concentration has to be performed in situations when a concentration-unbiased donor–acceptor distance is an object of interest (Lakowicz 1999 ; Murata et al. 2000b ). Several reports showed that the correction methods with a three-filter set for steady-state fluorescence microscopy could account for this effect, and it proved to be a good approach for accurate FRET efficiency and distance measurements in systems with multiple acceptors (Szollosi et al. 1987 ; Gordon et al. 1998 ). Fortunately, we have built a steady-state fluorescence microscopic system using a three-filter correction method published by Gordon et al. 1998 .

In this study we decided to extend our previous FLIM work and describe in more detail cell cycle-related changes of the DNA organization, while taking into account highly variable donor and acceptor concentrations in the nucleus. We have measured energy transfer between Ho and 7-AAD by steady-state fluorescence microscopy, and differences of the FRET signal before and after the Gordon's correction method were compared. Images of the cell cycle-dependent FRET signal were classified by texture analysis (Haralick et al. 1973 ; Haralick 1992 ), and the results were interpreted in terms of the spatial proximity of the AT- and GC-rich DNA regions.


  Materials and Methods
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Materials and Methods
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Cells and Fluorescence Staining
Bovine aortic endothelial cells were grown at 37C in 35-mm Falcon dishes (Becton Dickinson; Franklin Lakes, NJ) containing DME with 10% FCS. The unsynchronized cell colonies in the dishes were fixed in 70% ethanol (4C, at least 30 min). After a rinse, the cells were stained with 0.8 µM Ho and 1.6 µM 7-AAD, which were used as the donor and the acceptor, respectively. The concentrations of donor and acceptor were determined to be able to measure significant changes in FRET signal. The concentration of 7-AAD typically resulted in about a twofold decrease in the Ho fluorescence intensity. All experiments were carried out at room temperature in 10 mM Tris-HCl buffer, pH 7.5, containing 100 mM NaCl (Murata et al. 2000a , Murata et al. 2001b ). Ho and 7-AAD were obtained from Molecular Probes (Eugene, OR).

Image Acquisition
Images of the stained cell cultures were acquired using an epi-illumination fluorescence microscope BX50 (Olympus; Tokyo, Japan) with an UplanApo x40 oil immersion objective (NA 1.0). The DNA fluorescence was recorded by a scientific-grade cooled CCD camera, Sensys (Photometrics; Tucson, AZ) connected to a PC. The images were pre-processed by IPLab software (Scanalytics; Fairfax, VA). The illumination level of the sample was adjusted by neutral density filters to the level that the image acquisition time was in the order of seconds. No detectable photobleaching was observed under these conditions.

Fluorescence Intensity and FRET Measurements
Fluorescence intensity images of 137 cells on a single dish, double-stained by Ho and 7-AAD, were measured. Three different filter sets were used for the FRET measurement: (a) the donor filter set consisting of a 360–370-nm excitation filter, a 400-nm dichroic mirror, and a 420–460-nm emission filter; (b) the acceptor filter set consisting of a 530–550-nm excitation filter, a 590-nm dichroic mirror, and a 590-nm longpass emission filter; and (c) the FRET filter set consisting of a 360–370-nm excitation filter, a 400-nm dichroic mirror, and a 590-nm longpass emission filter. With a complete absence of non-FRET signals, a simple FRET measurement can be done by observing the acceptor emission while exciting the donor. Since the non-FRET signals cannot be completely eliminated, the FRET signal IFRET can be calculated from the following equation (Gordon et al. 1998 ):

where F(da) is a fluorescence signal from the cell in the presence of both donor and acceptor collected with the FRET filter set, D(da) is a signal in the presence of both donor and acceptor collected with the donor filter set, A(da) is a signal in the presence of both donor and acceptor using the acceptor filter set, F(d) is a signal from a donor-only stained cell using the FRET filter set, D(d) is a signal from a donor-only stained cell with the donor filter set, F(a) is a signal from the acceptor-only stained cell acquired with the FRET filter set, and A(a) is a signal from the acceptor-only stained cell using the acceptor filter set. The constant ratios F(d)/D(d) and F(a)/A(a) depend on the fluorophores and on the instrumentation.

The fluorescence intensity IFRET calculated from Eq. 1 is subtracted from the non-FRET signals but is not corrected for the concentration of the donor and acceptor. Corrected FRET signal IcFRET, where the bias caused by the concentration of donor and acceptor as well as by the fluorescence crosstalk is removed, can be calculated from the following equation (Gordon et al. 1998 ):

The constant factor G relates a FRET-induced loss of the donor emission intensity observed with the donor filter set to the gain of the acceptor fluorescence intensity observed with the FRET filter set (Gordon et al. 1998 ). We have assumed G=10-6 in this study, to transform pixel values of the IcFRET images into the 12-bit region. We have used F(d)/D(d)=0.34 and F(a)/A(a)=0.03. The ratios were measured for the donor-only- and the acceptor-only-stained cell, respectively.

Because the donor cannot be excited with the acceptor filter set, the overall signal from the double-stained nucleus contained fluorescence from the acceptor only and was related to the amount of the DNA in the nucleus. With the acceptor filter set we measured the total acceptor fluorescence from 137 double-stained cells, evaluated the amount of DNA in the nucleus, and constructed DNA histograms for the cell cycle analysis (Ashihara et al. 1986 ; Murata et al. 2000a , Murata et al. 2001a , Murata et al. 2001b ). Since we worked with unsynchronized cell colonies, cells from our ensemble were randomly distributed along the cell cycle.

Texture Analysis of Co-occurrence Matrices
The high-dimensional texture analysis of the co-occurrence matrices is a classical method sensitive enough to detect subtle changes in the DNA organization (Murata et al. 1993b ; Murata et al. 2001a ). The co-occurrence matrix was constructed and analyzed for each corrected FRET signal (IcFRET) image after reduction of the bit depth from 12 to 8 bits. Haralick described 14 parameters that quantify various texture patterns (Haralick et al. 1973 ; Haralick 1992 ). Haralick's parameters are classical methodologies, but they are more sensitive in detecting very subtle spatial changes of texture images than one-dimensional texture analysis. We have selected three parameters from the Haralick's parameter set as mathematically independent descriptors of our IcFRET images. Specifically, we have chosen the angular second moment (ASM), which shows spatial homogeneity of the image, the sum variance (SVar), which reveals spatial heterogeneity, and the difference variance (DVar), which characterizes contrast of the IcFRET images. These three Haralick's parameters are also independent of the size and shape of the cells, and can analyze pure texture feature of the images (Murata et al. 2002 ). Equations for these parameters have been described by Murata et al. 2001a , Murata et al. 2001b .


  Results
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Our donor–acceptor pair exhibits a good spectral match for the FRET experiments. Spectral overlap of the absorption and the emission spectra of the Ho (the AT-specific dye) and 7-AAD (the GC-specific dye), respectively, is shown in Fig 1. Using this pair of the fluorochromes, we examined FRET in the nuclei of 137 bovine aortal endothelial cells. The nuclear images typically displayed spatial heterogeneity of the fluorescence intensity with a number of brighter spots (Fig 2A and Fig 2B). We found differences in the distribution and size of the bright spots between the Ho-stained and the 7-AAD-stained cells. As previously found, the Ho-stained images exhibited higher heterogeneity and a greater number of bright spots compared to the images of the 7-AAD-stained cells (Murata et al. 2000a ). We attributed the effect to a different distribution of the AT- and GC-rich regions of the DNA in different cells and to local variability of the DNA concentration.



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Figure 1. Fluorescence emission (Fluo) and absorption (Abs) spectra of DNA-bound Ho and 7-AAD. The spectra of the dyes were measured in the presence of calf thymus DNA without sonication in 10 mM Tris-HCl buffer, pH 7.5, containing 100 mM NaCl. The DNA concentration was 200 µM (base pair: bp), while the concentration of Ho was 4 µM and the concentration of 7-AAD was 60 µM. Emission spectra were excited at the respective absorption maxim. The marked region represents the spectral overlap.



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Figure 2. Fluorescence intensity and FRET images of Ho and 7-AAD. (A) A fluorescence intensity image of Ho in the absence of 7-AAD taken with the donor filter set. (B) A fluorescence intensity image of Ho in the presence of 7-AAD taken with the acceptor filter set. Since with the acceptor filter set Ho was not excited, the signal from the nucleus contains only fluorescence from 7-AAD. (C) Uncorrected FRET image calculated from Eq. 1. (D) IcFRET image calculated from Eq. 2.

Fig 2C and Fig 2D show the FRET images before and after the three-filter-set correction, respectively. Eq. 1 separates the FRET signal from the non-FRET signals, but is not normalized for the concentration of the donor and acceptor. On the other hand, the corrected FRET image (IcFRET image) calculated from Eq. 2 was corrected for both non-FRET signal and the donor and acceptor concentrations. As expected, different patterns were observed before and after the correction. Both FRET images from Fig 2C and Fig 2D exhibited considerable spatial heterogeneity. Regions of high FRET signal in the uncorrected FRET image were correlated with regions of low values in the corrected FRET image. These apparently opposite results strongly stress the need for concentration corrections, since high local concentration of the donor–acceptor pairs can completely offset and even dilute the loss of the intensity-based FRET signal. Interestingly, the lowest corrected energy transfer was found in regions with the highest DNA concentration (Fig 2A, Fig 2B, and Fig 2D), indicating higher donor–acceptor distances and consequently larger separation of AT- and GC-rich DNA segments in these locations.

Fig 3A shows a histogram of the integrated fluorescence intensities collected from the nuclei of 137 cells. Because the integrated intensity is proportional to the DNA content, we could assign to each cell a position within the cell cycle. Having the histogram from Fig 3A, we compared the IcFRET images of cells from different stages. Fig 3B–3Q show 16 representative IcFRET images of cells in the G1- and S-G2/M-phases. Regions with a low FRET signal are shown as light and dark blue spots. Visually, the size of the blue regions appeared to be larger in the G2/M-phases compared to the G1-phase. The value of the IcFRET was lower in the G2/M-phases than in the G1-phase, as shown by a comparison of the mean IcFRET values calculated from whole nuclear areas (Fig 4). As seen in Fig 4, cells in the G2/M-phases exhibited about 40% lower cFRET signal than cells in the G1-phase.



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Figure 3. (A) DNA histogram was calculated from the fluorescence images of the 137 nuclei stained with Ho. IcFRET images of the nuclei in G1-phase (B–I), S-phase (J–M), and G2/M-phases (N–Q) were calculated from fluorescence intensity images using Eq. 2. Pixel values of the background were set to 0. To observe FRET signal heterogeneity, the images are normalized to optimal average value of the pixels.



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Figure 4. Cell cycle-dependent scattergram of the mean value of the cFRET signal (IcFRET) for the set of 137 nuclei. Each data point represents one nucleus double stained by Ho and 7-AAD. The mean IcFRET values were calculated as the overall IcFRET signal divided by the nuclear area. The cell cycle analysis was based on the total fluorescence intensity of the 7-AAD collected from the nucleus with the acceptor filter set.

To confirm the observation demonstrated in Fig 3, we employed methods of the texture analysis (Murata et al. 1993a , Murata et al. 1993b , Murata et al. 2000a , Murata et al. 2000a , Murata et al. 2000b ). Fig 5 shows cell cycle-dependent statistical analysis of the co-occurrence matrices associated with the IcFRET images of all 137 cells in our ensemble. Fig 5 reveals that the ASM (homogeneity) value is higher for nuclei in the G2/M-phases compared to the nuclei in the G1-phase. The nuclei in the G2/M-phases exhibited comparatively lower values of SVar (heterogeneity) and DVar (contrast) than nuclei in the G1-phase. The result suggests more homogeneous spatial distribution of the cFRET intensity (IcFRET) in the G2/M-phases. The higher homogeneity in the G2/M-phases arises from the increased size and number of the low FRET efficiency spots.



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Figure 5. Scattergrams of the texture parameters calculated from the IcFRET images of 137 cells. Closed circles represent individual cells.


  Discussion
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As reported by number of groups, the fluorescence intensity images provide informative data on the DNA condensation in nuclei (Bruno et al. 1991 ; Murata 1991 ; Colomb and Martin 1992 ; Santisteban and Brugal 1995 ; Rousselle et al. 1999 ). However, analysis of the fluorescence intensity only, without evidence from FRET experiments, can hardly reveal changes of the DNA organization, that occur on the scale below the diffraction limit of the microscope. Such information can be obtained by the FRET technique, which exhibits high sensitivity for subtle changes of the distance between the donor and acceptor. With the hope for a possible clinical application to obtain informative data of the DNA organization for cytological diagnosis, we used ethanol fixation, which is usually used for the Papanicolaou stain in medical cytological examination.

FRET in this study was measured between dyes bound to the double-helical DNA. In this case, multiple acceptors are distributed around donors on DNA, and the extent of FRET depends on donor and acceptor concentrations (Murata et al. 2000b ). We used Gordon's equation for correction of the dependence of FRET on the concentrations of the donor and acceptor (Gordon et al. 1998 ). We need to consider that the corrected FRET by Gordon's equation for donor concentration is not equal to or proportional to the exact value of FRET because the donor fluorescence intensity, which is used for the estimation of the donor concentration in the equation, is quenched by FRET. This quenching may vary considerably depending on distances between donors and acceptors. Gordon's equation is accurate only in approximation if the energy transfer efficiency of individual donors is small. However, the corrected FRET is a relative measure of the exact value of FRET as discussed by Gordon et al. 1998 . For the FRET measurement between DNA dyes on interphase cell nuclei, the corrected FRET by Gordon's equation makes the best use of the data collected using the three filter sets. Furthermore, we do not intend to quantitatively report any distances in angstroms. Our results are dealing with the DNA topology on the qualitative level. We used this method as a simple approach from practical purposes because the simplification does not change our conclusion.

Previously we demonstrated that addition of 7-AAD to Ho-stained nuclei results in a shortening of the donor fluorescence lifetime (Murata et al. 2000a , Murata et al. 2001b ), which proves the presence of the FRET phenomenon as a consequence of the spatial proximity of Ho and 7-AAD. Despite the same staining conditions for all cells on the dish, both donor and acceptor concentrations in every nucleus exhibit a spatial heterogeneity resulting from a non-uniform AT and GC base-pair distribution, respectively (Murata et al. 2001a ). The high local AT and GC concentrations, observed as bright spots, could be caused either by the presence of AT- and GC-rich segments of DNA, respectively, or it could reflect higher base-pair concentrations caused by local condensation of the nonspecific DNA. Since we have found differences between spatial distribution of AT and GC concentrations, as shown in Fig 2, local variation of the donor:acceptor ratio should exist in the nucleus. This prevented us from simply interpreting FRET-induced changes of the donor lifetime in terms of the donor–acceptor distance (Lakowicz et al. 1992 ; Murata et al. 2000a , Murata et al. 2000b ). We believed that local variation of the donor:acceptor ratio in nuclei was the cause for the uncorrected IFRET and the corrected IcFRET images to produce opposite results in this study. That is, the uncorrected IFRET images were influenced by the concentration of donor with FRET and by a variation of the distance between donors and acceptors. For example, the high concentration regions of donor with low FRET showed more loss of the donor signal than the low concentration regions of donor with high FRET. On the other hand, the IcFRET imaging, corrected for the influence of donor and acceptor concentrations, revealed the FRET efficiency to be affected only by a variation of the distance of AT- and GC-rich DNA in nuclei.

During the cell cycle, three-dimensional distribution of chromosomes changes in nuclei. Finally, the AT- and GC-rich DNA regions of each chromosome separate in the metaphase (Alberts et al. 1994 ). Studies with smeared metaphase chromosomes reported the low FRET efficiency in the AT-rich bands (Latt 1974 ; Latt et al. 1979 ). On the basis of our data, we propose that the lower overall FRET efficiency measured on the cells in the G2/M-phases represents an early detection of the separation process, which starts in late interphase. Our conclusion, supported by the statistical analysis, is illustrated on the representative cells shown in Fig 3. Regions with the low FRET efficiency are clearly larger in the G2/M-phases (Fig 3N–3Q), than analogous spots in the G1-phase (Fig 3B–3I). The quantitative texture analysis confirmed observed differences of the FRET efficiency in different stages of the cell cycle. The higher homogeneity in the G2/M-phases was shown by ASM and SVar, and the lower contrast was revealed by DVar. Based on the mathematical meaning of these three parameters, cells in the G2/M-phases exhibit higher spatial uniformity of the FRET efficiency, which arises from the increased size and number of the low FRET efficiency spots. We interpret these spots as separated regions of the AT- and GC-rich DNA.

In conclusion, we have demonstrated that corrected FRET imaging with texture analysis is a useful tool for analysis of the intracellular DNA organization and gives information inaccessible by non-FRET steady-state fluorescence microscopy. The corrected FRET technique can be applied as a helpful tool that will give us additional information about the chromatin pattern for medical cytological diagnosis of human tumors.


  Acknowledgments

Supported by the NIH, National Center for Research Resources, RR-08119.

We thank our colleagues Dr. T. Yamane and Dr. A. Ooi for the preparation of cell cultures and for the technical support of the microscopic and imaging equipment.

Received for publication August 12, 2002; accepted February 4, 2003.


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Introduction
Materials and Methods
Results
Discussion
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