ARTICLE |
Correspondence to: Juan C. Stockert, Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, Cantoblanco, E-28049 Madrid, España.
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Summary |
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We describe the fluorescence properties and cytochemical applications of the aromatic diamidine M&B 938. Treatment of cell smears (chicken blood, Ehrlich ascites tumor, rat bone marrow, mouse mast cells, and Trypanosoma cruzi epimastigotes) with aqueous solutions of M&B 938 (0.5-1 µg/ml at pH 6-7; UV excitation) induced bright bluish-white fluorescence in DNA-containing structures (interphase and mitotic chromatin, AT-rich kinetoplast DNA of T. cruzi ), which was abolished by previous DNA extraction. DNA was the unique fluorescent polyanion after staining with M&B 938 at neutral or alkaline pH, other polyanions such as RNA and heparin showing no emission. M&B 938-stained mouse metaphase chromosomes revealed high fluorescence of the AT-rich centromeric heterochromatin, and strong emission of heterochromatin in human chromosomes 1, 9, 15, 16, and Y was found after distamycin A counterstaining. On agarose gel electrophoresis, M&B 938-stained DNA markers appeared as fluorescent bands. The 1.635-KBP fragment from DNA ladder revealed a higher emission value than that expected from linear regression analysis. Spectroscopic studies showed bathochromic and hyperchromic shifts in the absorption spectrum of M&B 938 complexed with DNA, as well as strong enhancement of fluorescence at 420 nm. In the presence of poly(dA)-poly(dT), the emission of M&B 938 was 4.25-fold higher than with DNA; no fluorescence was observed with poly(dG)-poly(dC). Experimental results and considerations of the chemical structure suggest that the minor groove of AT regions of DNA could be the specific binding site for M&B 938, which shows interesting properties and useful applications as a new DNA fluorochrome. (J Histochem Cytochem 45:97-105, 1997)
Key Words: Diamidines, Cationic fluorochromes, Fluorescence microscopy, DNA cytochemistry, Cell polyanions, DNA minor groove
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Introduction |
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In the past few years there has been renewed interest in the design and development of aromatic diamidino compounds that exhibit specific DNA-binding modes as well as important biological and therapeutic applications (
It is well known that aromatic diamidines specifically bind to adenine-thymine (AT) sequences of DNA by a non-intercalative mechanism (
Interestingly, some DNA-binding aromatic diamidines such as berenil, 2-hydroxystilbamidine, and DAPI also exhibit striking fluorescence properties (
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At present, non-rigid DNA fluorochromes such as DAPI and the bisbenzimidazole Hoechst 33258 (both synthesized as trypanocidal drugs) are widely used in cytochemistry, cytogenetics and cell biology (
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Material and Methods |
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Smears of chicken and horse blood, Ehrlich ascites tumor cells, rat bone marrow, mast cells obtained from the peritoneal cavity of Balb/c mice (
For metaphase preparations, human lymphocytes were grown at 37°C for 3 days in RPMI 1640 medium (Sigma) supplemented with fetal bovine serum (10%), l-glutamine, antibiotics, and phytohemagglutinin (Sigma) as usual. To arrest cells at metaphase, colcemid (Sigma; final concentration 0.05 µg/ml) was added 3 hr before collection. Cells were centrifuged, resuspended in hypotonic solution (75 mM KCl) at 37°C for 5 min, fixed in methanol-acetic acid (3:1 v/v) for 1 hr, spread on slides, and air-dried. Bone marrow cells from mice previously injected with colcemid solution (0.01% in 0.9% NaCl) for 2 hr were subjected to the same hypotonic treatment, fixation, and spreading as described above.
The compound M&B 938 (4,4'-diamidinodiphenylamine dihydrochloride, batch 7451; Figure 1) was kindly provided by Rhône-Poulenc Rorer (Dagenham Research Centre, Essex, UK). A stock solution (1 mg/ml) was first made in distilled water (pH 6) and then diluted to the appropriate staining concentration (0.5-1 µg/ml) with distilled water. Stock solutions kept in the dark at -20°C for several months also proved to be suitable for the fluorescence reaction. Staining time with M&B 938 solutions at pH 6-7 was 5-15 min at room temperature (RT; 20°C).
Smears of bone marrow and peritoneal mast cells were also stained with 1 µg/ml solutions of M&B 938 adjusted to different pH values (from 1 to 9) with 0.1 N HCl or 0.1 N NaOH. All preparations were washed in distilled or tapwater, air-dried, and mounted either in distilled water, 20% or 50% glycerol (Merck), immersion oil (Zeiss; Oberkochen, Germany), or DePeX (Serva; Heidelberg, Germany). According to current methods to enhance fluorescent chromosome banding (
To confirm microscopic observations, standard 0.8% agarose gels in 45 mM Tris-borate-1 mM EDTA buffer at pH 8 were loaded with 2 µg of either DNA/Hind III fragments or 1 kbp DNA ladder marker (Bethesda Research Laboratories; Life Technologies, Gaithersburg, MD). After horizontal electrophoresis at 10 mA/cm for 30 min, gels were stained with 30 µg/ml M&B 938 in distilled water for 5 min at RT, washed in distilled water for 20-25 min, and then photographed using an Eagle Eye uv transilluminator (Stratagene; Cambridge, UK). Other DNA samples were run in the same way but incorporating 0.5 µg/ml of M&B 938 into the gel before electrophoresis. For comparative purposes, other gels were also stained with ethidium bromide (EB) as usual. Photographs of M&B 938-stained gels were subjected to densitometry using a color imaging scanner "La Cie" (model G 520 A; Seiko Epson, Tokyo, Japan) and the images were analyzed with the NIH image 1.55 f Macintosh software pack.
Spectroscopic studies were carried out by using freshly made solutions of M&B 938 (1 and 10 µg/ml), calf thymus DNA (Sigma), and the high molecular weight double-stranded homopolymers poly(dA)-poly(dT) and poly(dG)-poly(dC) (Boehringer, Mannheim, Germany; and Sigma). Solutions of DNA and synthetic polynucleotides (10 µg/ml) were made in distilled water to avoid the scattering contribution of ions and buffer co-solutes to the baseline of solvents when excited at uv wavelengths (
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Results |
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After treatment of cell smears with 0.5-1 µg/ml solutions of M&B 938 at pH 6, bright bluish-white fluorescence reaction was observed specifically in chromatin from interphase and mitotic cells under uv excitation. Violet and violet-blue excitations were not suitable because of their weaker emission and lower specificity. No appreciable difference was found between 5- and 15-min staining time. Compact chromatin masses in nuclei of chicken erythrocytes, Ehrlich tumor cells, and lymphocytes, as well as kinetoplasts of T. cruzi epimastigotes, showed the brightest fluorescence (Figure 2). No emission was found in other cell structures (e.g., heterophil, eosinophil and neutrophil leukocyte granules, erythrocyte cytoplasm, mast cell granules, and basophilic cytoplasm of lymphoblasts, lymphocytes, and Ehrlich tumor cells). Control (unstained) smears did not reveal appreciable fluorescence.
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DePeX was the optimal mounting medium for microscopic examination of cell smears stained with M&B 938. In this case, only a very low fading rate was observed, which permitted prolonged analysis as well as photography. Higher fading rates and diffusion artifacts were found with immersion oil or aqueous mounting media, respectively. Previous DNA extraction procedures (DNAse, TCA) abolished the chromatin fluorescence induced by M&B 938. A strong dependence of fluorescence intensity on the pH value was clearly observed. At pH 3, heparin-containing granules from mouse mast cells showed bright emission, whereas nuclei exhibited negligible fluorescence. On the contrary, bright nuclear emission in mast cells and lymphocytes and no fluorescence of mast cell granules occurred at pH 9. The effect of a gradual variation of pH on M&B 938 fluorescence of basophilic structures from rat bone marrow smears is shown in Figure 3, which reveals the striking specificity of M&B 938 for chromatin DNA at neutral and alkaline pH values, whereas no emission is detected in both basophil leukocyte granules and RNA-rich cytoplasm of lymphoblasts and lymphocytes.
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After M&B 938 staining, human metaphase chromosomes exhibited bright and rather homogeneous fluorescence (Figure 4a), the remaining cytoplasm showing no emission. In some cases, very weak G/Q banding and somewhat brighter centromeric regions could be detected. When counterstained with DA, human chromosomes revealed a typical fluorescence pattern, with the centromeric heterochromatin of chromosome pairs 1, 9, 15, and 16, and the chromosome Y appearing highly fluorescent (Figure 4b). The same results were obtained when AMD preceded M&B 938 staining. Clearly differentiated centromeric regions could be also observed in mouse chromosomes after staining with M&B 938 (Figure 4c). Centromeric heterochromatin of most chromosomes appeared brighter than the arms, in which weak G/Q banding was found in some cases. DA counterstaining or AMD pretreatment did not improve the fluorescence pattern induced by M&B 938 on mouse chromosomes.
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After agarose gel electrophoresis, M&B 938 clearly revealed the DNA markers as fluorescent bands (Figure 5a). Although previous incorporation of M&B 938 into the gel was also suitable, optimal results were obtained when gels were poststained with the compound. In this case, a higher concentration was necessary to achieve adequate penetration of M&B 938 into the bulky agarose matrix. Likewise, thorough washing in distilled water was required to produce the progressive loss of background gel emission and the precise visualization of DNA bands. After densitometric analysis, plotting the molecular length of 1 kbp DNA ladder fragments as a function of their fluorescence intensity revealed a point well above the best-fit regression line (Figure 5b). Preliminary computer analysis of the base sequences of the corresponding DNA ladder fragments showed that the band of 1.635 kbp that appears above the regression line contains a higher amount of AT clusters (e.g., four or five consecutive AT base pairs) than other DNA fragments. On the contrary, EB-stained gels showed a more regular fluorescence pattern of DNA bands.
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To analyze the emission characteristics of M&B 938 and to confirm its microscopic fluorescence reaction with DNA-containing structures, spectral studies were performed in vitro. A 10 µg/ml solution of the free compound revealed absorption peaks at 215 and 345 nm, with a shoulder at 270-290 nm; the emission maximum was found at 430 nm (Figure 6). Optimal exciting wavelength was 340 nm, although excitation in the range 340-370 was also suitable. In the presence of 10 µg/ml DNA, the near uv absorption peak of M&B 938 (1 µg/ml) showed bathochromic (345-358 nm) and hyperchromic shifts (Figure 7). Under instrumental conditions of very low sensitivity, strong emission at 420 nm was found for M&B 938 in the presence of DNA, whereas the free compound showed no fluorescence (Figure 7). In the presence of the synthetic polynucleotide poly(dA)-poly(dT), the fluorescence of M&B 938 strikingly increased and shifted from 430 to 415 nm, the emission intensity showing a value 4.25-fold higher than that observed with DNA (Figure 8). At the very low sensitivity level used for these comparative studies, no fluorescence was detected for either M&B 938 alone or in the presence of poly(dG)-poly(dC).
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Discussion |
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With the exception of DAPI, the use of aromatic diamidines has largely been overlooked in DNA cytochemistry, and only very few observations on the microscopic fluorescence induced by berenil (
In this work we show that the aromatic diamidine M&B 938 induces a bright fluorescence emission of chromatin that depends on the presence of DNA. The staining method is very simple and rapid, low fluorochrome concentration is required, and high specificity and stability of the fluorescence reaction can be easily achieved. Results from microscopic observation, DNA extraction, gel electrophoresis, and spectrofluorimetric analysis confirm the specific binding of M&B 938 to DNA. Interestingly, DNA was the unique cell polyanion that proved to be fluorescent at neutral or alkaline pH values. Under these conditions, other polyanions such as RNA (basophilic cytoplasm) and sulfated glycosaminoglycans (heparin-containing granules in mast cells and basophil leukocytes) revealed no fluorescence.
This advantage is not commonly found in fluorescence reactions induced by other non-rigid cationic fluorochromes which, in addition to chromatin DNA, also reveal different polyanionic and/or hydrophobic substrates. In fact, simultaneous orthochromatic (blue-white) and metachromatic (yellow-green) emission of nuclei and mast cell granules, respectively, has been described after staining with DAPI (
In addition to compact chromatin of interphase nuclei, kinetoplast DNA of T. cruzi and centromeric heterochromatin of mouse chromosomes reveal very bright M&B 938 fluorescence, possibly related to the abundance of AT sequences or AT clusters [e.g., oligo(dA)-oligo(dT) tracts] in these structures (
Characteristic spectral changes are observed when cationic dyes bind to polyanionic substrates. In the case of M&B 938, bathochromic and hyperchromic shifts in the absorption spectrum and strong enhancement of fluorescence occur in the presence of DNA. It is known that binding or solvent conditions that increase the molecular rigidity of non-planar (flexible) fluorophores result in a great enhancement of fluorescence (
Inspection of the chemical structure of M&B 938 reveals that its molecular geometry is well suited to interact with the DNA minor groove. As illustrated in Figure 1 and Figure 9a, M&B 938 has two cationic amidinium groups with hydrogen bonding capacity and shows a crescent-shaped and non-planar structure. The compound is the shortest-chain homologue of berenil, the triazene bridge (-NH-N=N-) of the latter being replaced by an amino group (-NH-). A torsion angle of about 45° between phenyl rings is necessary to avoid the steric clash that would be produced by the proximity of the C2 and C2' hydrogens. Although the central amino bridge is rather flexible (allowing rotation and some opening of the C1-N-C1' bond angle), the normal non-planar conformation of M&B 938 must prevent its intercalation between base pairs. This interpretation is also supported by the fact that no fluorescence enhancement of M&B 938 is found in the presence of poly(dG)-poly(dC). On the contrary, DAPI and other non-rigid (unfused) aromatic compounds have a more planar structure and they can also intercalate between GC base pairs of DNA and RNA duplexes (
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The diamidino compound used in this work shares some chemical features with AT-specific minor groove binders, such as the bowed shape, cationic status, hydrogen bonding possibilities, and non-rigid bonds between aromatic rings (
Studies of the selective interaction of drugs and dyes with DNA minor groove have taken on a new significance in recent years (
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Acknowledgments |
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This work was supported by grant PM95-0027 awarded by the Dirección General de Investigación Científica y Técnica, Spain.
We are indebted to Rhône-Poulenc Rorer (Dagenham Research Centre, Essex, UK) for providing the compound M&B 938. The valuable collaboration of J. Espada, O. Fernández-Cantero, G.E. Bertolesi, and M. Fresno is also acknowledged. JCS is a member of the Scientific Career (Consejo Superior Investigaciones Científicas), Spain.
Received for publication March 19, 1996; accepted August 15, 1996.
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