ARTICLE |
Correspondence to: Claude E. Gagna, Dept. of Pharmacology and Physiology, U. of Medicine and Dentistry of New Jersey-Medical School, Room H647, 185 S. Orange Ave., Newark, NJ 07103.
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Summary |
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We examined histochemically and immunohistochemically the distribution of B- and Z-DNA in the epithelium and terminally differentiating dog lens fiber cells. On the basis of anti-DNA antibody reactivity, qualitative and quantitative data on B- and Z-DNA in cells were determined. Anti-B-DNA immunoreactivity gradually declined throughout nucleated fibers, with a precipitous decrease at 90 µm. Anti-Z-DNA antibody binding decreased with a sudden loss of immunoreactivity at
90 µm. The pattern of anti-B- and Z-DNA staining correlates with the loss of
-crystallin immunoreactivity, the major lens crystallin, and decreased eosin staining of proteins. Germinative zone cell nuclei showed the highest DNA probe binding values, followed by the superficial fibers, central zone, middle fibers, and deep fibers. The presence of single-stranded (ss)DNA in deeper fibers was detected by anti-ss-DNA antibodies. This is indicative of DNA degradation. These observations suggest that a dramatic reorganization of lens fiber cells' supramolecular order occurs at
90 µm, the phase transition zone. (J Histochem Cytochem 45:1511-1521, 1997)
Key Words: B-DNA, Z-DNA, lens, immunohistochemistry, lens supramolecular order, terminal differentiation, crystallins
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Introduction |
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The refractive index gradient in all eye lenses is required to eliminate spherical aberration as light rays are focused on the retina. This gradient is created by an increasing concentration of the major lens proteins, the crystallins, as the elongated fiber cells are displaced inward. The avascular lens grows throughout life by continued division and differentiation of epithelial cells that cover its anterior surface. The addition of cell layers on the lens surface and the displacement of older fiber cell lamellae inward initiate the process of terminal differentiation. This is a specialized cell death that does not follow the classical route of apoptosis. However, it can be regarded as controlled or apoptotic in the kinetic sense (
The present study was designed to analyze the nuclear events relative to DNA content and conformation monitored by anti-B-DNA and anti-Z-DNA antibodies. Lens nuclear DNA content in single cells was measured by a new and innovative image analyzing system. DNA can exist in many different conformations, i.e., right-handed A- and B-DNA (
Previously, the presence of Z-DNA was demonstrated in the normal calf lens (
In this study, relative estimates of total DNA, and B- and Z-DNA were determined in epithelial cells and terminally differentiating fiber cell nuclei of the normal dog lens. These observations are correlated with immunohistochemistry of -crystallin and an eosin Y staining pattern. The process of DNA degradation was further defined by histochemical Feulgen reactions, histological stains, and immunohistochemical detection of single-stranded (ss-) DNA. These observations indicate that a drastic change in DNA content and structure correlates with altered intracellular protein supramolecular order, the phase transition zone (PTZ), at
90 µm.
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Materials and Methods |
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Histological Preparations of the Dog Lenses
Normal coonhound dog eyes (Adler Ridge Farms; Lakewood, PA) from 12 littermates, 1 year and 9 months, were provided by Mr. Tom M. Poandl (Department of Orthopedics, UMDNJ-Medical School, Newark, NJ). Dogs were housed, handled, and sacrificed by lethal injection that conforms to the practices established by the Institutional Animal Use Committee. After fixation, histological processing of the lens was performed as described previously (
Anti-DNA Polyclonal and Monoclonal Antibodies
The 2C10 anti-double-stranded (ds-) B-DNA IgG monoclonal antibody (MAb), which reacts with a wide range of BP, was provided by Dr. B.D. Stollar (Tufts University, Boston, MA) (
Immunohistochemistry of the Dog Lenses
Slides were deparaffinized and hydrated through graded alcohols and the lens tissue sections (2.5 µm) were stained with either the anti-DNA IgG polyclonal antibodies (10-100 µg/ml) or the anti-DNA IgG MAb (1-10 µg/ml) using the avidin-biotin-peroxidase Elite kit (Vector Laboratories; Burlingame, CA) (
Eosin Plasma Stain
Tissue sections of dog lenses were dipped into a solution of eosin Y plasma stain [200 ml of eosin Y (1% aqueous solution) (Polysciences), 600 ml of 95% ethyl alcohol, and 4 ml of glacial acetic acid (ultra pure grade reagents; Gallard-Schlesinger), pH 4.8, and then processed through 95% alcohol, absolute alcohol, xylene, and finally coverslipped.
Antibody-Antigen Competition Experiments
Validity of the antibody staining specificity for the anti- ds-B-DNA, anti-ds-Z-DNA, and anti-ss-DNA antibodies was determined by using right-handed ds-B-DNA, left-handed ds-Z-DNA and ss-DNA polynucleotides, and RNA competitors. For these studies the ss-DNA, calf liver RNA and poly[d(G-me5C)] were provided by Dr. John H. Chen. The polynucleotides, poly[d(G-C)], poly(G-T).poly(A-C), and poly(A-G).poly(C-T) were purchased from Pharmacia Biotech (Piscataway, NJ). The Z-DNA conformation was induced by adding polynucleotides to solutions containing high salt (3.8 M). To ensure full conversion, the solution was heated to 60C for 10 min. To stabilize the Z-DNA it was brominated by the addition of 5% saturated Br2 water and incubated for 3 min at RT. After this procedure the Z-DNA conformation persists even in low salt (150 mM NaCl). To determine the presence and to quantitate the Z-DNA, UV spectroscopy was employed at A295/A260. For Z-DNA this ratio should be between 0.33 and 0.38. Finally, the preparation was dialyzed overnight against 10 mM Tris, 1 mM EDTA and 50 mM NaCl. The quantitation of Z-DNA was then repeated (
Immunohistochemical Nuclease Digestion
The following procedures were used to determine the effects of ss nicks on B-DNA and Z-DNA immunostaining. Fixed dog lens tissue sections were incubated in a moisture chamber with DNase I, RNase Free (Boehringer Mannheim; Indianapolis, IN). Full DNase I digestion consisted of 0.5 mg/ml of DNase I in 50 mM Tris pH 7.5, 10 mM MgSO4, 0.1 mM dithiothreitol, 50 µg/ml bovine serum albumin (BSA) for 20 min to 1 hr at 20C (data not shown). The nicking digestion solution contained only 5 ng/ml of DNase I. Full DNase I digestion treatment either reduced or completely abolished anti-B-DNA antibody reactivity, depending on the enzyme concentration and exposure time. The nicking DNase I had essentially no effect on anti-B-DNA immunoreactivity. Both nicking and full digestion concentrations of DNase I enzyme treatment prevented anti-Z-DNA immunoreactivity. RNase ONE (Promega; Madison,WI) used at a concentration of 0.5 mg/ml in PBS for 1-6 hr at 37C, had no effect on either B- or Z-DNA immunoreactivity. This indicates that the anti-DNA antibodies were binding DNA and not RNA. After enzyme digestion, slides were washed with PBS and incubated separately with anti-DNA antibodies (
Topoisomerase I Enzyme Treatment
Enzyme (Promega) treatment was performed to examine the effects of negative supercoiling on B- and Z-DNA immunoreactivity. The use of topoisomerase I or DNase I, as mentioned above, prevents anti-Z-DNA antibody binding by inhibiting torsional strain in the DNA. Lens tissue sections were processed at 37C for 1-3 hr in 50 mM NaCl, 50 mM Tris-HCl, pH 8.0, 0.5 mM dithiothreitol, and 30 µg/ml of BSA. The topoisomerase I was used at a concentration of 1.5 U/µl. The use of topoisomerase I treatment of fixed tissue sections before immunohistochemical staining had no effect on binding of any of the anti-B-DNA antibodies, but prevented anti-Z-DNA immunoreactivity.
Anti--Crystallin Antibody Production, Histological Preparations and Immunohistochemistry of the Human Lens
An anti--crystallin polyclonal IgG antibody was produced in rabbits (
Feulgen Reaction
Both the modified and standard Feulgen reactions and controls (
Nuclear and Chromosome Stains
Cell nuclei were examined with Harris's hematoxylin nuclear stain (
Image Acquisition and Analysis
Data were obtained by analyzing 14 serial cross-sections of dog lens tissue, each of which was stained by one of 14 different probes. The arrangement of lens fiber cells in successive lamellae dictates that in the cross-sections studied the long axis of the cell is parallel to the plane of each section. Therefore, the orientation of each nucleus was approximately parallel to the plane of the section. Human lenses were separately immunostained with an anti--crystallin antibody. The reaction products of the eight anti-DNA antibodies (DAB substrate), the anti-
-crystallin antibody (fluorescein), the two Feulgen reactions, and the three histological dyes were analyzed for lens tissue sections as follows. The image analysis of nuclear morphology was performed using a Leitz DM-RB compound microscope and Leica Quantimet 500+ (Q 500+) image analysis system (Leitz; Cambridge, UK). A 20 x /1.00-0.50 PL Fluotar objective (200 x) was used for this application. A live video image of nuclei was acquired through a CCD-72S Black and White Camera (DAGE-MTI; Michigan City, IN) and digitized into running Microsoft Windows in the IBM 486/66 MHz computer for further analysis. The nuclei were illuminated with incident light at the aperture setting of 3.0. The quantitative measure for nuclear topology of the reaction products of DNA and
-crystallin staining was generated in the Quantimet Performer Interactive Programming System (QUIPS) in the Q500+ system. The measurement was performed with the black-and-white mode set so that the white level was close to one and the black level to zero. Individual black-and-white levels for each reaction product were carefully adjusted to reach this requirement. This ensured the best quality of video images presented for measurement. The gray level of each nucleus stained with respective reactions, as mentioned above, was accordingly presented to its corresponding binary image. With the aid of drawing and erasing of binary editing functions in the software, the amended binary images of nuclear morphology were thus accurately delineated to present as a real-time live image of nuclear contour. The positive immunocytochemical results and extent of nuclear DNA histological staining were microphotometrically measured in individual nuclei. Results for each nucleus were expressed as mean optical density (MOD) values [the mean value, on a linear scale of 0 (white) to 1.0 (black), of the optical density of each of the pixels within the image of each nucleus (0.411 pixels per µm2)], and integrated optical density (IOD) (the product of the mean optical density of a nucleus and the area of that nucleus in µm2). Unstained slides were analyzed to determine MOD and IOD control values: 3 ± 1 MOD and 27 ± 7 IOD, respectively. Quantitation of the intensity of eosin Y staining was achieved by using editing functions to analyze square areas of lens fiber cell cytoplasm within the SF (prior to the PTZ) and the MF immediately adjacent to the PTZ. On the other hand, to ensure the consistency of the lens sections studied and the measurement of the depth of the PTZ, two measurements were made. The actual depth of the PTZ was a perpendicular line to the outer surface of the lens capsule from the last highly stained nuclear center of individual fiber cells. The second line, the line of orientation, extends from the same nucleus to the most posterior nucleus at the apex of the bow region. This serves as a check on the consistency of cellular architecture of the lens section. Measurements were made in µm. For each epithelial cell, the nuclear area (the total number of detected pixels in the field that fall within the measure frame, adjusted to µm2) was determined. The live nuclear images and their subsequent binary images, were printed in a CP1000 Dye Sublimation Printer (Mitsubishi Electronic America; Somerset, NJ), and the resultant spreadsheet data were printed in HP Laser Jet 4/4M (Hewlett-Packard; Boise, ID). Photographs were made using a Ricoh 35-mm camera with a Autocam exposure detector (Kramer Scientific; Elmsford, NY) and Kodak Pro 400 MC color film.
Statistical Analysis
Data from serial sections of a single dog lens, as well as serial sections from age-matched dogs, were highly reproducible. The staining of a single probe was repeated six times; therefore, n = 6 for the analysis of results for each probe. Data were analyzed by one-way ANOVA (SigmaStat; Jandel, IL). Results are expressed as the mean ± standard deviation (SD) and the statistical significance of each experiment was in the range of p<0.05.
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Results |
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Image Acquisition and Analysis of the Normal Ocular Lens
Figure 1 shows a diagrammatic cross-section of the mammalian lens, which defines groups of cells that are histologically and functionally similar. The average equatorial diameter was 8.7 ± 0.2 mm and the average anterior-to-posterior thickness was 6.4 ± 0.3 mm. In Figure 2 and Figure 3, ds-B- and Z-DNA immunoreactivity and Feulgen reactions are presented. The order of the highest to least staining intensities is as follows: germinative zone of the epithelium (GZ) (region of DNA synthesis, cell division, and differentiation into lens fiber cells); superficial secondary fiber cells (SF) (0-100 µm) (elongating cortical lens fiber cells starting at the capsule of the bow region); central zone epithelium (CZ) (region of nonproliferating cells withdrawn from the cell cycle in the G0 state); middle fiber cells (MF) (101-738 ± 15 µm) (containing degenerating nuclei; these fibers are located in the inner cortex); and deep fiber cells (DF) (739-1475 ± 13 µm) (fibers that contain some DNA staining; located in the deep inner cortex and extending into the adult nucleus). No probe reactivity occurred in the anucleated fiber cells (AF) (1476 ± 51 to 4350 ± 45 µm) (fiber cells of the inner portion of the adult nucleus, plus the infantile, fetal, and embryonic nuclear regions). The anatomic orientation of the lens cortex and nuclear regions correlates well with zones described by -crystallin antibodies. Table 1 shows the delineation of lens regions based on the average of all anti-B- and Z-DNA antibodies and both Feulgen reactions. The precipitous decrease in fiber staining by all these techniques at a depth of
90 µm denotes a significant molecular reorganization, the PTZ.
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DS-B-DNA and SS-DNA Immunoreactivity
The B-103 (Figure 2 and Figure 4; Table 1), B-11 (Table 1), and 2C10 anti-ds-B-DNA antibodies (Figure 5; Table 1) all produced similar qualitative binding patterns. Anti-B-DNA immunoreactivity occurred in all epithelial cells and nucleated fiber cells, with a gradual decrease in antibody binding as fiber cells are displaced deeper into the lens, from the SF to DF (Figure 4 and Figure 5). An average of quantitative binding values for all anti-B-DNA antibodies from highest to least intensities for MOD was (Figure 2A) GZ, SF, CZ, MF, and DF. From these data, it is clear that similar relative results were obtained with MOD or IOD measurements of nuclei of cells in different histological regions (Figure 2 and Figure 3). This indicates that the size and shape of the nuclei did not significantly affect the results. The dramatic drop in anti-ds-B-DNA immunoreactivity was observed at 92 ± 4.0 µm perpendicular to the outer surface of the lens capsule (bow region) and 145 ± 5.3 µm from the line of orientation. This region includes 27 ± 4.0 stained fiber cells. This abrupt change in ds-B-DNA content denotes a molecular reorganization, the PTZ (Figure 4 and Figure 5; Table 1). Staining for ds-B-DNA persisted to a depth of 1326 ± 69 µm (140 ± 4.6 stained fiber cells).
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Anti-ss-DNA antibody (B11) binding was detected only in the MF and DF. The intensity of ss-DNA immunoreactivity first appeared in the MF (85 ± 4 MOD; 2245 ± 21 IOD). However, the immunoreactivity in the DF (178 ± 8 MOD; 4461 ± 38 IOD) increased by twofold. Control values for MOD and IOD were 5 ± 2 MOD and 23 ± 6 IOD.
B-DNA antibody binding was not affected by postfixation. Immunoreactivity persisted after DNase I nicking and moderate exposure to full digestion (data not shown), which implies that negative supercoiling is not a factor for B-DNA immunoreactivity. In addition, these results show that undigested B-DNA segments retain anti-B-DNA antibody reactivity. Localization of B-DNA by immunostaining persisted deeper into the DF cell layers than either the Feulgen reactions or histological stains. This is undoubtedly due to its superior binding abilities, especially in highly degraded DNA. B-DNA staining of lens fiber cells ended within the adult nuclear region.
Anti-Z-DNA Immunoreactivity
Anti-Z-DNA antibody binding (Z22, Figure 2A, and Z44, Table 1) was maximal in the GZ, followed by the SF and the CZ epithelial cells, and was absent in the MF, DF, and AF (Figure 2A and Figure 6; Table 1). Of all monoclonal and polyclonal anti-Z-DNA antibodies used, the Z22 MAb produced the most intense Z-DNA immunoreactivity. However, the Z44 (Table 1), 4255 (Figure 6), and 2122 (Table 1) anti-Z-DNA antibodies produced about the same Z-DNA immunoreactivity. All the anti-Z-DNA antibodies produced the same qualitative results.
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An average of quantitative binding values for all anti-Z-DNA antibodies from highest to least intensities for MOD was (Figure 2A) GZ, SF, and CZ. Anti-Z-DNA immunoreactivity was visualized to a depth of 87 ± 4.5 µm perpendicular to the outer surface of the lens capsule (bow region) and 138 ± 6.8 µm from the line of orientation. This region includes 22 ± 4.5 stained fiber cells (Table 1). The loss of immunoreactivity between the SF and MF is designated as the PTZ, which denotes a change in molecular organization. The staining of Z-DNA gradually decreased within the SF. The relative staining intensities (MOD) of anti-Z-DNA antibody binding, anti-B-DNA antibody binding, and the Feulgen reactions are consistent within the CZ, GZ, and SF (Figure 2A and Figure 3A). The relative staining intensities (MOD) of anti-B-DNA antibody reactivity and Feulgen reactions in the remaining deeper fiber cells (MF and DF) are also consistent (Figure 2A and Figure 3A).
Anti--Crystallin Immunoreactivity
To correlate the observations of both Z-DNA and B-DNA with protein organization, anti--crystallin antibodies were used to localize this protein in human fiber cells. GZ and SF revealed the highest anti-
-crystallin immunoreactivity, followed by the CZ. The staining by anti-
-crystallin antibody persisted to a depth of 102 ± 2.4 µm perpendicular to the outer surface of the lens capsule (bow region) and 152 ± 4.4 µm from the line of orientation. These image-analyzed data include 27 ± 5.8 stained SF (Table 1). The absence of anti-
-crystallin staining beyond 102 ± 2.1 µm suggests that the epitopes are unavailable for interaction with the antibody. These results support the concept that a molecular reorganization (PTZ) has occurred (Figure 7). These data are in accord with significant changes in Z-DNA and B-DNA immunoreactivity in the dog lens. Also similar to the DNA data, the anti-
-crystallin antibody binding within the SF gradually produced a weaker fluorescence.
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Histochemical-Histological DNA Staining
Both types of Feulgen reactions produced similar staining patterns for DNA and similar quantitative results. Therefore, the results for both reactions were included in our calculations (Figure 3; Table 1). An average of quantitative binding values for both Feulgen reactions from highest to least staining intensities for MOD was (Figure 3A) GZ, SF, CZ, MF, and DF. Heavy reactivity was observed to a depth of 92 ± 4.7 µm perpendicular to the outer surface of the lens capsule (bow region) and 143 ± 7.2 µm from the line of orientation, before a precipitous drop in staining. This PTZ includes 23 ± 5.9 stained cells (Table 1). Staining for DNA persisted to a depth of 1141 ± 39 µm (109 ± 3.8 stained fiber cells) (Table 1).
The Harris's hematoxylin, aceto-orcein, and Loffer's methylene blue stains also provided evidence for the PTZ. Because the results are quantitatively similar, data are presented as an average of all three stains. The qualitative staining of lens cells was essentially identical to the other ds-DNA probes (highest to least intensities): GZ (250 ± 9 MOD; 7149 ± 1771 IOD); SF (180 ± 7 MOD; 5877 ± 1054 IOD); CZ (115 ± 7 MOD; 3804 ± 987 IOD); MF (75 ± 4 MOD; 2946 ± 791 IOD); and DF (39 ± 2 MOD; 1533 ± 379 IOD). Control data measured 4 ± 2 MOD and 24 ± 9 IOD. Heavy staining was visualized to a depth of 87 ± 3.7 µm perpendicular to the outer surface of the lens capsule (bow region) and 140 ± 7.5 µm from the line of orientation, before a precipitous drop in reactivity. This region, the PTZ, includes 22 ± 3.9 stained cells. Histological staining for DNA persisted to a depth of 930 ± 71 µm (105 ± 5.5 stained fiber cells). The anti-B-DNA antibodies stained deeper than the Feulgen reactions and the histological stains. The more specific anti-B-DNA antibody probes are required to differentiate cell types deep within the lens.
Eosin Protein Staining
Eosin Y-stained tissue sections were analyzed microscopically and revealed a sudden and drastic decrease in the intensity of staining at a depth of 95 ± 5.7 µm (23 ± 4.8 stained cells), which further supports the concept of a PTZ. Eosin density data measured 98 ± 3 MOD before the PTZ and 64 ± 3 MOD after the PTZ. Control data measured 3 ± 2 MOD.
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Discussion |
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The results provide evidence for a significant precipitous change in the supramolecular order of dog lens fiber cells at a depth of 90 µm from the outer equatorial surface of the lens capsule. The absence of staining for Z-DNA at this depth and the sudden decrease in staining of B-DNA suggest an altered nuclear function. This correlates well with the decrease and then sudden loss of
-crystallin staining in the human lens at
102 µm, which denotes an overall reorganization of this major lens crystallin within the fiber cell cytoplasm. In addition, in the dog lens the staining intensity of eosin is dramatically decreased at
95 µm. These events are accompanied by a sudden increase in protein concentration (
These observations are also most interesting considering the early work of Warburg (100 µm. These observations correlate with the shift from aerobic to anaerobic metabolism in the lens SF. This shift in metabolism is undoubtedly related to the limit of oxygen penetration. In addition, this is in agreement with the observation by
100 µm. In the same region of the frog lens, the depth of active elongating fiber cells is also
100 µm (
100 µm. The PTZ at
90 µm may be an important event in the process of fiber cell terminal differentiation that leads to the dissolution of the nucleus and virtually all cell organelles and finally results in relatively metabolically inert inner fiber cells.
In the metabolically intact cells, the epithelial and SF cells, the ratio of B-DNA to Z-DNA is about 8. This is in agreement with
Although the functions of Z-DNA have yet to be determined, several previous observations led to the speculation that Z-DNA functions as a transcriptional enhancer (
The degradation of DNA parallels a shift from aerobic to anaerobic metabolism at about 90 µm (
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Acknowledgments |
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We wish to thank all of the investigators who so generously provided anti-B- and Z-DNA antibodies. In addition, we would like to acknowledge the considerable intellectual and material support by Ormond Mitchell, PhD, Dept. of Human Anatomy, and John H. Chen, PhD, Dept. of Biochemistry, New York University Basic Medical Sciences. Many thanks to Henry Gibson, PhD, Kamalendra Singh, PhD, and Helen Gabrysiak for their assistance in finalizing the manuscript.
Received for publication December 13, 1996; accepted June 5, 1997.
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