Journal of Histochemistry and Cytochemistry, Vol. 45, 1511-1522, Copyright © 1997 by The Histochemical Society, Inc.


ARTICLE

Localization of B-DNA and Z-DNA in Terminally Differentiating Fiber Cells in the Adult Lens

Claude E. Gagnaa, W. Clark Lambertb, Hon-Reen Kuob, and Patricia N. Farnswortha
a Department of Ophthalmology, University of Medicine and Dentistry of New Jersey-Medical School, Newark, New Jersey
b Department of Laboratory Medicine and Pathology, University of Medicine and Dentistry of New Jersey-Medical School, Newark, New Jersey

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.


  Summary
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Summary
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Materials and Methods
Results
Discussion
Literature Cited

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 {alpha}-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


  Introduction
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 (Kokileva 1994 ). For the most part, earlier studies of terminal differentiation of the lens fiber cells have focused on embryonic (Modak et al. 1969 ; Kuwabara and Imaizumi 1974 ; Yamamoto et al. 1990 ; Chaudun et al. 1994 ) and postnatal lenses (Modak and Bollum 1970 , Modak and Bollum 1972 ). Similar to apoptosis, terminally differentiating lens fiber cells undergo preprogrammed cellular denucleation (Broglio and Worgul 1982 ).

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 (Dickerson et al. 1982 ), and left-handed Z-DNA (Rich 1994 ). In 1979, Rich and co-workers (Wang et al. 1979 ) resolved the X-ray crystal structure of a 6-base pair (BP) oligonucleotide that exists as a left-handed helix. Left-handed Z-DNA, a high energy state of B-DNA, is the most dramatic type of helical conversion (Herbert and Rich 1996 ). Z-DNA is stabilized by negative supercoiling in vivo (Rahmouni and Wells 1989 ) and is induced by many conditions in vitro (Herbert and Rich 1996 , and references therein). The conversion of B- to Z-DNA is preferred in sequences with alternating purine-pyrimidine bases (Herbert and Rich 1996 , and references therein). It has been proposed that Z-DNA may regulate transcription, act as a molecular switch for gene function, control the level of supercoiling, and affect nucleosome positioning on promoters (Sinden 1994 , and references therein).

Previously, the presence of Z-DNA was demonstrated in the normal calf lens (Gagna et al. 1991a , Gagna et al. 1991b ). Transcriptionally induced Z-DNA segments have been observed in the human c-myc gene (Wölfl et al. 1995 ).

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 {alpha}-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.


  Materials and Methods
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 (Gagna et al. 1991b ). The NANOpure UV reagent grade water system (Barnstead; Dubuque, IA) was used to obtain deionized water to ensure consistent solution composition for all procedures in these studies. Successful fixation was achieved at 7 hr at room temperature (RT) in Carnoy's solution (60 ml absolute alcohol, 30 ml chloroform, and 10 ml glacial acetic acid: ultra pure grade reagents) (Gallard-Schlesinger; Carle Place, NY). To enhance anti-Z-DNA immunoreactivity, fixed lens tissue sections were postfixed by placing 2 ml of 45% acetic acid fixative (ultra pure grade; Gallard-Schlesinger) on each section for 10 min at RT. This procedure did not affect B-DNA results but provided excellent Z-DNA immunoreactivity.

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) (Jang and Stollar 1990 ). The B103 anti-ds-B-DNA IgG MAb (Jang and Stollar 1990 ), B11 anti-ds-B-DNA IgG polyclonal antibody (Zouali and Stollar 1986 ; Ausubel et al. 1991a ; Ausubel et al. 1991b ) and B14 anti-ss-DNA IgG polyclonal antibody (Zouali and Stollar 1986 ; Ausubel et al. 1991a ; Ausubel et al. 1991b ) were developed by and obtained from Dr. J.H. Chen (New York University, New York, NY). These antibodies are reactive with a variety of BP. Two anti-Z-DNA IgG MAbs (Z22 and Z44) were provided by Dr. E. Lafer of Dr. A. Rich's laboratory (Massachusetts Institute of Technology, Cambridge, MA) (Brigido and Stollar 1991 , and references therein). The Z44 MAb is sequence-specific and recognizes only G-C BP. However, the Z22 MAb binds a variety of BP and recognizes higher-order DNA structures. Two anti-ds-Z-DNA polyclonal IgG antibodies, 4255 (Gagna et al. 1991a ; Gagna et al. 1991b ) and 2122 (Lafer et al. 1981 ), developed and supplied by Dr. J.H. Chen, are specific for G-C BP and methylated and unmethylated G-C BP, respectively.

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) (Carson 1990 ). The avidin-biotin staining was developed with 3,3'-diaminobenzidine (DAB) chromogen (Polysciences; Warrington, PA). Appropriate controls were used to verify the specificity (Carson 1990 ). Half of the avidin-biotin-treated tissue sections were counterstained with 1% alcoholic eosin Y (no acetic acid) (Gagna et al. 1991b ) for definition of other cellular components and half were used for image analysis. To test the possible induction of Z-DNA conformation by anti-Z-DNA antibodies, serially diluted concentrations were used and no change was observed in the degree of Z-DNA immunoreactivity (data not shown). Histological processing, fixation time, postfixation time, temperature, reagent age, staining, color development, chemicals and image analysis of serial sections were standardized to ensure the accuracy of comparative quantitative results.

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 (Johnston 1992 ). Before immunohistochemical staining of lens tissue, the anti-B-DNA IgG antibody fractions (0.5-2000 µg/ml) and the anti-Z-DNA IgG antibody fractions (0.5-20 µg/ml) were separately preincubated with antigen competitors in low NaCl (150 mM) containing either B-DNA or bromine-stabilized Z-DNA and in high NaCl (4M), which is known to stabilize the Z-DNA conformation. Antibody-antigen mixtures were incubated for 30 min at 37C. This was followed by incubation at 4C for 1-17 hr and then by centrifugation for 40 min at 9000 x g. The supernatants were incubated with lens tissue sections. Anti-B- and Z-DNA immunohistochemical staining was clearly abolished after prior absorption of antibody with B- and Z-DNA, respectively. No reduction of immunohistochemistry occurred after incubation of antibodies with either RNA or ss-DNA. These results indicate the specificity of the B- and Z-DNA antibodies.

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 (Carson 1990 ).

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-{alpha}-Crystallin Antibody Production, Histological Preparations and Immunohistochemistry of the Human Lens
An anti-{alpha}-crystallin polyclonal IgG antibody was produced in rabbits (Ausubel et al. 1991a ) and fully characterized for the determination of antibody titer (Ausubel et al. 1991b ) and for its binding properties (Towbin et al. 1979 ). Normal adult eyebank human lenses were obtained, from the Lions Eye Bank of New Jersey (Newark, NJ). Lenses were fixed for 7 hr at RT in Triple Fix, pH 7.2 (10 ml of 10% EM grade glutaraldehyde, 10 ml of 10% EM grade acrolein, 16.5 ml of 6% EM grade paraformaldehyde, and 13.5 ml of H2O). All fixatives were purchased from Polysciences. Tissues were histologically processed and sectioned (2.5 µm) as previously described (Gagna et al. 1991b ). This polyclonal antibody was used to immunostain paraffin-embedded tissue sections employing the immunofluorescence technique, along with the appropriate controls (Dorsett and Ioachim 1978 ).

Feulgen Reaction
Both the modified and standard Feulgen reactions and controls (Schulte 1991 , and references therein) were used to determine DNA content as a standard for comparing the immunohistochemical and histological (nuclear chromosome) staining results. All chemicals were purchased from Sigma (St Louis, MO) and dyes from Polysciences.

Nuclear and Chromosome Stains
Cell nuclei were examined with Harris's hematoxylin nuclear stain (Sheehan and Hrapchak 1980 ), and one synthetic nuclear stain, Loffler's methylene blue (Sheehan and Hrapchak 1980 ). All materials were purchased from either Sigma or Polysciences. Staining of chromosomes was performed by using synthetic aceto-orcein (Sigma) (Sheehan and Hrapchak 1980 ). The tissue sections were stained with nuclear dyes, and half of these were counterstained with 1% alcoholic eosin Y (no acetic acid) (Polysciences) for definition of other cellular components (data not shown). The noncounterstained tissue sections were used for image analysis studies.

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-{alpha}-crystallin antibody. The reaction products of the eight anti-DNA antibodies (DAB substrate), the anti-{alpha}-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 {alpha}-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.


  Results
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Materials and Methods
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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 Garland et al. 1996 . In the present study these regions and the PTZ (Figure 2 and Figure 3; Table 1) are defined by their B-DNA and Z-DNA content determined by immunohistochemistry, and the staining patterns of DNA by the Feulgen reactions, nuclear histological stains, protein stains (eosin), and anti-{alpha}-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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Figure 1. Diagrammatic cross-section of a normal adult mammalian ocular lens defining the PTZ and lens cellular organization. Superficial fibers (SF) are elongating nucleated cortical lens fiber cells starting at the capsule of the bow region. Middle fibers (MF) contain degenerating nuclei and are located in the inner cortex. Deep fibers (DF) contain some DNA staining, are located in the deep inner cortex, and extend into the adult nucleus.



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Figure 2. Computerized image analysis of regional relative B-DNA and Z-DNA content as defined by the anti-ds-B-DNA (B103) and anti-ds-Z-DNA (Z22) immunoreactivity. (A) Mean optical density. (B) Integrated optical density.



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Figure 3. Computerized image analysis of regional relative DNA content as defined by the Feulgen reactions. (A) Mean optical density. (B) Integrated optical density.


 
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Table 1. Delineation of lens regional differences by the staining pattern of lens fiber cell DNA

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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Figure 4. Lens epithelium (E), capsule (C), and bow region, showing a precipitous decrease of anti-B-DNA antibody (B103) staining at the PTZ (~90 µm). Note light immunostaining persists in deeper fibers. C and E are astride the capsule and epithelium, respectively. Bar = 50 µm.



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Figure 5. Computerized image analysis. (A) The perpendicular distance from the outer surface of the lens capsule (bow region) to the center of fiber cell nuclei of the PTZ ~ 90 µm. (B) The distance of the line of orientation extends from the PTZ to the most posterior fiber cell nucleus at the apex of the bow region (~157 µm). These data were derived from staining with the 2C10 anti-B-DNA MAb.

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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Figure 6. Lens bow region showing the sudden loss of anti-Z-DNA polyclonal (4255) antibody staining at the PTZ (~90 µm). Bar = 100 µm.

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-{alpha}-Crystallin Immunoreactivity
To correlate the observations of both Z-DNA and B-DNA with protein organization, anti-{alpha}-crystallin antibodies were used to localize this protein in human fiber cells. GZ and SF revealed the highest anti-{alpha}-crystallin immunoreactivity, followed by the CZ. The staining by anti-{alpha}-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-{alpha}-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-{alpha}-crystallin antibody binding within the SF gradually produced a weaker fluorescence.



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Figure 7. Anti-{alpha}-crystallin IgG polyclonal antibody staining in the bow region of superficial fibers (SF) and epithelium (E). The depth at which staining abruptly ceases corresponds to the phase transition zone (PTZ) at about 102 µm from the capsule (C). Bar = 50 µm.

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.


  Discussion
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Materials and Methods
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Discussion
Literature Cited

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 {alpha}-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 (Fagerholm et al. 1990 ). Because eosin is a negatively charged protein stain, it is expected to interact with the more positively charged beta-gamma superfamily of crystallins. A decrease in eosin staining probably reflects an altered charge and/or protein-protein interaction of these crystallins. An increase in anti-ss-DNA immunoreactivity and a decrease in anti-ds-DNA antibody binding in the MF and DF are indicative of terminal differentiation, which is considered apoptotic in the kinetic sense (Kokileva 1994 ). A possible mechanism for such a drastic shift in supramolecular order is a phase transition (PTZ) and/or syneresis resulting from a number of changes in the internal environment of the fiber cells.

These observations are also most interesting considering the early work of Warburg (Lipinski 1989 , and references therein) on oxygen uptake of tissue blocks in vitro. He determined that the limit of oxygen penetration into tissue blocks was ~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 Bassnett 1992 that mitochondria are present in Rhesus monkey lens equatorial elongating fiber cells to a depth of ~100 µm. In the same region of the frog lens, the depth of active elongating fiber cells is also ~100 µm (Kuszak et al. 1986 ). The work of Fagerholm et al. 1990 is also similar to our data, in that they noticed a precipitous increase in protein concentration at ~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 Soyer-Gobillard et al. 1990 , whose data suggested that this ratio is about 7. The strong Z-DNA immunoreactivity in the SF parallels active crystallin synthesis as these fiber cells begin terminal differentiation. The high intensity of Z-DNA immunostaining in the SF and the absence of ss-DNA implies that the DNA is functionally intact. The sudden loss of anti-Z-DNA antibody binding immediately after the PTZ (MF + DF) suggests a molecular reorganization. A major event in the mechanism for terminal differentiation in the normal adult lens fiber cells occurs at the PTZ. Enhanced activity of endogenous lens DNase (Torriglia et al. 1995 ) may result in ss nicking of functionally intact high molecular weight DNA. This is followed by a cascade of events leading to DNA degradation by the production of ds-DNA fragments within which some ss nicks may exist. The ds-DNA fragmentation may be due to the close proximity of ss nicks on opposite strands in the linker region of chromatin. The production of denatured ss-DNA may result from additional nicking within ds fragments. It has been demonstrated that two ss nicks on opposite DNA strands within 14 BP of linker DNA is sufficient to dissociate the DNA molecule, producing denatured ss-DNA (Peitsch et al. 1993 ). In the lens, from the cortical to inner fiber cells, our data show a progressive loss of ds-DNA, which correlates with a progressive increase in denatured ss-DNA.

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 (Rich 1994 ; Herbert and Rich 1996 ). It should be noted that negative supercoiling of DNA is a normal in vivo process in eukaryotic chromatin (Vogelstein et al. 1980 ). DNA negative supercoiling is induced by transcription upstream from the RNA polymerase (Liu and Wang 1987 ). Others have shown that the level of Z-DNA formation was increased by stimulation of transcription, which induces an increase in RNA synthesis (Wittig et al. 1991 ). In the present study, our data also support this concept because Z-DNA exists in the SF that are actively synthesizing crystallins (Bloemendal 1981 ) and not in the MF, DF, and AF, where little or no crystallin is synthesized. This implies that Z-DNA may play a role in protein synthesis of crystallins.

The degradation of DNA parallels a shift from aerobic to anaerobic metabolism at about 90 µm (Lipinski 1989 , and references therein). The absence of survival factors (Bowen 1993 ), decreased glucose and enzyme activities (Farnsworth et al. 1989 ; Zhang and Augusteyn 1995 ), decreased H2O (Fagerholm et al. 1981 ), pH (Bassnett and Duncan 1986 ), ATP (Pirie 1962 ; Greiner et al. 1985 ), and increased protein concentration (Fagerholm et al. 1981 ) may activate endoproteases, endonucleases, or topoisomerase II (Kokileva 1994 ), which then triggers the genome detachment, cleavage, and disintegration. Therefore, the degradation of B-DNA and Z-DNA structure within the total lens chromatin probably inactivates the machinery necessary for nuclear function, and thereby the synthesis and turnover of lens proteins. However, after the loss of nuclear function, several well-defined regional differences within the lens suggest that post-translational changes in lens proteins are linked to a continuing reorganization of lens supramolecular order, i.e., the transition from a solution phase in the cortex to the coexistence of a solid-like and a solution phase in the nuclear region (Morgan et al. 1989 ). The biological phenomena associated with fiber cell terminal differentiation have potential for developing new approaches for diagnosis, prevention, and treatment of cataractogenesis.


  Acknowledgments

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.


  Literature Cited
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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