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(The FASEB Journal. 2001;15:221-229.)
© 2001 FASEB

Lens epithelial cells derived from {alpha}B-crystallin knockout mice demonstrate hyperproliferation and genomic instability

U. P. ANDLEY*,{dagger}1, Z. SONG*, E. F. WAWROUSEK, J. P. BRADY,2, S. BASSNETT*,{ddagger} and T. P. FLEMING*,§

Departments of
* Ophthalmology and Visual Sciences,
{dagger} Biochemistry and Molecular Biophysics,
{ddagger} Cell Biology and Physiology and
§ Genetics, Washington University School of Medicine, St. Louis, Missouri 63110, USA; and
National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892, USA

1Correspondence: Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, 660 S. Euclid Ave., Campus Box 8096, St. Louis, MO 63110, USA. E-mail: andley{at}vision.wustl.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
{alpha}B-crystallin is a member of the small heat shock protein family and can act as a molecular chaperone preventing the in vitro aggregation of other proteins denatured by heat or other stress conditions. Expression of {alpha}B-crystallin increases in cells exposed to stress and enhanced in tumors of neuroectodermal origin and in many neurodegenerative diseases. In the present study, we examined the properties of lens epithelial cells derived from mice in which the {alpha}B-crystallin gene had been knocked out. Primary rodent cells immortalize spontaneously in tissue culture with a frequency of 10-5 to 10-6. Primary lens epithelial cells derived from {alpha}B-crystallin-/- mice produced hyperproliferative clones at a frequency of 7.6 x 10-2, four orders of magnitude greater than predicted by spontaneous immortalization (1) . Hyperproliferative {alpha}B-crystallin-/- cells were shown to be truly immortal since they have been passaged for more than 100 population doublings without any diminution in growth potential. In striking contrast to the wild-type cells, which were diploid, the {alpha}B-crystallin-/- cultures had a high proportion of tetraploid and higher ploidy cells, indicating that the loss of {alpha}B-crystallin is associated with an increase in genomic instability. Further evidence of genomic instability of {alpha}B-crystallin-/- cells was observed when primary cultures were infected with Ad12-SV40 hybrid virus. In striking contrast to wild-type cells, {alpha}B-crystallin-/- cells expressing SV40 T antigen exhibited a widespread cytocidal response 2 to 3 days after attaining confluence, indicating that SV40 T antigen enhanced the intrinsic genomic instability of {alpha}B-crystallin-/- lens epithelial cells. These observations suggest that the widely distributed molecular chaperone {alpha}B-crystallin may play an important nuclear role in maintaining genomic integrity.—Andley, U. P., Song,, Z., Wawrousek, E. F., Brady, J. P., Bassnett, S., Fleming, T. P. Lens epithelial cells derived from {alpha}B-crystallin knockout mice demonstrate hyperproliferation and genomic instability.


Key Words: molecular chaperone • nuclear • immortalization • ploidy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
{alpha}B-crystallin, a major protein of lens fiber cells, is a stress-inducible chaperone that is constitutively expressed at low levels in the lens epithelium and in numerous tissues, particularly heart, kidney, skeletal muscle, lung, and brain (2 3 4 5) . However, the functions of {alpha}B-crystallin in these tissues are unknown. Several reports suggest a general cellular function of {alpha}B-crystallin linked with growth (4 5 6 7 8 9 10 11 12 13) . Nearly 20% of {alpha}B-crystallin polypeptides isolated from the lens are phosphorylated in vivo, which suggests its association with signal transduction pathway(s) (4 , 11) . Chinese hamster ovary cells induced to express {alpha}B-crystallin change its localization from the cytoplasm to the nucleus during interphase (5) , and {alpha}B-crystallin expression increases in mitotically arrested NIH 3T3 fibroblasts (6) . {alpha}B-crystallin also accumulates in the developing central nervous system (particularly in glial cells), in fibroblasts transformed by the v-mos and Ha-ras oncogenes, and in brain tumors of neuroectodermal origin (7 8 9 10 11 12 13 14) . Enhanced expression of {alpha}B-crystallin is also observed in numerous neurodegenerative diseases such as Alexander’s, Alzheimer’s, Creutzfeldt-Jakob, multiple sclerosis, and Parkinson’s diseases (11 12 13) . {alpha}B-crystallin-/- mice develop muscle abnormalities but do not develop cataracts (15) . The major phenotype in these mice, lacking both {alpha}B-crystallin and small heat shock protein HSPB2 (encoded by an adjacent gene that was also disrupted), is a progressive muscular dystrophy that destroys a specific subset of skeletal muscles, particularly those in the head and perivertebral regions. Since HSPB2 is not expressed in the lens (16) , its deletion is not expected to contribute to lens phenotypes. Lens epithelial cells derived from mice lacking {alpha}A-crystallin, a protein normally associated with {alpha}B-crystallin in the lens, have a diminished growth potential (17) . To study the physiological role of {alpha}B-crystallin, lens epithelial cells were cultured from {alpha}B-crystallin-/- mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Cell culture
Wild-type (129SvJ) and {alpha}B-crystallin-/- lens epithelial cells were used in this study. {alpha}B-crystallin-/- mouse lenses were obtained from mice with targeted disruption of the {alpha}B-crystallin gene and the adjacent HSPB2 gene (15) . The latter gene is not expressed in the lens and is not expected to give lens phenotypes (15 , 16) . To obtain primary lens epithelial cells, capsule epithelial explants of wild-type (129SvJ) or {alpha}B-crystallin-/- mouse lenses (6–8 wk old) were placed in 35 mm tissue culture plates containing Eagles minimum essential medium-20% fetal bovine serum and cultured for 1–2 wk until growth of cells was detectable (17) . Primary cells were passaged and plated on glass coverslips to facilitate microscopic examination. Cultures from a pair of lenses were routinely placed together in one tissue culture plate. To identify differences in growth potential of fellow lenses from a given mouse, in some experiments each of the two capsule epithelial explants from a mouse was cultured separately. Once it was determined that the fellow lens epithelial explants of a particular animal had the same proliferation behavior, one epithelium of a pair of lenses was placed in fixative and the other one was cultured. {alpha}A-crystallin-/- cells described previously (17) were used as a control for some experiments. Primary cultures of wild-type, {alpha}A-crystallin-/-, and {alpha}B-crystallin-/- cells were infected with Ad12-SV40 hybrid virus as described previously (18) .

Cell growth
Primary cultures of wild-type or {alpha}B-crystallin-/- lens epithelial cells in passage 1 were subcultured into 24-well plates (2x103 cells per well) for a period of 2 wk. Attachment efficiency determined 2 h after plating was found to be 85%, and was the same for wild-type and {alpha}B-crystallin-/- cells. Cultures were fed twice weekly. Cell numbers were measured on indicated days after trypsinization and counting with a Coulter counter (17) . Hyperproliferative {alpha}B-crystallin-/- cells were plated at low densities to examine clonal growth (17) . To determine whether the hyperproliferative {alpha}B-crystallin-/- cells were immortal, they were subcultured every week, the number of cells was counted, and the population doubling level was determined as described previously (18) .

Cell cycle distribution
Wild-type or {alpha}B-crystallin-/- cells (106 cells) were washed with phosphate-buffered saline (PBS) and cell pellets were labeled for 30 min on ice with propidium iodide (50 µg/ml) in 0.1% sodium citrate containing 0.3% Nonidet P-40 and 20 µg/ml ribonuclease A (pH 8.3). The percentage of cells in each phase of the cell cycle was determined using a flow cytometer (Becton-Dickinson FACScan, Rutherford, N.J.) and analyzed using the Cell Quest software (17) .

Immunofluorescence
Cells were fixed in 4% paraformaldehyde/PBS for 30 min at room temperature, permeabilized in 0.1% triton X-100 for 30 min, and blocked with 10% goat serum. To visualize the distribution of {alpha}B-crystallin, cells were incubated overnight at 4°C in a 1:50 dilution of a polyclonal antibody raised against bovine {alpha}B-crystallin (a kind gift from Dr. Joseph Horwitz). This primary antibody was used because it gave a very low background in immunocytochemistry with the {alpha}B-crystallin-/- mouse lens slices. A fluorescein-conjugated goat anti-rabbit immunoglobulin G (IgG) was used as the secondary antibody. To visualize the distribution of {alpha}A-crystallin, cells were incubated overnight at 4°C in a 1:100 dilution of a monoclonal antibody against bovine {alpha}A-crystallin (a kind gift from Dr. Paul Fitzgerald). This primary antibody was used because it has been shown to react with a high specificity with {alpha}A-crystallin in immunocytochemical detection (17) and gave no background with {alpha}A-crystallin-/- mouse lens slices. A lissamine-rhodamine-conjugated goat anti-mouse IgG was used as the secondary antibody. To visualize the organization of f-actin, fixed and permeabilized cells were incubated in a 1:50 dilution in PBS of a fluorescein phalloidin (Molecular Probes Inc., Eugene, Oreg.) methanolic stock solution (100 U/ml of methanol). Cells were stained with fluorescein phalloidin for 20 min, washed 3 x 5 min in PBS, and viewed. Lens epithelial cells were viewed using a Zeiss LSM 410 confocal microscope equipped with an Argon-Krypton laser.

Western blotting
Western immunoblotting was used to examine the expression of {alpha}A- and {alpha}B-crystallin in primary mouse lens epithelial cultures (17 , 19) . The antibody used for immunoblot analysis of {alpha}A-crystallin was a monoclonal antibody to bovine {alpha}A-crystallin (at a dilution of 1:100), which has been shown to react with high specificity with mouse {alpha}A-crystallin (17) . For immunoblotting analysis of {alpha}B-crystallin, a polyclonal antiserum raised against the 21 amino acid carboxyl-terminal peptide of human {alpha}B-crystallin was used at a dilution of 1:1000. This antiserum showed a high specificity for mouse {alpha}B-crystallin. Immune complexes were detected using 125I-protein A.

Cytogenetics
Karyotype analyses were done in the Cell Culture Laboratory of Dr. Bharati Hukku, Children’s Hospital of Michigan (Detroit, Mich.) using standard methods. Chromosome counts were determined in 51 metaphases, and 16 Giemsa-banded karyotypes were examined from each culture (20 , 21) .


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSION
 REFERENCES
 
{alpha}B-Crystallin was detected as a single protein band of ~20 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis in wild-type (129SvJ) mouse lens epithelial cells, but was absent from {alpha}B-crystallin-/- epithelial cells derived from homozygous {alpha}B-crystallin-/- mouse lenses (Fig. 1A ). {alpha}B-crystallin was readily detectable in the cytoplasm of wild-type mouse lens epithelial cells by immunofluorescence but was absent in the cultures derived from {alpha}B-crystallin-/- lenses (Fig. 1C , E ). Very low background staining seen in the nuclei of the {alpha}B-crystallin-/- cells is most likely to be due to the interaction of the rabbit antiserum used with nucleolar antigen and not to {alpha}B-crystallin. Since {alpha}B-crystallin is normally associated in the lens with {alpha}A-crystallin in vivo, we also examined the expression of {alpha}A-crystallin in cultured lens epithelial cells. {alpha}A-crystallin expression can be detected in the cytoplasm of wild-type and {alpha}B-crystallin-/- cells. The levels of {alpha}A-crystallin were similar in wild-type and {alpha}B-crystallin-/- lens epithelial cells (Fig. 1B , D ). Distribution of {alpha}A-crystallin within the wild-type and {alpha}B-crystallin-/- cells was also similar, indicating that knocking out {alpha}B-crystallin does not affect the expression of {alpha}A-crystallin. The organization of the actin cytoskeleton in wild-type and {alpha}B-crystallin-/- lens epithelial cells was also examined. As shown in Fig. 1 , there were no significant differences in the actin cytoskeleton of wild-type and {alpha}B-crystallin-/- cultured lens epithelial cells.



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  		Figure 1. Expression of {alpha}A-crystallin and {alpha}B-crystallin in mouse lens epithelial cells derived from wild-type and {alpha}B-crystallin-/- lenses. A) Cell lysates were prepared from 2 x 105 wild-type or {alpha}B-crystallin-/- cultured mouse lens epithelial cells and analyzed by SDS-PAGE and Western blotting. Left panel: the primary antibody used was a monoclonal antiserum to {alpha}A-crystallin. WT, lysates of wild-type lens epithelial cell cultures. The higher molecular weight band represents the {alpha}A insert protein, a variant form of {alpha}A-crystallin present in rodents; {alpha}B-/-, lysates of {alpha}B-crystallin-/- lens epithelial cell cultures. Note that both wild-type and {alpha}B-crystallin-/- lens epithelial cells continued to express both forms of {alpha}A-crystallin in the culture. Right panel: the primary antibody was a polyclonal antiserum raised to the 21 amino acid carboxyl-terminal peptide of human {alpha}B-crystallin, which does not cross-react with {alpha}A-crystallin. WT, lysates of wild-type lens epithelial cell cultures; {alpha}B-/-, lysates of {alpha}B-crystallin-/- lens epithelial cell cultures. Note the absence immunoreactive bands in the lysates from {alpha}B-crystallin-/- cells, demonstrating the specificity of the antiserum in Western blotting. B–E) Distribution of {alpha}A-crystallin, {alpha}B-crystallin, and actin in primary cultures of mouse wild-type and {alpha}B-crystallin-/- lens epithelial cells by immunofluorescence. B, D) Localization of {alpha}A-crystallin (red), using a monoclonal antibody to bovine {alpha}A-crystallin; f-actin (green) was localized using fluorescein-phalloidin. B) Lens epithelial cells from wild-type lenses; D) lens epithelial cell from {alpha}B-crystallin-/- lenses. C, E) Localization of {alpha}B-crystallin (red) using an antibody to bovine {alpha}B-crystallin; f-actin (green) was localized using fluorescein-phalloidin. C) Lens epithelial cells from wild-type lenses; E) lens epithelial cells from {alpha}B-crystallin-/- lenses. Note the low background staining seen in the nuclei of the {alpha}B-crystallin-/- cells, which is most likely to be due to the interaction of the rabbit antiserum used with nucleolar antigen and not due to {alpha}B-crystallin. F, G) Comparison of {alpha}A-crystallin localization in normal-growing and hyperproliferative {alpha}B-crystallin-/- lens epithelial cells by immunofluorescence. (Only {alpha}B-crystallin-/- lenses produced primary cultures having a high growth potential at an increased frequency; Table 1 .) F) Normal-growing {alpha}B-crystallin-/- cells; G) hyperproliferative {alpha}B-crystallin-/- cells. Note that {alpha}A-crystallin is expressed in the cytoplasm of both normal-growing and hyperproliferative {alpha}B-crystallin-/- cells. Also note the variation in {alpha}A-crystallin level in the cells of a given culture.

Of 263 {alpha}B-crystallin-/- explanted capsule epithelia, 7.6% of the explants exhibited a high proliferative ability as compared with wild-type cultures, producing a frequency of hyperproliferation of {alpha}B-crystallin-/- cells of 7.6 x 10-2 (Table 1 ). In contrast, the frequency of spontaneous immortalization of rodent cells in culture is estimated to be 10-5 to 10-6 (1) . The faster growing colonies were observed only in the cells cultured from the {alpha}B-crystallin-/- lenses, and 289 wild-type primary lens epithelial cultures produced no hyperproliferative clones. Differences between proliferation of the wild-type and {alpha}B-crystallin-/- cells were observed within 7 days of initiation of the culture. Examination of 173 {alpha}A-crystallin-/- cultures also produced no hyperproliferative cultures. These observations are consistent with the fact that immortalization of mouse lens epithelial cells in culture is a very uncommon event. No spontaneously immortalized mouse lens epithelial cell lines are now available (22 23 24) .


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  		Table 1. Frequency of hyperproliferation of wild-type (129SvJ) and {alpha}B-crystallin-/- mouse lens epithelial cells

The hyperproliferative {alpha}B-crystallin-/- cells were shown to grow at a higher rate, with a doubling time of 1.5 days as compared with 3 days for the wild-type cells. Ten days after culture, the number of hyperproliferative {alpha}B-crystallin-/- cells was eightfold higher than the wild-type or normal-growing {alpha}B-crystallin-/- cells (Fig. 2A ). The hyperproliferative cells can be considered to be truly immortal, since they have already been passaged to >100 population doublings with no observable diminution in their proliferative ability (Fig. 2B ). Expression of {alpha}A-crystallin in the hyperproliferative {alpha}B-crystallin-/- cells was observable at levels similar to that in the normal-growing {alpha}B-crystallin-/- cells (Fig. 1F , G ). Since growth rates can affect cell cycle distribution, we examined propidium iodide-labeled cells by flow cytometry. As shown in Fig. 2C , the proportion of cells in the G2/M phases was 9.6 ± 1.6% in wild-type and 8.5 ± 2.0% in normal-growing {alpha}B-crystallin-/- cells, but increased to 27.7 ± 1.8% in the hyperproliferative {alpha}B-crystallin-/- cells.



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  		Figure 2. Growth parameters of wild-type, {alpha}B-crystallin-/-, and hyperproliferative {alpha}B-crystallin -/- cultured lens epithelial cells. A) First passage cells were cultured in minimum essential medium containing 20% serum, trypsinized, and counted in a Coulter counter at the indicated times. WT, lens epithelial cells from wild-type lenses; {alpha}B-/-, lens epithelial cells from {alpha}B-crystallin-/- lenses; Hyperproliferative {alpha}B-/-, hyperproliferative {alpha}B-crystallin-/- lens epithelial cells. (Note that the y axis is plotted on a logarithmic scale.) The doubling time of hyperproliferative {alpha}B-crystallin-/- cells was determined to be 1.5 days, whereas that of the wild-type and the normal-growing {alpha}B-crystallin-/- cells was 3 days. B) Population doubling level vs. passage number of wild-type, {alpha}B-crystallin-/- cells with normal growth potential, and hyperproliferative {alpha}B-crystallin-/- cells. The hyperproliferative {alpha}B-crystallin-/- cells maintained their growth potential over >100 population doubling levels, whereas the wild-type and normal-growing {alpha}B-crystallin-/- cells had a low growth potential and did not grow beyond population doubling levels {cong} 6. C) Flow cytometric analysis of propidium iodide-labeled wild-type, normal-growing {alpha}B-crystallin-/-, and hyperproliferative {alpha}B-crystallin -/- cells. The bars designated as M1, M2, and M3 were used to determine the proportion of cells in G1, S, and G2/M phases of the cell cycle, respectively. The proportion of cells in the G2/M phase of the cell cycle increased by approximately threefold in the hyperproliferative {alpha}B-crystallin-/- cells.

Analysis of karyotypes of {alpha}B-crystallin-/- cells indicated the presence of cells with above normal chromosome counts per metaphase (Fig. 3 ). Of the 51 metaphases examined from a single hyperproliferative {alpha}B-crystallin-/- cell line, 60% were diploid (2N), 28% were tetraploid, and 12% were of higher ploidy. Higher ploidy metaphases were observed at an even greater level in the {alpha}B-crystallin-/- cells having normal growth potential, with 23% diploid, 73% tetraploid, and 4% higher ploidy metaphases. In contrast, wild-type cells were 91% diploid. These observations suggest that {alpha}B-crystallin has an important role in maintaining genomic stability.



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  		Figure 3. Karyotypic changes in {alpha}B-crystallin-/- cells. Primary cultures of wild-type (WT) or {alpha}B-crystallin-/- cells ({alpha}B-/-) were treated with 0.04 µg/ml Colcemid for 1–2 h, fixed, and stained with 4% Giemsa solution. Chromosome counts per 51 metaphases were analyzed. The wild-type cells were 91% diploid and 9% tetraploid. In contrast, {alpha}B-crystallin-/- cells with normal growth potential were 23% diploid, 73% tetraploid, and 4% polyploid. Hyperproliferative {alpha}B-crystallin-/- cells were 60% diploid, 28% tetraploid, and 12% polyploid.

We next determined whether the hyperproliferative {alpha}B-crystallin-/- cells were capable of growing on soft agar. A melanoma cell line, C8161, was used as positive control and NIH 3T3 fibroblasts were used as a negative control. Figure 4 shows that hyperproliferative {alpha}B-crystallin-/- cells (FGAB-1) did not grow on soft agar in 3 wk, similar to NIH 3T3 fibroblasts (not shown). In contrast, the C8161 cell line produced colonies in soft agar. This observation suggests that the genomic instability introduced by knocking out the expression of {alpha}B-crystallin is in its early stages of cellular evolution from normal to cancerous, and cells have not acquired the ability to grow on soft agar (25) . Consistent with this observation, the hyperproliferative {alpha}B-crystallin-/- cells did not produce tumors when injected subcutaneously into nude mice. In contrast, the C8161 cells produced well-developed tumors (data not shown).



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  		Figure 4. Tumorigenic potential of hyperproliferative {alpha}B-crystallin-/- lens epithelial cells. Cells were plated on soft agar and their ability to form colonies was determined over a period of 3 wk. Top: C8161 melanoma cell line, used as a positive control. Bottom: hyperproliferative {alpha}B-crystallin-/- lens epithelial cells. Note that hyperproliferative {alpha}B-crystallin-/- lens epithelial cells did not produce colonies in soft agar.

To determine whether fellow lenses of a given animal produced hyperproliferative clones, lens epithelial tissue from each capsule epithelial explant of 39 {alpha}B-crystallin-/- mice was cultured in individual tissue culture plates, of which three pairs of lens epithelial cultures produced hyperproliferative cells. Moreover, whenever one culture from a given mouse produced hyperproliferative cells, the epithelium from the contralateral lens also produced hyperproliferative cells. This suggests that genomic instability associated with loss of {alpha}B-crystallin occurs early in mouse development and at the level of individual animals, not individual cells of a particular mouse. Furthermore, clonal cultures of hyperproliferative primary cells plated at low density indicated that each attached cell produced a hyperproliferative colony, suggesting that most, if not all, lens epithelial cells of such an animal had aberrant proliferative activity. To examine the morphology of the lens producing hyperproliferative cells, tissue from one lens was placed in culture and the fellow lens was fixed and sectioned. Examination of the germinative region of the {alpha}B-crystallin-/- lenses giving rise to hyperproliferative lens epithelial cells indicated a morphology that was not significantly altered from age-matched wild-type or normal-growing {alpha}B-crystallin-/- lenses (Fig. 5 ). The {alpha}B-crystallin-/- mice did not get cataracts and had no significant change in lens morphology during the period examined from postnatal to 40 wk. Thus, it appears from the observed ploidy changes and frequency of hyperproliferation of {alpha}B-crystallin-/- lens epithelial cells that we have unmasked a phenotype of the {alpha}B-crystallin-/- lens that could not be detected in vivo. This is consistent with reports that the mitotic activity of the lens epithelium is enhanced by the removal of fiber cells from the capsule epithelium (26) . It also suggests that epithelial–fiber interaction (27) as well as the presence of factors essential for normal growth and differentiation masked the genetic instability of the {alpha}B-crystallin-/- cells in vivo.



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  		Figure 5. Morphology of wild-type and {alpha}B-crystallin-/- mouse lenses. Lenses were fixed and mounted in methacrylate. Mid-sagittal sections were stained with toluidine blue and viewed by brightfield microscopy. Morphology of the germinative region is shown. A) Wild-type lens; B) {alpha}B-crystallin-/- lens producing cells of normal growth potential; C) {alpha}B-crystallin-/- lens producing hyperproliferative cells. Note that no significant differences in morphology of the lenses were detected.

Nontransformed {alpha}B-crystallin-/- lens epithelial cells (both normal-growing and hyperproliferative), like wild-type and {alpha}A-crystallin-/- cells, could be maintained in confluent culture for extended periods (>60 days) and remained viable. However, further evidence of enhanced genomic instability of {alpha}B-crystallin-/- lens epithelial cells was obtained when primary cultures of wild-type and {alpha}B-crystallin-/- lens epithelial cells were infected with the hybrid virus Ad12-SV40 to increase their ability to proliferate. Although the wild-type, {alpha}B-crystallin-/-, and {alpha}A-crystallin-/- (used as a control) lens epithelial cells produced cell lines that grew rapidly, only the lines derived from wild-type and {alpha}A-crystallin-/- epithelial cells could be maintained in confluent cultures for periods longer than 30 days without substantial cell death. In striking contrast, {alpha}B-crystallin-/- cells expressing SV40 T antigen exhibited a widespread cytocidal response 2 to 3 days after attaining confluence (Fig. 6 ), indicating that SV40 T antigen-induced genetic instability results in cell death of the {alpha}B-crystallin-/- cells only. To determine whether hyperproliferative primary cultures of {alpha}B-crystallin-/- lens epithelial cells also displayed a cytocidal response in response to SV40 T antigen expression, the hyperproliferative cells were infected with Ad12-SV40 hybrid virus. As observed with the normal-growing {alpha}B-crystallin-/- cells, expression of SV40 T antigen also induced a cytocidal response in the hyperproliferative {alpha}B-crystallin-/- cells, although the cell death occurred over a more protracted period of 10, instead of 3, days after gaining confluence (data not shown).



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  		Figure 6. Cell death in postconfluent cultures of Ad12-SV40 infected wild-type, {alpha}A-crystallin-/-, and {alpha}B-crystallin-/- mouse lens epithelial cells as detected by TUNEL labeling. Ad12-SV40-infected mouse lens epithelial cells were grown to confluence and cultured for 3 days thereafter. Cultures were TUNEL-labeled and examined by confocal microscopy. A) Confluent culture of wild-type lens epithelial cells. B) Confluent culture of {alpha}A-crystallin-/- lens epithelial cells. C) Confluent culture of {alpha}B-crystallin-/- lens epithelial cells showing extensive TUNEL-labeled cells. Blue: cellular morphology was imaged with differential interference contrast; green: TUNEL-labeled cells in the culture. Note that wild-type and {alpha}A-crystallin-/- cells showed very few TUNEL-positive nuclei (arrows). In contrast, {alpha}B-crystallin-/- cells showed high levels of TUNEL-labeled cells. D) Higher magnification of TUNEL-labeled cells in a confluent culture of hyperproliferative {alpha}B-crystallin-/- lens epithelial cells. Green: TUNEL-labeled cells; red: Texas red-phalloidin staining for actin in {alpha}B-crystallin-/- lens epithelial cells. E) Percent cell death in postconfluent cultures of wild-type (WT), {alpha}A-crystallin-/- ({alpha}A-/-), and {alpha}B-crystallin-/- ({alpha}B-/-) Ad12-SV40-infected lens epithelial cells. The number of cells in each culture was counted 1 and 3 days after cells achieved confluence. Percent cell death was determined as the ratio of the number of cells 1 day after confluence to the number of cells 3 days after confluence multiplied by 100.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSION
 REFERENCES
 
{alpha}B-crystallin is a major lens protein expressed in numerous non-lens tissues of vertebrates, consistent with its putative nonrefractive cellular functions (3 , 11 12 13 14) . It has been shown to prevent the aggregation of denaturing proteins in vitro, similar to other molecular chaperones, and its expression increases under stress conditions (11 , 28 , 29) . However, the in vivo cellular functions of {alpha}B-crystallin have not been elucidated. {alpha}B-crystallin normally is associated with {alpha}A-crystallin in the in vivo lens. During murine ocular development, the transcription of {alpha}B-crystallin precedes that of {alpha}A-crystallin (30) . Recently, mice lacking the {alpha}A-crystallin gene were shown to develop cataracts (31) and cultured lens epithelial cells derived from {alpha}A-crystallin-/- mice were shown to have a decreased ability to proliferate (17) . Unlike {alpha}A-crystallin-/- mice, the {alpha}B-crystallin-/- mice do not develop cataracts (15) . {alpha}B-crystallin is particularly abundant in muscle cells. {alpha}B-crystallin-/- mice are also deficient in the muscle-specific small heat shock protein HSPB2, which is not expressed in the lens (16) . [Using primers specific to the HSPB2 gene and reverse transcriptase-polymerase chain reaction, we did not detect any message in wild-type murine lens epithelial cells (Z. Song and U. Andley, unpublished observations).] These mice develop skeletal muscular dystrophy often characterized by increased intracellular levels of desmin (15) . Similarly, patients who carry a missense mutation in {alpha}B-crystallin (R120G) develop desmin-related myopathy and cataracts (32) .

The present work demonstrated that deletion of the murine {alpha}B-crystallin gene permits the emergence of lens epithelial cells with an increase in polyploidy. However, a significant proportion (23%) of normal-growing {alpha}B-crystallin-/- cells maintained normal ploidy. In contrast, 91% of the wild-type cells were diploid. This suggests that the genomic instability of the {alpha}B-crystallin-/- cells is a property of the culture population as a whole, perhaps due to a subtle defect in the {alpha}B-crystallin-/- cells. Note also that hyperproliferation was never observed in the wild-type cultures, but the {alpha}B-crystallin-/- lens epithelial cells demonstrated a wide variation in proliferative capacity. These results suggest that the loss of {alpha}B-crystallin may predispose cells to undergo ploidy changes and hyperproliferation. During the cell cycle, genomic changes such as spindle errors or spindle pole errors produce aneuploidy or tetraploidy respectively in cells, whereas replication errors produce chromosome aberrations (33) . p53 monitors genomic integrity at the G1 and G2/M cell cycle checkpoints and cells lacking p53 show polyploidy or aneuploidy typical of many tumors (33 34 35) . The presence of tetraploid and higher ploidy cells in {alpha}B-crystallin-/- cultures suggests a role of {alpha}B-crystallin associated with cell cycle. Similarly, the reported increase in expression of {alpha}B-crystallin in mitotic fibroblasts and its transient expression in the nucleus during interphase of transfected CHO cells also suggest its involvement with the cell cycle machinery (5 , 6) . The phosphorylation of {alpha}A- and {alpha}B-crystallin in vivo, their autokinase activity, and the existence of enzymes in the lens, which can dephosphorylate {alpha}-crystallin, have previously suggested an association of {alpha}-crystallin with cellular signal transduction pathways (36 37 38) .

Maintenance of genomic integrity is important for cell survival, and the tumor suppressor protein p53 is involved in this function (39) . Preservation of genomic stability encompasses many factors, such as the maintenance of primary DNA sequence and the preservation of chromosomal ploidy and structure (33) . Karyotypic alterations, including ploidy changes observed in the {alpha}B-crystallin-/- lens epithelial cells, are common in cancer cells. It is well established that the expression of oncogenes creates genomic instability (40 , 41) . The combination of p53 inactivation and oncogene expression markedly accelerates the development of polyploidy, cell death, DNA amplification, and tumor formation (33 34 35) . Since genomic instability is a hallmark of cancer cells, the overexpression of {alpha}B-crystallin during oncogene expression (9 , 10) and in tumors of neuroectodermal origin (7 , 8) suggests that the protein may accumulate as a stress response to maintain chromosomal integrity during cellular evolution from a normal to a cancer cell. This notion is consistent with the current finding that {alpha}B-crystallin-/- cells demonstrate genomic instability.

The present work further supports a link between {alpha}B-crystallin and nuclear functions. When we introduced T antigen into the {alpha}B-crystallin-/- primary mouse lens epithelial cells, it was found that unlike the nontransformed {alpha}B-crystallin-/- or wild-type lens epithelial cells, SV40 T antigen-transformed cells could not survive confluence, since >95% of the cells died by apoptosis a few days after gaining confluence. This indicates that although the T antigen is normally a growth enhancing protein, it triggers an apoptotic pathway in postconfluent cells derived only from mice lacking {alpha}B-crystallin. Even though the mechanism by which cell death in the SV40 T-antigen expressing {alpha}B-crystallin-/- cell lines occurs is not known, it is well established that oncogene expression creates genomic instability (42 , 43) . Together with other oncoproteins or in the absence of tumor suppressor gene products, oncogenes contribute to tumor formation by supporting accelerated proliferation and deregulating cell cycle control and apoptosis (44) . The SV40 T antigen very likely enhances the genomic instability inherent in {alpha}B-crystallin-/- lens epithelial cells. T antigen expression may also deregulate HSP25 expression, thus decreasing its level below a threshold necessary for cell survival (45) . The mechanism by which the loss of {alpha}B-crystallin enhances genomic instability needs further investigation.

The hyperproliferation of {alpha}B-crystallin-/- lens epithelial cells that we observed in culture did not produce a detectable in vivo lens phenotype, as shown by the lack of significant differences in size and morphology of the lenses producing hyperproliferative and normal-growing {alpha}B-crystallin-/- lens epithelial cells. Previous studies suggest that the presence of contacts between lens epithelial and fiber cells decreases the mitotic index of lens epithelial cells in vivo (26 , 27) . This may explain the finding that in the {alpha}B-crystallin-/- lenses, genetic instability or cataract formation did not occur in vivo. Aberrant proliferation and apoptosis have been demonstrated in vivo in the developing lenses of Rb-deficient mice (46) and in lenses of transgenic mice expressing polyoma large T antigen (47) , although these lenses showed no in vivo lens tumor formation. Transgenic mice expressing viral oncoproteins E6 and E7 showed lens tumor formation (48) . In the current work, no tumors were noted in animals <40 wk old, but the formation of tumors in older {alpha}B-crystallin-/- mice could not be evaluated (because the animals developed a severe skeletal muscular dystrophy by ~40 wk and were killed). Our studies indicate that we have unmasked a subtle predisposition of {alpha}B-crystallin-/- lens epithelial cells to undergo ploidy changes and hyperproliferation in culture. However, the genomic instability we observed appears to be at early stages of cellular changes that accompany evolution of normal to cancerous cells, since the hyperproliferative {alpha}B-crystallin-/- cells did not grow on soft agar or produce tumors in nude mice. This suggests that the number of genetic changes needed to cause tumorigenicity has not occurred in the {alpha}B-crystallin-/- lenses.

The present studies indicate that culturing lens epithelial cells from mice lacking {alpha}B-crystallin resulted in effects that were extremely different from those observed in cells derived from mice lacking {alpha}A-crystallin. The expression of {alpha}A-crystallin is more lens specific than that of {alpha}B-crystallin, but is not induced by stress, and {alpha}A and {alpha}B are complexed in 3:1 stoichiometry in lens fiber cells (4 , 11) . In contrast to the hyperproliferation and ploidy changes observed in {alpha}B-crystallin-/- lens epithelial cells, the absence of {alpha}A-crystallin decreased the in vitro growth potential of cultured lens epithelial cells (17) , indicating that knocking out {alpha}A or {alpha}B-crystallin affected the in vitro growth of lens epithelial cells in opposite ways. This suggests that {alpha}A and {alpha}B-crystallin have distinct and independent cellular functions in the lens. It will be important in future work to determine whether {alpha}A or {alpha}B-crystallin directly alter the regulation of the cell cycle in vivo. Since secondary cataracts are primarily a growth problem, it would be significant to understand whether the increase in lens epithelial cell growth in secondary cataracts is associated with change in expression of {alpha}A or {alpha}B-crystallins.


   CONCLUSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
REFERENCES
 
In summary, lens epithelial cells derived from {alpha}B-crystallin-/- mice suggest that {alpha}B-crystallin may be an important component of the cellular machinery involved in maintaining genomic stability. By culturing lens epithelial cells, a novel effect of knocking out a chaperone-like molecule was unmasked. The unique phenomenon described here for the stress-inducible chaperone {alpha}B-crystallin may be particularly significant in growing tissues, tumors, and neurodegenerative diseases. Genomic instability has been associated with other dystrophic diseases such as myotonic dystrophy (49 , 50) , but it is not known whether it is involved with the reported in vivo phenotype of the {alpha}B-crystallin-/- mice (15) . It is likely that maintaining genomic integrity is important in the lens, since the anterior lens epithelial cells are held in G0 phase of the cell cycle throughout life (even though they are capable of mitosis when subjected to injury) and cell division is restricted to the germinative zone (51 , 52) . The proliferation and ploidy changes of the {alpha}B-crystallin-/- cells provide a basis for future work on {alpha}B-crystallin interactions with cell cycle components, including p53, the retinoblastoma protein Rb, cyclins, and cyclin-dependent kinases.


   ACKNOWLEDGMENTS
 
This work is supported by National Eye Institute, NIH grants R01EY05681 (U.P.A.), R01EY09852 (S.B.), and EY02687 (Core grant), and by Research to Prevent Blindness, Inc.


   FOOTNOTES
 
2 Current address: MetaMorphix, Inc., Baltimore, MD 21227, USA.

Received for publication May 15, 2000. Revision received June 15, 2000.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 

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