|Molecular Vision 2006;
Received 11 March 2006 | Accepted 11 September 2006 | Published 13 September 2006
Expression and characterization of the catalytic subunit of telomerase in normal and cataractous canine lens epithelial cells
Carmen Maria Helena Colitz,1
Curtis Andrew Barden,1 Ping Lu,1
Heather Lynn Chandler2
Department of 1Veterinary Clinical Science and 2Veterinary Biosciences, The Ohio State University, Columbus, OH
Correspondence to: Carmen M. H. Colitz, DVM, PhD, Department of Veterinary Clinical Science, The Ohio State University, Columbus, OH, 43210; Phone: (614) 292-3551; FAX: (614) 688-8566; email: firstname.lastname@example.org
Purpose: To determine whether the catalytic subunit of telomerase reverse transcriptase (TERT) is regionally distributed in canine lens epithelial cells (LEC), compare TERT and the RNA subunit of telomerase (TR) mRNA expression and TERT protein expression in normal and cataractous LEC, and to evaluate whether telomerase activity is present in the cytoplasm and nucleus from normal LEC. Finally, the expression of p23 and heat shock protein 90 (hsp90), coactivators of TERT in neoplastic cells, were evaluated in normal and cataractous LEC.
Methods: TERT protein was detected by imunohistochemical staining and western immunoblotting in normal and cataractous LEC. Quantitative RT-PCR (qRT-PCR) was used to measure TERT and TR expression. Separated cytoplasmic and nuclear extracts from primary cultures of normal canine LEC were evaluated for TERT protein expression and telomerase activity. Western immunoblotting was performed on normal and cataractous LEC for p23 and hsp90, and coimmunoprecipitation was used to determine whether p23 and hsp90 were interacting with TERT in LEC.
Results: TERT expression in normal lens capsule whole mounts varied by region in normal LEC. All cataractous LEC demonstrated more intense TERT immunostaining in both the nucleus and cytoplasm when compared to normal LEC. Normal LEC expressed less TERT protein and less TERT and TR mRNA than cataractous LEC. Normal LEC expressed hsp90 while cataractous LEC did not; p23 was not significantly expressed in either normal or cataractous LEC. Neither hsp90 nor p23 interacted with TERT.
Conclusions: The localization of TERT in normal LEC corresponded with the LEC's regional functions. There was more cytoplasmic TERT in the central region that corresponds with the need for inhibited apoptosis and for proliferative capabilities; there was more nuclear TERT in the germinal and equatorial regions corresponding with the need for proliferative capabilities. In addition, cataractous LEC demonstrated increased TERT protein and increased TERT and TR mRNA expression than normal LEC corresponding with their increased proliferative potential. However, the telomerase coactivators, p23 and hsp90, are not overexpressed and do not associate with TERT in cataractous LEC, suggesting that telomerase regulation in cataractous LEC, a somatic cell type, differs from that in neoplastic cells.
The adult crystalline lens is lined on its anterior surface with a monolayer of lens epithelial cells (LEC) with variable replicative potential. Central LEC are capable of undergoing mitosis, though this occurs in less than 1% of the cells at any given time but increases substantially following traumatic injury [1-4]. The germinative LEC undergoes a controlled amount of mitosis thoughout life that contributes to the continuous growth of the lens, and the equatorial LEC undergoes terminal differentiation into lens fiber cells [1-10]. Despite having varied mitotic capabilities, the three regions have equivalent amounts of telomerase activity . Telomerase is a ribonucleoprotein complex responsible for extending eukaryotic telomeric ends. This prevents chromosomal degradation, recombination, and helps to repair DNA strand breaks [12,13]. Studies in other cell types indicate that these activities are important in preventing cell senescence and oxidative stress-induced apoptosis [14-18]. Telomerase activity is present in some normal proliferating stem and germ cells [19-21], as well as most cancers [22-24], However, telomerase is absent, or found at extremely low levels, in most normal somatic cells, except for the gastrointestinal epithelia, developing neuronal cells, and LEC [11,25,26].
The telomerase complex is composed of two major subunits, an RNA subunit (TR) and the catalytic subunit of telomerase reverse transcriptase (TERT) . TR is expressed in most cells, including those that lack telomerase activity , but TERT expression directly correlates with telomerase activity [29-32]. We previously showed that normal LEC have equivalent amounts of telomerase activity in all three regions of the lens and that cataractous LEC have significantly increased telomerase activity . However, we did not evaluate the regional or intracellular localization of telomerase or the relationship between TR and TERT mRNA levels and telomerase activity. In the present study, we used immunohistochemical staining techniques to determine the regional distribution of TERT as well as real time RT-PCR to evaluate expression of TERT and TR mRNA in normal and cataractous LEC. Though not quantitative, immunohistochemical staining is an accepted and established method for evaluating telomerase activity in archived human tissue, as TERT expression correlates with telomerase activity [33-35]. Due to its role in proliferation, telomerase activity would be expected to result in positive TERT immunoreactivity in the nucleus. However, TERT has recently been shown to be located in the cytoplasm in some cell types, possibly linking it to an antiapoptotic role . Primary cultures of canine LEC were evaluated for cytoplasmic and nuclear telomerase activity to see if TERT was active. In addition, expression of p23 and heat shock protein 90 (hsp90), co-activators of the telomerase holoenzyme in neoplastic cells, were evaluated in LEC to determine whether they interacted with TERT.
Normal globes were obtained by enucleation from dogs in good general health that were euthanized at a local animal shelter for population control purposes. All dogs used in this study were estimated to be between 1 and 8 years of age, based on dentition and thickness of the anterior lens capsule. The animals were humanely euthanized, and the globes were collected within 1 h of death. Eyes were immediately placed in 2% betadine solution and then rinsed and immersed in 1X phosphate-buffered saline solution (PBS, pH 7.2) until dissection, which was performed within 2 h of enucleation.
Normal whole lenses
Dissection of the lenses was performed following a previously described procedure . Whole lenses were immediately fixed in neutral-buffered 10% formalin, embedded in paraffin, and sectioned. Routine hematoxylin and eosin (H&E) staining was performed to assess the orientation of the specimens, and six specimens were used in immunohistochemical staining experiments. In addition, anterior lens capsules were harvested using a previously described technique and fixed, embedded in paraffin, and sectioned (n=20) or snap frozen and stored at -70 °C until protein (n=16) or RNA extraction (n=16) could be performed.
Anterior lens capsule mounts
Fine scissors were used to incise around the equator of five normal adult canine lenses, estimated to be between 3 and 8 years of age. Each anterior capsule was gently separated from the underlying lens fiber mass and placed on a Probe-On Plus (Fisher Scientific, Pittsburgh, PA) slide, cell side up, and dried for 3 min at room temperature. Four perpendicular cuts were made at the edges of the capsule to allow the capsule to flatten on the slide. Fixation with 4% paraformaldehyde for 10 min was followed by a 5 min wash in 1X PBS prior to immunostaining.
Primary canine lens epithelial cell cultures
Anterior lens capsules with adherent LEC were incubated in trypsin (1X EDTA, Invitrogen, Carlsbad, CA) for 5 min at 37 °C, centrifuged to pellet the LEC, then resuspended and cultured on laminin-coated flasks (BD Biosciences, San Jose, CA). The LEC were evaluated seven days post-plating to ensure they were growing, then just prior to reaching confluence, they were trypsinized, pelleted, then protein was extracted as will be described in the next section.
Cataractous anterior lens capsules
Anterior capsulorrhexis specimens from dogs with naturally developing cataracts were obtained prior to routine phacoemulsification cataract extraction. This tissue is routinely disposed of and the Ohio State University College of Veterinary Medicine's Hospital Board did not require further consent. Samples were obtained by continuous curvilinear capsulorrhexis and either immediately placed in 10% neutral-buffered formalin (n=20) or snap frozen and stored at -70 °C until protein (n=18) or RNA extraction (n=30) could be performed. Whole cataractous lenses (n=8) from patients with anterior lens luxations were immediately fixed in 10% neutral-buffered formalin. Fixed samples were embedded in paraffin, sectioned, and stained with H&E, before being examined by light microscopy to assess whether there were sufficient cells for immunohistochemical staining. For immunohistochemical staining, samples were sectioned onto charged slides (ProbeOn Plus, Fisher Scientific).
Quantitative reverse transcriptase polymerase chain reaction for TERT and TR
RNA was extracted according to the suggested protocol using Absolutely RNA Microprep Kit (Stratagene, La Jolla, CA). The ImPromII Reverse Transcriptase kit (Promega, Madison, WI) was used to synthesize the first strand cDNA. Quantitative RT-PCR was performed using the Mx3000p Multiplex Quantitation System (Stratagene) as follows: 95 °C for 15 min, then 45 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s, using QuantiTect SYBR Green PCR kit (Stratagene). Primers to amplify sequence of TR were designed based on the cloning and sequence data obtained in our laboratory (submitted to GenBank AY833720). The primers to amplify TERT and hypoxanthine phosphoribosyl-transferase (HPRT; housekeeping control) were based on previously published sequence data (TERT AF380351; HPRT NM_001003357). Employed primers were as follows: TERT/F (canine) GAA CTG ACG TGG AAG ATG AA; TERT/R (canine) CCT GGC CAG GAT CTC CTC TC; TR/F (canine) GAG GCC GCG GCC GGC CCG GG; TR/R (canine) GCG GCC CGC GGC TGA CAG AGC C; HPRT/F (canine) GGT GGA GAT GAT CTC TCA AC; HPRT/R (canine) GGT CCT TTT CAC CAG CAA GCT.
The threshold cycle value was calculated for each sample by the instrument software. The relative amount of TR, TERT, and HPRT mRNA was calculated using a previously described procedure . The results were expressed as the ratio of the target gene to the HPRT housekeeping gene. Statistical analysis was performed with Prism software (GraphPad Prism® version 4, San Diego, CA) using a student t-test with Wilcoxon signed rank test, p<0.05.
Immunocytochemical and immunohistochemical staining for TERT
Standard immunohistochemical staining was performed following guidelines described in reference . Anti-TERT (Calbiochem) antibody was used at a dilution of 1:400. Stained samples were examined under light microscopy and photographed (Olympus, Melville, NY). A canine lymph node sample was used as the positive control, and the negative control omitted the primary antibody.
Four digital photographs were taken in each of the three regions from three of the five normal lens capsule whole mounts. Each image was then evaluated by counting the number of cells with (1) positive immunostaining only in the cytoplasm, (2) positive immunostaining only in the nucleus, (3) positive immunostaining in both cytoplasm and nucleus, and (4) no immunostaining. The percentage of cells with cytoplasmic only, nuclear only, both cytoplasmic and nuclear, or no immunostaining, was calculated for each anatomical region of the lens epithelium. The cataract samples were not evaluated in a similar manner because these samples only had the central and a portion of the germinative regions represented, and they were paraffin-embedded and examined in cross-section. Statistical analysis of the normal anterior capsule whole mounts was performed using ANOVA with Tukey's test of multiple means. The cross-sections of normal and cataractous samples were not evaluated for statistical differences in cellular compartment staining. The cytoplasm and nuclear compartments of all cataractous cells stained positively.
Protein from normal and cataractous anterior lens capsules with adherent LEC was extracted following manufacturer's instructions (Chemicon International Inc., Temecula, CA) with minor adaptations for our tissue. Lens capsules were minced using fine scissors while still frozen and while on ice. Scissors were maintained RNAse free and cleaned between each sample. Ice-cold CHAPs lysis buffer (10 mM Tris-HCl, 1 mM MgCl2, 1 mM EGTA, 0.1 mM benzamidine, 5 mM β-mercaptoethanol, 0.5% CHAPs, 10% glycerol; Chemicon International, Inc.) was added to each sample and vortexed for 15 s, then incubated on ice for 30 min. The lysate was centrifuged for 20 min at 16,000x g at 4 °C, then the supernatant was transferred to a new RNAse free tube. The protein concentration of each sample was determined using the Bio-Rad CD protein Assay (Bio-Rad Laboratories, Hercules, CA).
Separation of cytoplasmic and nuclear protein was performed using a commercially available kit (N-PER; Pierce, Rockford, IL) as per the manufacturer's instructions. Protein fractions from LEC adhered to the lens capsule could not be separated using this method; therefore, only primary cultures of canine LEC were evaluated in this manner. Separation of nuclear and cytoplasmic proteins was confirmed with western blot analysis using anti-hsp90 antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA; 1:500). Hsp90 is restricted to the cytoplasm and routinely used to assess separation as per the Pierce Biotechnology brochure.
Western blot analysis of TERT, p23, and hsp90
Western blot analysis was used to compare protein expression as well as to confirm the specificity of the anti-TERT antibodies used in immunostaining experiments using previously described methods . Primary antibodies used were as follows: anti-TERT antibody (Santa Cruz Biotechnology Inc.; H-231, 1:2500), anti-p23 antibody (Abcam, Cambridge, MA; 1:1000), and anti-hsp90 antibody (Santa Cruz Biotechnology Inc.; 1:500). Protein signals were detected by chemiluminescence (Femto Western blotting system; Pierce Biotechnology) using Kodak X-OMAT AR film. Membranes were stripped (Restore stripping solution; Pierce) and the technique was repeated using anti-β-actin antibody (Sigma-Aldrich, St. Louis, MO; 1:5000) to control for gel loading. Control samples for anti-TERT antibodies included whole protein extract from a canine mast cell tumor and a melanoma positive for telomerase activity.
Immunoprecipitation and immunoblotting of TERT/p23/hsp90
Six normal and six cataractous canine samples were evaluated. Two kits were used to perform this experiment separately to ensure results were consistent. The Seize Mammalian Immunoprecipitation Plate-based kit (Pierce Biotechnology) and the Catch & Release version 2.0 kit (Upstate Cell Signaling Solutions, Chicago, IL) were used following manufacturers' instructions. Briefly, the Catch and Release kit required incubation of the capture antibody (anti-TERT H-231; Santa Cruz Biotechnology Inc.), 100 ng of the sample protein, affinity ligand, and wash buffer overnight in the supplied spin column. The control for this kit was protein incubated with rabbit IgG. The Seize kit's initial step bound the capture antibody (anti-TERT H-231; Santa Cruz Biotechnology Inc.) to the protein G coated wells. Protein samples (10 μg) were then immunoprecipitated and, following appropriate incubation and washes, the antigen:antibody complex was eluted. Controls for the Seize kit included a well without either antigen or antibody. Elutions from both kits were applied to a SDS-PAGE gel for electrophoresis and immunoblotting with anti-TERT antibody (TERT L-20; Santa Cruz Biotechnology Inc.), anti-p23 antibody (Abcam, Cambridge, MA), or anti-hsp90 antibody (Santa Cruz Biotechnology Inc.). Results were scanned and evaluated using the Kodak EDAS 290 digital imaging system.
Telomeric repeat amplification protocol
The telomeric repeat amplification protocol (TRAP) assay, modified by Intergen Company (Chemicon International Inc.), is an extension of the original method . In the first step, telomerase, if present in the protein lysate, adds a number of telomeric repeats onto the 3' end of a biotinylated Telomerase Substrate oligonucleotide (b-TS). In the second step, the extended products are amplified by PCR using Taq polymerase, the b-TS and RP (reverse) primers, and a deoxynucleotide mix containing dCTP labeled with dinitrophenol, generating a ladder of products with six base increments starting at 50 nucleotides (Chemicon International, Inc.). Briefly, a master mix was prepared with 5X TRAP Reaction mix, Taq polymerase (Panvera Takara; Fisher Scientific), and RNAse-free water. One μg of protein was added to the master mix for a final volume of 50 μl; all set-up steps were performed on ice. Primer extension was then performed at 30 °C for 30 min, followed by 33 cycles of amplification as follows: denaturation for 30 s at 94 °C and annealing for 30 s at 53 °C. Samples were held at 4 °C until detection using (1) hybridization and ELISA (absorbance measured at 450 nm with reference wavelength of 690 nm) following manufacturer's protocol and (2) electrophoresis on a 10% nondenaturing polyacrylamide gel that was subsequently stained with SYBR® gold (1:10,000; Invitrogen, Carlsbad, CA). By comparing the PAGE gel method of evaluating TRAP results with the ELISA methods, we have set 0.100 units as the lowest amount of detectable telomerase activity.
Quantitative reverse transcriptase polymerase chain reaction for TR and TERT in lens epithelial cells
TR mRNA expression was increased in cataractous LEC when compared to normal LEC, but this difference was not statistically significant (Figure 1A). Canine TERT mRNA was expressed at significantly higher levels in cataractous LEC than in normal LEC (p<0.05; Figure 1B).
Immunocytochemical and immunohistochemical staining
Cells in the normal anterior lens capsule whole mounts and paraffin-embedded sections had positive immunostaining in the cytoplasm, nucleus, or both, varying by region (Figure 2A,B,C,G,H,I). Quantitation of immunostaining for TERT in normal whole mounts revealed that 81.02% of the cells in the central region had only cytoplasmic staining, 18.24% had both nuclear and cytoplasmic staining, 0.39% had only nuclear immunostaining, and 0.35% had no immunostaining (Figure 2A,D). All comparisons were statistically significant (p<0.001), except for the comparison between nuclear staining and negative staining (p>0.05). In the germinative region, 49.10% of cells had only cytoplasmic staining, 50.71% had both nuclear and cytoplasmic staining, no cells had only nuclear immunostaining, and 0.21% showed no immunostaining for TERT (Figure 2B,E). The comparisons between cytoplasmic staining and cytoplasmic plus nuclear staining, and between nuclear staining and negative staining were not significant (p>0.05). All other comparisons (nuclear versus cytoplasmic, nuclear versus both, negative versus cytoplasmic, and negative versus both) were significantly different (p<0.001). In the equatorial region, a total of 21.95% of cells had only cytoplasmic staining, 76.61% had both nuclear plus cytoplasmic staining, no cells had only nuclear immunostaining, and 0.14% had no immunostaining for TERT (Figure 2C,F). All comparisons were statistically significant (p<0.001) except for the comparison between nuclear staining and negative staining (p>0.05).
Quantitative evaluation of cataractous anterior lens capsules could not be performed using the whole mount method because the LEC are extremely flattened and often multilayered (Figure 3). Collection during surgery does not provide the entire anterior capsule; only an axial button encompassing approximately 40% of the capsule is collected. The axial anterior capsulotomies removed at the time of cataract surgery were evaluated by immunohistochemistry as were whole cataractous lenses collected from patients with anterior lens luxations. They were compared to normal whole lenses and anterior capsules from normal lenses using standard immunohistochemical staining methods. LECs in all regions from cataractous samples had intense nuclear immunoreactivity and diffuse cytoplasmic immunoreactivity (Figure 4A,B); both were more intense than in the normal LEC (not shown).
Western blot analysis and immunoprecipitation of TERT in canine LEC
Western blot analysis confirmed the presence of TERT protein in normal and cataractous canine LEC. It also confirmed the specificity of the antibody used in the immunostaining experiments (Figure 5). Results shown are representative of eight separate western blot analyses evaluating two normal, four cataract, and two control samples per blot. There was greater TERT expression in the cataractous LEC samples than in the normal LEC. The canine mast cell tumor, used as a positive control, showed exceptionally high expression of TERT compared to the cataractous LEC samples. A faint band was evident in normal LEC; in cataract and mast cell tumor samples, a strong band was evident; and, in normal lens fibers, the band was absent. To confirm TERT expression in canine LEC, we used immunoprecipitation with anti-TERT (H-231) antibody followed by immunoblotting with the anti-TERT (L-20) antibody identified TERT in canine LEC (Figure 6). The opposite was also performed with similar results. This was not performed to quantitate protein expression, only to confirm western data.
Western blot of p23 and hsp90 in canine LEC
Normal LEC demonstrated higher hsp90 expression than cataractous LEC (Figure 7). Neither normal nor cataractous LEC demonstrated significant p23 expression. Coimmunoprecipitation experiments between TERT and p23 or hsp90 did not demonstrate an interaction between TERT and p23 or hsp90 (not shown).
Western blot analysis confirms subcellular localization of canine TERT expression
Once the cytoplasmic and nuclear protein fractions from primary cultures of normal LEC were separated, Western blotting detected a faint band cytoplasmic fraction, similar in intensity to the nuclear fraction even though the nuclear fraction had demonstrable telomerase activity and the cytoplasmic fraction did not (Figure 8A). Hsp90 was used to demonstrate that separation of protein fractions had occurred (Figure 8A). This separation procedure worked well for cultured cells, but did not effectively separate nuclear and cytoplasmic protein fractions of normal or cataractous LEC still adherent to the lens capsule, even with trypsin incubation. For this reason we could not confirm the immunostaining findings in normal LEC that identified regional differences in TERT expression. However, the detection of TERT in both the nuclear and cytoplasmic protein fractions of primary cultures of canine LEC supports the immunohistochemical staining results.
Telomerase activity in lens epithelial cells
All samples evaluated by western blot analysis also underwent TRAP evaluation for the presence or absence of telomerase activity. The canine mast cell tumor had high telomerase activity, the fiber cells were negative for telomerase activity, and the normal LEC had less telomerase activity than the cataractous LEC, as expected and previously reported (not shown) . The separation method yielded acceptable protein yield from the primary cultures of canine LEC but the level of telomerase activity seen in freshly dissected LEC was not observed. We have previously shown that primary cultures of canine LEC gradually lose telomerase activity when they are put into culture and passaged . The reason for this is unknown, but since TERT protein is observed by Western immunoblotting and immunohistochemical staining, loss of phosphorylation may be responsible; this was not evaluated in this study.
Though extremely low, telomerase activity in the separated fractions demonstrated that the nuclear fraction had telomerase activity, and the cytoplasmic fraction was negative (Figure 8B,C).
We previously reported that telomerase activity was significantly higher in cataractous LEC than in normal canine LEC , and we speculate that this was due to increased transcription of TERT, the first level of telomerase regulation . In the present study, qRT-PCR clearly demonstrated that TERT transcription levels were correlated with telomerase activity in both normal and cataractous canine LEC. Since TR is constitutively expressed in all cells, no significant change was expected nor seen.
One of the goals of this study was to localize the catalytic subunit of telomerase, TERT, in normal and cataractous LEC. The central LEC require protection from ultraviolet light and other oxidative stressors that could lead to cataractogenesis. Therefore, we hypothesized that TERT would be relegated to the cytoplasm or nucleus or both, depending on the regional needs of those cells. We discovered that the central region had significantly more cytoplasmic TERT expression than nuclear plus cytoplasmic expression. TERT, a ribonucleoprotein, was originally thought to be restricted to the nuclei of cells [33,34]; however, recently it has also been found in the cytoplasm of cells that have telomerase activity [27,41]. Cytoplasmic TERT, not involved in telomerase activity, has been shown to inhibit apoptosis at a premitochondrial step in the apoptotic pathway  but can also be recruited to the nucleus when needed. In nonneoplastic lymphocytes following stimulation with TNFα, telomerase activity was evident and increased in the cytoplasm prior to translocating to the nucleus. This implies that proteins involved in increasing telomerase activity interact in the cytoplasm then move into the nucleus for DNA repair and proliferation . A recent study by Jagadeesh, et al.  also showed that translocation of TERT from the cytoplasm to the nucleus is one of the methods by which telomerase is regulated. These centrally located LECs also require the ability to undergo proliferation in situations such as traumatic injury. Under normal circumstances, the central region of LEC are rarely mitotic. Rat LEC have less than 1% of cells in the cell cycle at any given time , most being in the G1 phase of the cell cycle . LEC in the central region of the rabbit lens have an estimated mitotic index of 0.02%  and mature human central LEC have a mitotic index of 0.016%  which decreases with age. Upon explantation or mechanical wounding, the cells reenter the cell cycle and undergo mitosis by 24 h post-wounding , and by three days post-explantation, the cells have achieved a high rate of mitosis . In rabbits, injury to the central LEC results in proliferation within 48 h . Therefore, cytoplasmic TERT has numerous roles including protection against oxidative stress-induced apoptosis, and when necessary, translocation to the nucleus for telomerase activation.
Nuclear TERT functions to maintain the telomeres in proliferating cells and a linear relationship between telomerase activity and the rate of cellular proliferation has been shown . The germinative and equatorial regions have LEC undergoing a controlled slow rate of mitosis [5,9,10] which explains why these cells have an increase in nuclear TERT immunoreactivity and a detectable amount of telomerase activity . Our studies show that while there was regional variation in cytoplasmic versus nuclear TERT immunoreactivity in normal LEC, cataractous LEC had more nuclear staining in all regions. It is probable that in response to injury, explantation of the peripheral LEC, or during cataractogenesis, cytoplasmic TERT translocates to the nucleus in a reparative response to increase proliferation. In clinical cases of peripheral lens capsule rupture in dogs, the LEC proliferate rapidly and are seen to encircle the entire outside of the anterior and posterior lens capsules within days of rupture, histologically . Immunohistochemically, the LEC on both sides of these lens capsules have increased expression of TERT in the nucleus as well as the cytoplasm. Cataractous LEC undergo increased proliferation [38,45], and as shown previously, they have increased telomerase activity compared to normal LEC . Conventional thought regarding telomerase activity denotes an immortal more rapidly proliferating phenotype and though cataractous LEC are not immortal, the increase in TERT expression correlates with increased telomerase activity and increased proliferation.
Lens fiber cells did not express TERT and are negative for telomerase activity  as they should be since they are unable to proliferate and undergo terminal differentiation and denucleation under normal conditions [46-48]. However, under experimental conditions, transgenic mice overexpressing the oncogenes E6 and E7 showed hyperproliferation in the transitional zone of the fiber cell compartment . Therefore, TERT expression might have been maintained in these cells for unusual circumstances. A recent study demonstrated that the lens is able to differentially regulate the processes of terminal differentiation and apoptosis, depending on the situation . Perhaps, the loss of TERT expression is a step in the terminal differentiation process resulting in critically shortened telomeres that induces permanent senescence.
Many telomerase-associated proteins have been identified thus far in neoplastic cells and include hsp90 and p23 . Hsp90 and p23 are molecular chaperones necessary for the assembly of a functional telomerase complex and they are the first telomerase-associated proteins to contribute to telomerase activity in neoplastic cells . We have shown that hsp90 and p23 are not associated with TERT in normal or cataractous LEC and their expression is decreased in cataractous LEC. This shows that telomerase in LEC differs from neoplasia-associated telomerase. We do not have an explanation for this, though it may be one of the reasons that LEC do not undergo naturally occurring neoplastic transformation. There is still an incredible amount of knowledge that is unknown about telomerase regulation in nonneoplastic cells.
We have shown that TERT transcription correlates with telomerase activity in normal and cataractous LEC, which is consistent with TERT transcription being the first level of regulation in telomerase activity . However, this is not the only method by which telomerase activity is regulated; phosphorylation is also important . Preliminary data from our laboratory suggest that phosphorylation also plays a role in the increased telomerase activity seen in catararactous LEC . Further studies are in progress to confirm this observation and to identify the kinase(s) responsible for phosphorylation of TERT in normal and cataractous LEC.
In summary, the three regions of normal LECs showed specific TERT localization patterns that correlate with the regional differences and needs of normal LEC. The LEC in the central region express more cytoplasmic TERT, allowing them to retain potential proliferative capabilities and also for inhibition of oxidative stress-induced apoptosis. The germinative region has increased nuclear TERT expression compared to the central region indicating ongoing proliferation. Further investigation into the roles of TERT and telomerase in the cytoplasm and nucleus of normal LEC appears warranted. TERT expression correlates with telomerase activity and with the increased proliferative capacity necessary in cataractous LEC. In addition, LEC have different telomerase regulation compared to neoplastic cells as evidenced by negligible expression of p23 and hsp90 in cataractous LEC. Furthermore, neither p23 nor hsp90 interact with TERT in normal or cataractous LEC. We are presently attempting to determine the functional differences between nuclear and cytoplasmic TERT as well as to evaluate TERT's transcriptional and posttranslational regulation.
The authors would like to sincerely thank Dr. Anne Gemensky-Metzler, Dr. David Wilkie, Dr. Dineli Bras, Dr. Terah Robbin, Dr. Vanessa Kuonen, Dr. Ellen Belknap, Ms. Chris Basham, and Ms. Kelley Norris for help in collecting cataractous canine anterior lens capsules. This work was supported by NIH EY-00414.
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