Bovine Lens Epithelial Cells Research Articles

Protective Action of Hydrogen Sulfide-Releasing Compounds against Oxidative Stress-Induced Cataract Formation in Cultured Bovine Lenses

ABSTRACT Purpose The gaseous signalling molecule, hydrogen sulfide (H2S) has antioxidant, anti-inflammatory and anti-apoptotic properties. Since oxidative stress has been implicated in the pathogenesis of cataracts and lenticular hydrogen peroxide (H2O2) is elevated in some cataract patients, the present study investigated the ability of H2S-releasing compounds to prevent H2O2-induced cataract formation in cultured bovine lenses. Methods Lenses were cultured in either Dulbecco's Modified Eagle Medium (DMEM; control); H2O2 (50 mM); ascorbic acid (AA; 3 mM) (positive control); and the H2S-releasing compounds (diallyl trisulfide [DATS] or GYY4137) in the presence of H2O2 (50 mM). Lens opacity was determined using a plate reader to measure transmittance. Lens glutathione content (GSH), superoxide dismutase (SOD) activity and lactate dehydrogenase (LDH) cytotoxicity were assessed before and after treatment with the H2S-releasing compounds. Results Both DATS (10−7M – 10−4M) and GYY4137 (10−7M – 10−4M) significantly (p < .001) attenuated H2O2 (50 mM)-induced loss in transmittance, with DATS (10−4M) and GYY4137 (10−7M) achieving a maximal reversal of opacity by 56.86 ± 0.01% (n = 6) and 8.39 ± 0.11% (n = 6) after 120 hours, respectively. These observations were corroborated by photographic evaluation, where DATS (10−5M – 10−4M) and GYY4137 (10−7M – 10−5M)-treated lenses had relatively clear grids after 120 hours, compared to H2O2 (50 mM)-treated lenses. The H2O2 (50 mM)-induced decline in total GSH content and total SOD activity were significantly (p < .001; n = 5) reversed by DATS (10−4M) and GYY4137 (10−7M). After 24 hours, DATS (10−4M) and GYY4137 (10−7M) significantly (p < .001; n = 4) reduced cytotoxicity of primary bovine lens epithelial cells by 33.88 ± 4.59% and 36.19 ± 10.53%, respectively. Conclusion Both H2S-releasing compounds protected cultured bovine lenses against oxidative stress-induced cataract formation. The slow-releasing H2S compound, GYY4137 was more potent than DATS in restoring lenticular total GSH content and total SOD activity along with reducing H2O2 (50 mM)-induced cytotoxicity

Oxidative Modifications in Crystallin Proteins and Lens Epithelial Cells Associated with Photosensitized Reactions Mediated by the Major Chromophore Arising from Glucose Degradation

Deleterious effect mediated by glucose degradation, as a parallel pathway to Maillard reaction, was analyzed in terms of the feasibility of inducing photo-crosslinking in isolated bovine crystalline proteins when exposed to ultraviolet A (UVA)-visible light. These experiments showed evidence supporting the generation of a glucose-derived chromophore (GDC). The ability of this chromophore to induce oxidative damage in lens protein and bovine lens epithelial cells (BLEC) was further assessed. The analysis of dityrosine and carbonyl levels in lens proteins irradiated at 5 and 20% O 2 indicates the occurrence of mixed type I/type II photosensitizing mechanisms. When BLEC were exposed to photosensitized reactions induced by GDC a decrease in cellular viability and intracellular reduced (GSH) and oxidized (GSSG) glutathione ratio was observed, as well as an increase in the amount of intracellular reactive oxygen species. Our data suggest a major effect of type I photosensitizing mechanism in both lens proteins photo-oxidation and oxidative stress induced in BLEC.

Linolenic and Linoleic Acid Induce the Opening of Connexin 43, 46 and 50 Hemichannel in Human Hela Cells

Abstract: Connexin (Cx) hemichannels (HC) located at the plasma membrane have a very low open probability, but under physiological conditions they can briefly open, allowing passage of both ions and small signaling molecules, thereby participating in the control of diverse cellular responses. Cx43, 46 and 50 are expressed in human lens and are essential to maintaining its transparency, since mutations in the genes coding for these Cxs have been identified, inducing the development of different types of cataracts. However, non-congenital cataracts may result from consumption of polyunsaturated fatty acids (PUFAs). While the consumption of omega-6 PUFAs, such as linoleic acid (ω-6LA) has been associated with development of cataracts, intake of omega-3linolenic acid (ω-3LA) has been associated with their decline. Here, we report the effects of ω-6LA and ω-3LA on HCs in HeLa cells that express Cx43, Cx46 or Cx50. Bath application of ω-6LA or ω-3LA induces opening of HCs (measured as Ethidium uptake) formed by Cx43, Cx46 or Cx50in HeLa cells and also in human lens epithelial cell line (HLE) that expresses endogenously Cx46 and Cx50 HCs, in a concentration- dependent manner and inhibited by HC blockers such as Lanthanum. Both PUFAs also increased the membrane current mediated by these HCs measured by patch clamp assays in the whole cell configuration in both cell types. Since PUFAs have demonstrated cytotoxic effects in HLE and also in organotypic cultures of bovine lens epithelial cells, their effect on HCs might be relevant to development and prevention of cataracts.

The effects of TiO_2 nano thin film on the migration and adherence of BLEC in vitro

Objective The generate of posterior capsular opacification(PCO)is associated with the adhensive and migration of residual subcapsular lens epithelial cells(LECs).Titanium dioxide(TiO_2)nanometer is proved to have the ability of killing tumor cells and cultured bovine LECs.This study tried to observe the effects of TiO_2 nanometer thin film provoked by light on adhesiveness and migration of bovine LECs in vitro.MethodsThe fresh bovine lenses were obtained and cultured in DMEM containing 10% of newborn bovine serum.The second to fifth generation of cells were used in this experiment.The slide modified by TiO_2 photocatalyst film was prepared by sol-gel method.Cultured cells were seeded in filmed or unfilmed slides respectively and exposed to ultraviolet(wavelength 365 nm)for 20 or 40 minutes.The contact angle between water drop and slide was measured by droping method and the cells adhered to slides were calculated after 24 and 48 hours of culture.The growth status and migration distance of bovine LECs were assayed and compared between filmed and unfilmed groups.ResultsThe contact angle between water drop and slide was 0°±2°and 18.825°±2.342° in filmed and unfilmed group respectively,indicating a obviously smaller contact angle in TiO_2 filmed group than unfilmed one.The numbers of bovine LECs adhered to filmed slide was considerably reduced in TiO_2 filmed group compared with unfilmed group in different UVA exposure time(t_(0 min)=5.492,P=0.001;t_(20 min)=6.031,P=0.000;t_(40 min)=6.828,P=0.000).However,no significant difference was found in the numbers of adhensive cells among 3 UVA irradiation time points(F=1.278,P=0.297).The migration distance of the cells was significantly shorter in TiO_2 filmed group in comparison with unfilmed group in 24 and 48 hours after UVA irradiation(F_(group)=14.965,P=0.000;F_(time)=38.033,P=0.000).ConclusionThe TiO_2 nanometer thin film is characterized by the superhydrophilic property.So it can effectively impede the adhesion and migration of bovine LECs in vitro. Key words: lens epithelium cell; after cataract; titanium dioxide; photocatalyst film

The effects of connective tissue growth factor on bovine lens epithelial cells in vitro

Objective This study was to observe the effects of connective tissue growth factor(CTGF) on migration and transdifferentiation of bovine lens epithelial cells(BLECs). MethodsThe culture and identification of BLECs adopted the method of Hu(reference 1).The 2-3 passages of BLECs were collected and used in this experiment at the concentration of 1×10~6 cells/hole.The free-serum DMEM containing 0.1 ng/L,0.5 ng/L,1.0 ng/L of CTGF was added into medium for 24 hours in different experimental group respectively,and only equal volume of free-serum DMEM was added in control group.Expression of α-smooth muscle actin(α-SMA) mRNA and protein in the BLECs were examined by semiquantitative RT-PCR and Western blot,respectively.The transwell inserts were used to evaluate the migration ability of BLECs. ResultsThe expression of α-SMA mRNA in cultrued BLECs was gradually increased in different concentrations of CTGF groups.Compared with control group,the expression of α-SMA mRNA in experimental group was significantly enhanced (F=66.56,P＜0.01).The expression of α-SMA protein followed the same pattern(F=65.43,P＜0.01).The migration ability of BLECs was obviously elevated after CTGF stimulation under the light microscope.The migration rate of BLECs was considerably increased in experimental group compared with blank control group (t=51.7,P＜0.01).ConclusionCTGF promotes the migration and transdifferentiation of BLECs at a dose-dependent manner in vitro.CTGF plays an important role in the formation of posterior capsule opacification. Key words: connective tissue growth factor; lens epithelial cells; migration; smooth muscle actin; cataract

The apoptosis of bovine lens epithelial cells induced by proteasome inhibitor MG132

To investigate the effect of proteasome inhibitor MG132 on the apoptosis of bovine lens epithelial cells (BLECs), the cells were treated with MG132 at different concentrations for 12, 24 and 36 h. The cell viability was analyzed by MTT assay and the effect of MG132 on the apoptosis of BLECs was assessed by flow cytometry (FCM). The results showed that after treatment for the same period, the inhibitory effect of MG132 on BLECs proliferation was enhanced with the increment of the concentration of MG132 (0, 2, 5, 10, mumol/L) (P<0.05). The 50% inhibiting concentration (IC(50)) was 2.03 mumol/L when the BLECs were treated with MG132 for 36 h. MG132 also induced the apoptosis of BLECs obviously. FCM showed that the apoptosis index of the cells treated by MG132 at 2 mumol/L for 12 h was (20.24+/-1.51)%, and that of the control was (0.98+/-0.20)% respectively (P<0.01, n=3). It was concluded that MG132 could lead to apoptosis of BLECs. The decrease of proteasome activity may play an important role in the formation and development of cataract.

The Apoptosis of Bovine Lens Epithelial Cells Induced by Proteasome Inhibitor MG132

目的 观察蛋白酶体抑制剂MG132对晶状体上皮细胞(LECs)凋亡的诱导作用.方法 用不同浓度的MG132分别处理牛LECs 12、24、36h,通过MTT法检测细胞活力,流式细胞术(FCM)检测MG132对牛LECs凋亡的影响.结果 在作用时间相同的条件下,随着MG132浓度的增高(0、2、5、10μmol/L),对牛LECs增生的抑制作用逐渐增强(P＜0.01).当作用时间达36h时,半效抑制浓度IC50为2.03μmol/L.同时,MG132能够明显诱导牛LECs凋亡:FCM分析结果表明,浓度为2μmol/L的MG132作用12h,细胞早期凋亡率为20.24%±1.51%,对照组为0.98%±0.20%(P＜0.01,n=3).结论 蛋白酶体抑制剂MG132能够诱导牛LECs凋亡,蛋白酶体活性的丧失可能在白内障的形成中起重要作用。

Effects of lutein on the growth and migration of bovine lens epithelial cells in vitro

The effects of lutein on the growth and migration of bovine lens epithelial cells (BLECs) in vitro were observed in an attempt to find a drug that can prevent after-cataract. BLECs were cultured in vitro and different concentrations of lutein were added to the BLECs cultures of the second and third generations. The effects of lutein on the proliferation of BLECs in vitro were examined by the MTT method, and the migration of BLECs was evaluated by a scratch wound assay. The results showed that: (1) Lutein at concentrations of 1 to 16 micromol/L could inhibit the proliferation of BLECs in a dose-and time-dependent manner (P<0.01): (2) The migration of BLECs was evaluated by wound healing rate. As compared with the control group, the wound healing rate in the experimental groups was decreased from 0.672+/-0.164 to -0.234+/-0.144 and -0.597+/-0.063 (P<0.01) at 1 and 2 micromol/L lutein, respectively. It was concluded that lutein at concentration of >or=1 micromol/L inhibited the proliferation and migration of BLECs in vitro. Lutein may become an effective drug to prevent after-cataract.

Expression and localisation of apical junctional complex proteins in lens epithelial cells

The lens epithelium possesses an apical junctional complex (AJC) comprising adherens and tight junctions (AJs and TJs) and yet several key structural components and associated regulatory proteins have not been identified or localised in these cells. Here we determine the subcellular distribution of the archetypal TJ markers (ZO-1, claudin-1, and occludin) and TJ-associated cell polarity proteins (aPKC, Par3 and Par6β) with AJ markers, E- and N-cadherin. As seen in other polarised epithelia, all these markers were located by confocal immunofluorescence microscopy to the apical ends of the lateral plasma membranes of bovine lens epithelial cells at sites of cell–cell interaction. Using immunoelectron microscopy, we show that ZO-1 concentrated at “kissing points” between neighbouring cells and these data, when taken in the context of our confocal immunofluorescence microscopy and blotting data, suggest the presence of TJs within the AJC. Likewise, immunogold labelling for E-cadherin identified AJs within these AJCs. We also report aPKC immunogold labelling localised to the AJC. These data show that the AJC of lens epithelial cells are a composite of TJs and AJs.

Status of caveolin-1 in various membrane domains of the bovine lens

Recent studies of the distribution and relative concentration of caveolin-1 in fractions of bovine lens epithelial and fiber cells have led to the novel concept that caveolin-1 may largely exist as a peripheral membrane protein in some cells. Caveolin-1 is typically viewed as a scaffolding protein for caveolae in plasma membrane. In this study, membrane from cultured bovine lens epithelial cells and bovine lens fiber cells were divided into urea soluble and insoluble fractions. Cytosolic lipid vesicles were also recovered from the lens epithelial cells. Lipid-raft domains were recovered from fiber cells following treatment with detergents and examined for caveolin and lipid content. Aliquots of all fractions were Western blotted for caveolin-1. Fluorescence microscopy and double immunofluorescence labeling were used to examine the distribution of caveolin-1 in cultured epithelial cells. Electron micrographs revealed an abundance of caveolae in plasma membrane of cultured lens epithelial cells. About 60% of the caveolin-1 in the epithelial-crude membrane was soluble in urea, a characteristic of peripheral membrane proteins. About 30% of the total was urea-insoluble membrane protein that likely supports the structure of caveolae. The remaining caveolin was part of cytosolic lipid vesicles. By contrast, most caveolin in the bovine lens fiber cell membrane was identified as intrinsic protein, being present at relatively low concentrations in caveolae-free lipid raft domains enriched in cholesterol and sphingomyelin. We estimate that these domains occupied 25–30% of the fiber cell membrane surface. Thus, the status of caveolin-1 in lens epithelial cells appears markedly different from that in fiber cells.

Changes in gap junction organization and decreased coupling during induced apoptosis in lens epithelial and NIH-3T3 cells

We demonstrate that global induction of apoptosis in primary bovine lens epithelial (LEC) or fibroblastic mouse NIH-3T3 cells by staurosporine, puromycin, cycloheximide, or etoposide is accompanied by a decrease in coupling by gap junctions. Cell coupling as tested by neurobiotin spreading was maintained when the LEC or NIH-3T3 cells were pre-incubated with the pan-caspase inhibitor zVAD or the caspase-3 inhibiting tetrapeptide DEVD. Immunohistochemistry using anti-connexin-43 antibodies showed a reduction of plasma membrane integrated connexin-43 in both cell lines when undergoing apoptosis. Western blotting indicated degradation of connexin-43 that was inhibited by zVAD or DEVD. Cell coupling at single cell level was tested by direct microinjecting into LEC apoptosis-inducing agents of low molecular mass like staurosporine, etoposide and puromycin or the high molecular mass proteins caspase-3 and -8 in activated state. Microinjection of puromycin or etoposide induced apoptotic morphological changes of only the injected cell within 90 or 180 min, but did not affect adjacent cells. In contrast, microinjection of staurosporine led to a rapid induction of apoptosis of the injected and a number of adjacent cells suggesting spreading of staurosporine most probably through gap junction pores held open by dephosphorylation of connexin-43 as verified by immunoblotting and staining using a phospho-serine368-specific anti-connexin-43 antibody. Microinjection of active caspase-8 led after 3 h to morphological apoptotic alterations of only the injected cell, but did not inhibit spreading of co-injected neurobiotin to neighboring cells during the first hour. In contrast, microinjection of active caspase-3-induced apoptosis only of the injected cell after 60 min and rapidly and completely suppressed coupling to neighboring cells.

Effect of acrylamide on the cytoskeleton and apoptosis of bovine lens epithelial cells

Acrylamide, a known disrupter of intermediate filaments, has been used to produce the collapse of vimentin filaments in bovine lens epithelial (BEL) cells, and its potential modulation of staurosporine-induced apoptosis has been investigated. In BEL cells, short treatments with acrylamide caused the collapse of vimentin filaments and microtubules and the almost complete disappearance of stress fibers, with thick f-actin bundles remaining in the cell periphery. Actin organization was less affected in cells pretreated with colchicine and in spreading cells, suggesting that extended microtubules and vimentin filaments are required for acrylamide to produce its maximal effects. Acrylamide alone slightly increased apoptosis compared to controls. However, simultaneous exposure to acrylamide and staurosporine for 8h produced significantly less apoptosis than staurosporine alone, and preincubation with acrylamide followed by staurosporine markedly reduced apoptosis at 8 and 24h of treatment. Acrylamide seems therefore to have a dual effect on BEL cell survival.

Modification of the surface properties of a lens material to influence posterior capsular opacification

To study the effect of surface properties of materials on cellular behaviour and the formation of posterior capsular opacification (PCO). Polymethylmethacrylate, silicone and a hydrophobic acrylic were plasma treated and used in tissue culture. The changes in surface properties were quantified by dynamic contact angle measurements. Bovine lens epithelial cells (BLECs) were seeded onto these materials and cultured for 1 month. Serial photographs were taken. The cells were then fixed and stained to facilitate counting. Plasma treatment significantly increased the hydrophilicity of surfaces. BLECs grew on all surfaces but significantly more cells adhered to the treated than the untreated surfaces. On the untreated surfaces the BLECs had a fibroblastic morphology whereas on the treated surfaces the cells maintained their epithelial morphology. Posterior capsular opacification is a form of wound healing and the behaviour of lens epithelial cells is central to its progression. Emphasis has been on the elimination of residual lens epithelial cells to combat PCO. This study demonstrated that the phenotype of BLECs was influenced by the surface properties of the intraocular lens materials. Gas plasma treatment of the materials increased their hydrophilicity and allowed the adhered BLECs to maintain their normal epithelial morphology. We believe that controlled growth of lens epithelial cells may reduce the incidence of PCO.

PTEN Deletion Leads to Up-regulation of a Secreted Growth Factor Pleiotrophin

Tumor suppressor gene PTEN is highly mutated in a wide variety of human tumors. To identify unknown targets or signal transduction pathways that are regulated by PTEN, microarray analysis was performed to compare the gene expression profiles of Pten null mouse embryonic fibroblasts (MEFs) cell lines and their isogenic counterparts. Expression of a heparin binding growth factor, pleiotrophin (Ptn), was found to be up-regulated in Pten-/- MEFs as well as Pten null mammary tumors. Further experiments revealed that Ptn expression is regulated by the PTEN-PI3K-AKT pathway. Knocking down the expression of Ptn by small interfering RNA resulted in the reduction of Akt and GSK-3beta phosphorylation and suppression of the growth and the tumorigenicity of Pten null MEFs. Our results suggest that PTN participates in tumorigenesis caused by PTEN loss and PTN may be a potential target for anticancer therapy, especially for those tumors with PTEN deficiencies.

Signal transduction mechanism of curcumin in inhibiting the proliferation of bovine lens epithelial cell induced by recombinant human epidermal growth factor

To investigate the signal transduction mechanism of curcumin in inhibiting the proliferation of bovine lens epithelial cell (LEC) induced by recombinant human epidermal growth factor (rhEGF). There were three groups in this experiment, which were normal control group, untreated group and curcumin-treated group. Proliferation of LEC was induced by rhEGF (50 microg/L). The concentration of intracellular Ca(2+) ([Ca(2+)](i)) in LEC was measured with Fura-2/AM by spectrofluorimetry. The contents of intracellular cAMP and cGMP were assayed by radioimmunoassay. The [Ca(2+)]i in LEC was obviously increased in the untrated group as compared with that in the normal control group (P<0.01), and the [Ca(2+)](i) in LEC in the curcumin-treated group was highest among three groups (P<0.01). The content of intracellular cAMP in LEC was decreased while the content of intracellular cGMP was obviously increased in the untreated group as compared with those in the normal control group (P<0.01). The content of intracellular cAMP in LEC was higher in the curcumin-treated group than that in the untreated group, while the content of intracellular cGMP was lower than that in the untreated group (P<0.01). The antiproliferation effects of curcumin on LEC may relate to the regulations of multiple processes of signal transduction.

Fatty Acid Cytotoxicity to Organ-Cultured Bovine Lenses

High sensitivity of cultured bovine and human lens epithelial cells to unsaturated free fatty acids has been reported in previous papers. Here we show that micromolar concentrations of unsaturated free fatty acids also impair lens cells during organ culturing, a system which resembles the in vivo situation much better than cell cultures do. This added weight to the assumption that fatty acid cytotoxicity might be causally related to senile cataract as well as to cataracts associated with systemic diseases.

Multiple forms of 22 kDa caveolin-1 alpha present in bovine lens cells could reflect variable palmitoylation

Two-dimensional immunoblots of immunoprecipitated caveolin-1 from cultured bovine lens epithelial cells revealed four to five-22 kDa forms of caveolin-1 alpha with isoelectric points of between pH values 5.5 and 6.6. Fibre cell membrane recovered from fresh bovine lenses displayed an even greater number of multiforms, some with isoelectric point pH values as low as about 4. Caveolin-1 can be both phosphorylated and palmitoylated. None of the caveolin-1 alpha multiforms were labelled following culture of the lens epithelial cells with 32P-orthophosphate nor were they recognized by either caveolin-specific phosphotyrosine antibody or protein anti-phosphoserine antibody and treatment of lens fibre cell membrane with phosphatase did not alter the two-dimensional profile of immunoreactive caveolins. However, short-term incubation of BLEC with 3H-palmitate labelled some of the immunoprecipitated caveolin-1 multiforms. We suggest that the observed spectrum of caveolin multiforms could reflect variable palmitoylation of its three cysteine residues and result in populations of caveolin-1 alpha molecules with separate physical and functional properties.

Comparative characterization of rat deoxyribonuclease 1 (Dnase1) and murine deoxyribonuclease 1-like 3 (Dnase1l3)

Deoxyribonuclease 1 (DNASE1, DNase I) and deoxyribonuclease 1-like 3 (DNASE1L3, DNase gamma, DNase Y, LS-DNase) are members of a DNASE1 protein family that is defined by similar biochemical properties such as Ca2+/Mg2+-dependency and an optimal pH of about 7.0 as well as by a high similarity in their nucleic acid and amino acid sequences. In the present study we describe the recombinant expression of rat Dnase1 and murine Dnase1l3 as fusion proteins tagged by their C-terminus to green fluorescent protein in NIH-3T3 fibroblasts and bovine lens epithelial cells. Both enzymes were translocated into the rough endoplasmic reticulum, transported along the entire secretory pathway and finally secreted into the cell culture medium. No nuclear occurrence of the nucleases was detectable. However, deletion of the N-terminal signal peptide of both nucleases resulted in a cytoplasmic and nuclear distribution of both fusion proteins. Dnase1 preferentially hydrolysed 'naked' plasmid DNA, whereas Dnase1l3 cleaved nuclear DNA with high activity. Dnase1l3 was able to cleave chromatin in an internucleosomal manner without proteolytic help. By contrast, Dnase1 was only able to achieve this cleavage pattern in the presence of proteases that hydrolysed chromatin-bound proteins. Detailed analysis of murine sera derived from Dnase1 knockout mice revealed that serum contains, besides the major serum nuclease Dnase1, an additional Dnase1l3-like nucleolytic activity, which, in co-operation with Dnase1, might help to suppress anti-DNA autoimmunity by degrading nuclear chromatin released from dying cells.

Human Telomerase Reverse Transcriptase Immortalizes Bovine Lens Epithelial Cells and Suppresses Differentiation through Regulation of the ERK Signaling Pathway

Telomerase is a specialized reverse transcriptase that extends telomeres of eukaryotic chromosomes. The functional telomerase complex contains a telomerase reverse transcriptase catalytic subunit and a telomerase template RNA. We have previously demonstrated that human telomerase reverse transcriptase (hTERT) catalytic subunit is functionally compatible with a telomerase template RNA from rabbit. In this study, we show that hTERT is also functionally compatible with a telomerase template RNA from bovine. Introduction of hTERT into bovine lens epithelial cells (BLECs) provides the transfected cells telomerase activity. The expressed hTERT in BLECs supports normal growth of the transfected cells for 108 population doublings so far, and these cells are still extremely healthy in both morphology and growth. In contrast, the vector-transfected cells display growth crisis after 20 population doublings. These cells run into cellular senescence due to shortening of the telomeres and also commit differentiation as indicated by the accumulation of the differentiation markers, beta-crystallin and filensin. hTERT prevents the occurrence of both events. By synthesizing new telomere, hTERT prevents replicative senescence, and through regulation of MEK/ERK, protein kinase C, and protein kinase A and eventual suppression of the MEK/ERK signaling pathway, hTERT inhibits differentiation of BLECs. Our finding that hTERT can suppress RAS/RAF/MEK/ERK signaling pathway to prevent differentiation provides a novel mechanism to explain how hTERT regulates cell differentiation.

Comparison of effects of U18666A and enantiomeric U18666A on sterol synthesis and induction of apoptosis

Treatment of animals or cells with the amphipathic tertiary amine U18666A {3beta-[2-(diethylamino) ethoxy]androst-5-en-17-one} provides models for several human diseases (e.g., cataracts, Niemann-Pick disease, and epilepsy). Although U18666A can inhibit several enzymes in the cholesterol synthesis pathway, we hypothesized that induction of these varied conditions was due to physical effects of the amine rather than to inhibition of specific proteins. To test this possibility we compared the capacity of U18666A and its enantiomer, ent-U18666A, to inhibit net sterol synthesis and induce apoptosis in cultured bovine lens epithelial cells. Nonenantiospecific actions dependent on the physical properties of these mirror image molecules would be identical, but effects dependent upon enantiospecific interactions would be different for the enantiomers. At the same concentrations, both forms of the compound equally inhibited sterol synthesis and induced apoptosis. These observations supported a generalized mechanism of enzyme inhibition such as perturbation of the microenvironment of endoplasmic enzymes and alteration of membrane order, perhaps of the mitochondrial membrane, to explain induction of apoptosis.