the human lens epithelial line crl-1142 ( atcc , manassas , va , usa ) was used for this study . these human lens epithelial cells were cultured in dulbecco 's modified eagle 's medium ( sigma , st louis , mo , usa ) , which is composed of 10% fetal bovine serum , penicillin ( 100 u / ml ) , streptomycin ( 100 u / ml ) , and nystatin ( 25 ug / ml ) at 37 in humidified atmosphere , containing 5% co2 . the human lens epithelial cells were irradiated for 30 seconds with an irradiance of 0.6 mw / cm after removal of the serum - free growth medium . uv irradiance was modulated by adjusting the lamp output , calibrated with a uv sensor ( fs-20 t12-uvb ; national biological corp . , the uvb spectrum was between 280 nm and 320 nm with peak irradiance at 312 nm . after 0 , 2 , 4 , 6 , 12 , and 24 hours incubation , cell viability was evaluated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) assay . a solubilization solution is added to dissolve the insoluble purple formazan product into a colored solution . the mtt assay was then carried out using a standard protocol and the optical density was measured at 570 nm using a spectrophotometer . we compare the control group ( not exposed to uv irradiation ) with the experimental group ( 4 hours after 18 mj / cm uv shot ) . the cells were washed with phosphate buffer saline ( pbs ) and resuspended to 1  10 cells / ml in pbs . an equal volume of hoechst dye was then added ( working solution 5 mm ) . hoechst dye allows easy observation of the changes in nuclei by producing a bright fluorescence at 465 nm . we incubated the cells for 15 min at 37 , and then either analyzed the cells for fluorescence or washed the cells and fixed them in paraformaldehyde ( in 1% pbs ) for future analysis . we compare the control group ( not exposed to uv irradiation ) with the experimental group ( 4 hours after 18 mj / cm uv shot ) . the cells , which were cultured on the cover glass , were fixed in 4% paraformaldehyde at normal temperature for 30 minutes and then washed with pbs ( ph 7.4 ) . in order to increase the permeability , 0.1% sodium citrate ( ph 6.0 ) , which contains 0.1% triton x-100 , was added and allowed to react for 2 minutes . the primary antibody , caspase 3 ( 1:50 ) , was allowed to react at room temperature for 120 minutes and the cells were then washed with pbs . the secondary antibody , alex flour goat anti - rabbit 488 ( 1:200 ) , was allowed to react at room temperature for 60 minutes , washed with pbs , and visualized under a fluorescent microscope ( leica el6000 ; leica microsystems , wetzlar , germany ) . for western blot analysis , membranes were incubated overnight at 4 with the rabbit primary antibodies ( cell signaling technology , beverly , ma , usa ) diluted in 3% skim milk . the blots were then incubated for 60 minutes at room temperature ( rt ) with rabbit primary antibodies against phospho - akt ( ser473 ) ( 1:1,000 ) , akt ( 1:1,000 ) , phospho - fkhr ( ser256 ) ( 1:1,000),and phospho - gsk-3 ( ser9 ) ( 1:1,000 ) . after washing three times ( 20 minutes per wash ) using tris - buffered saline tween-20 , the blots were incubated with anti - rabbit secondary antibodies conjugated with horseradish peroxidase ( 1:5,000 in 2% milk ; jackson immunoresearch laboratories inc . , immunoreactive bands were visualized using an enhanced chemiluminescence light detection kit ( amersham , piscataway , nj , usa ) . , san antonio , tx , usa ) was used as an internal control . in all of the figures with densitometry data , optical density refers to the integrated density . to increase the reliability of the data the data was analyzed for significance using repeated measures , followed by duncan 's multiple range tests . the data are expressed as a mean percentage of the control value plus standard error measurement the human lens epithelial line crl-1142 ( atcc , manassas , va , usa ) was used for this study . these human lens epithelial cells were cultured in dulbecco 's modified eagle 's medium ( sigma , st louis , mo , usa ) , which is composed of 10% fetal bovine serum , penicillin ( 100 u / ml ) , streptomycin ( 100 u / ml ) , and nystatin ( 25 ug / ml ) at 37 in humidified atmosphere , containing 5% co2 . the human lens epithelial cells were irradiated for 30 seconds with an irradiance of 0.6 mw / cm after removal of the serum - free growth medium . uv irradiance was modulated by adjusting the lamp output , calibrated with a uv sensor ( fs-20 t12-uvb ; national biological corp . , the uvb spectrum was between 280 nm and 320 nm with peak irradiance at 312 nm . after 0 , 2 , 4 , 6 , 12 , and 24 hours incubation , cell viability was evaluated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) assay . a solubilization solution is added to dissolve the insoluble purple formazan product into a colored solution . the mtt assay was then carried out using a standard protocol and the optical density was measured at 570 nm using a spectrophotometer . we compare the control group ( not exposed to uv irradiation ) with the experimental group ( 4 hours after 18 mj / cm uv shot ) . the cells were washed with phosphate buffer saline ( pbs ) and resuspended to 1  10 cells / ml in pbs . an equal volume of hoechst dye was then added ( working solution 5 mm ) . hoechst dye allows easy observation of the changes in nuclei by producing a bright fluorescence at 465 nm . we incubated the cells for 15 min at 37 , and then either analyzed the cells for fluorescence or washed the cells and fixed them in paraformaldehyde ( in 1% pbs ) for future analysis . we compare the control group ( not exposed to uv irradiation ) with the experimental group ( 4 hours after 18 mj / cm uv shot ) . the cells , which were cultured on the cover glass , were fixed in 4% paraformaldehyde at normal temperature for 30 minutes and then washed with pbs ( ph 7.4 ) . in order to increase the permeability , 0.1% sodium citrate ( ph 6.0 ) , which contains 0.1% triton x-100 , was added and allowed to react for 2 minutes . the primary antibody , caspase 3 ( 1:50 ) , was allowed to react at room temperature for 120 minutes and the cells were then washed with pbs . the secondary antibody , alex flour goat anti - rabbit 488 ( 1:200 ) , was allowed to react at room temperature for 60 minutes , washed with pbs , and visualized under a fluorescent microscope ( leica el6000 ; leica microsystems , wetzlar , germany ) . for western blot analysis , membranes were incubated overnight at 4 with the rabbit primary antibodies ( cell signaling technology , beverly , ma , usa ) diluted in 3% skim milk . the blots were then incubated for 60 minutes at room temperature ( rt ) with rabbit primary antibodies against phospho - akt ( ser473 ) ( 1:1,000 ) , akt ( 1:1,000 ) , phospho - fkhr ( ser256 ) ( 1:1,000),and phospho - gsk-3 ( ser9 ) ( 1:1,000 ) . after washing three times ( 20 minutes per wash ) using tris - buffered saline tween-20 , the blots were incubated with anti - rabbit secondary antibodies conjugated with horseradish peroxidase ( 1:5,000 in 2% milk ; jackson immunoresearch laboratories inc . , immunoreactive bands were visualized using an enhanced chemiluminescence light detection kit ( amersham , piscataway , nj , usa ) . , san antonio , tx , usa ) was used as an internal control . in all of the figures with densitometry data to increase the reliability of the data , all experiments were repeated 3 times and the average values were obtained . spss ver . 10.1 ( spss inc . , chicago , il , usa ) was used to compute routine statistics . the data was analyzed for significance using repeated measures , followed by duncan 's multiple range tests . the data are expressed as a mean percentage of the control value plus standard error measurement after the human lens epithelial cells were exposed to ultraviolet light , the survival rate was measured by mtt assay at 0 , 2 , 4 , 6 , 12 , and 24 hours later . the mtt reduction ratios for the control group and at6 hours and 12 hours after uv exposure were 100 , 72  4.9 , and 21.5  6.7% respectively ( p < 0.05 ) ( fig . 1 ) . in order to investigate the appearance of apoptosis , hoechst staining was performed . 2a ) . the experimental group ( 4 hours after uv exposure ) showed abnormal nuclei appearance with nuclear fragmentation ( fig . it appeared that the fragments moved to neighboring cells or that neighboring cells moved to embrace the fragments . in order to investigate the apoptotic phenotype , the control group showed no fluorescent signal corresponding to active caspase 3 protein ( fig . the signal became significantly stronger and more distributed throughout the cytoplasm and nuclei with a spotted character after uv exposure . in the nuclei , the fluorescence exhibited a clustered pattern . in order to investigate apoptosis gene expression , we once again compared the control group with the experimental group 4 hours after uv exposure . on the western blot examination , an immune band was observed around 53kda ( fig . the expression of p53 , caspase 3 and noxa relative to beta - actin ( genetex inc . ) , used as an internal control , was analyzed . we found that p53 decreased from 86 to 40 , caspase 3 increased from 25 to 103 and noxa increased from 24 to 194 ( fig . apoptosis was first defined by kerr et al . in 1972 . in the last step of apoptosis , a cell divides into several smaller cells that are called " apoptotic bodies " . genetically normal genes make apoptosis , and it is a controllable , positive cell death process . this process is accompanied by a reduction of a cell 's specific gravity ; destruction of cell membranes ; condensation of chromosomes ; and transformation of apoptotic bodies into cysts which then undergo phagocytosis . in an experiment using a large mouse , researchers found that apoptosis caused by ultraviolet light reached its maximum within 24 hours of exposure . while removal of cells may be beneficial to an organism , in certain conditions , apoptosis can kill cells that are essential for continued function . in the case of lenses , proteases such as caspases are involved in cell death , and these enzymes might be activated after the oxidative damage to the lens . cataracts usually start on the lens epithelium . the lens epithelium grows as a cell layer under the front bag and has a generally low capability for proliferation , especially in the central layer . the cells there have a very low capability for proliferation , but they can proliferate very quickly under abnormal circumstances . several medical experiments on living bodies have shown that the lens epithelium , after exposure to ultraviolet light , promptly removes damaged cells ( such as apoptotic cells ) and proliferates normal cells which replace the damaged cells . therefore , it is important to study the role of the lens epithelium in protecting against ultraviolet light and to understand how the lens epithelium may recover after being damaged . although uv irradiation causes various kinds of modifications in lens epithelial cells , the earliest consequences are probably damage to dna and membrane pumps . it is conceivable that uv - damaged membranes would allow an influx of calcium into lens epithelial cells , which is known to activate apoptosis . the quick death of lens epithelial cells through apoptosis following uv irradiation would eliminate homeostatic epithelial cell control of the underlying fiber cells , leading to impairment of the integrity of these underlying fiber cells . such reactions , plus direct damage at the dna and membrane levels , may lead to the observed loss of epithelial cell viability . the death of the epithelial cells plus the uv - induced damage to the fiber cell proteins may lead to rapid lens opacification . in the human body , there are genes that suppress cancers ( called " cancer or tumor suppressing genes " ) which are being investigated using genomic research techniques . one such gene is p53 , which is found on chromosome 17 and which produces p53 protein to kill cancer cells . p53 is one of the most commonly studied genes in apoptosis research , as it prevents various types of cells from proliferating and may lead them to apoptosis . a targeted gene that receives a signal from p53 becomes unified with p53 to make various proteins that recover dna and fight cancers . additionally , p53 also has a strong relationship with apoptosis in human lens epithelial cells , and there is some evidence that the p53 pathway may be involved in lens development . expression of p53 has been demonstrated in the lens epithelial cells of the central and pre - equatorial zones and in the lens fibre nuclear bow region , while increased p53 expression in rat lens epithelial cells following exposure to uv light has been found to be associated with apoptosis and cataracts . furthermore , temporally distinct patterns of p53-dependent apoptosis have been identified during mouse lens development . however , once the p53 gene is damaged , it gets to lose the ability of apoptosis and can no longer prevent the proliferation of cancer cells . cellular stresses , such as growth factor deprivation and dna damage , lead to stabilization and activation of the p53 tumor suppressor protein . depending on the cellular context , this can result in one of two different outcomes : cell cycle arrest or apoptotic cell death . cell death induced through the p53 pathway is executed by caspase proteinases , which , by cleaving their substrates , lead to the characteristic apoptotic phenotype . caspase activation by p53 occurs through the release of apoptogenic factors from the mitochondria , including cytochrome c. released cytochrome c allows for the formation of a high - molecular weight complex , the apoptosome , which consists of the adapter protein apaf-1 and caspase 9 , which is activated following recruitment into the apoptosome . active caspase 9 then cleaves and activates the effector caspases , such as caspases-3 and -7 , which execute the death program . the release of mitochondrial apoptogenic factors is regulated by the pro- and anti - apoptotic bcl-2 family proteins , which either induce or prevent the permeabilization of the outer mitochondrial membrane . some of the pro - apoptotic family members , such as bax , noxa or puma , are transcriptional targets of p53 . the elucidation of the p53-dependent pathway , resulting in mitochondrial outer membrane permeabilization through the pro - apoptotic bcl-2 family of proteins , is key to understanding the mechanism of stress - induced apoptosis . noxa is a pro - apoptotic bh3-only member of the bcl-2 family of proteins that is up regulated at a transcriptional level by the nuclear protein p53 in response to cellular stresses such as dna damage or growth factor deprivation . noxa is able to interact with anti - apoptotic members of the bcl-2 family and causes the release of cytochrome c into the cytosol , leading to the activation of caspases and induction of apoptosis . a research group at the medical system in tokyo university revealed that noxa was a newly - targeted gene of p53and clarified that noxa leads apoptosis . the expressed noxa moves to the mitochondria of cells and promotes the discharge of cytochrome c , an inducer of apoptosis . if this noxa gene is removed , apoptosis does not occur ( even partially ) , even though p53 is forcibly expressed . however , it has recently been shown that there is an additional pathway for noxa expression that works independently of p53 . have discovered that mg132 induces noxa expression via a p53-independent mechanism that leads to caspase - dependent apoptosis . a research team led by armstrong jl of northern institute for cancer research , school of clinical and laboratory sciences , newcastle university , uk , has shown that noxa is important in p53-independent fenretinide - induced apoptosis of neuroectodermal tumors . these results demonstrate the importance of noxa induction in determining the apoptotic response to fenretinide and emphasize the role of noxa in p53-independent apoptosis . this study also suggests that there may be a noxa protein that appears independently of p53 ( in addition to noxa that arises directly from increases in p53 ) . we found that uv light caused apoptosis and an increase in cell toxicity on cultured human lens epithelial cells , and that this apoptosis promoted the expression of noxa independently of p53 . our study was designed to understand the possible mechanisms of apoptosis on cultured human lens epithelial cells after exposure to ultraviolet light . we found that exposure to ultraviolet light increased cellular toxicity and induced apoptosis , although apoptosis only occurred after at least 6 hours post - exposure . it is possible that the concentrations of apoptosis initiators are low initially , and increase with time . we also found increased expression of noxa and caspase 3 without increased expression of p53 . this may suggest that uv induced apoptosis in human lens epithelial cells follows a p53 independent pathway .
purposethe purpose of this study is to understand the mechanism of apoptosis occurring on a cultured human lens epithelial cell line after exposure to ultraviolet ( uv ) light . 
 we intended to confirm the presence of cellular toxicity and apoptosis and to reveal the roles of p53 , caspase 3 and noxa in these processes.methodscells were irradiated with an ultraviolet lamp . 
 cellular toxicity was measured by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay . 
 hoechst staining and fluorescent anti - caspase 3 antibodies were used for apoptosis investigation . 
 the quantities of p53 , caspase 3 , and noxa were measured by western blotting for to investigate the apoptosis pathway.resultscellular toxicity on the human lens epithelium markedly increased with time after uv exposure . on hoechst staining , we found that apoptosis also remarkably increased after exposure to ultraviolet light , compared with a control group . in the immunochemical study using anti - caspase 3 antibodies , active caspase 3 significantly increased after exposure to ultraviolet light . on western 
 blotting , p53 decreased , while caspase 3 and noxa increased.conclusionsexposure of cultured human lens epithelial cell lines to ultraviolet light induces apoptosis , which promotes the expression of noxa and caspase 3 increases without increasing p53 . 
 this may suggest that uv induced apoptosis is caused by a p53-independent pathway in human lens epithelial cells .