|Molecular Vision 2000;
Received 3 October 2000 | Accepted 5 December 2000 | Published 13 December 2000
Gene expression in the mouse retina: The effect of damaging light
Charlotte E. Remé
Department of Ophthalmology, University Eye Clinic, Zurich, Switzerland
Correspondence to: Christian Grimm, Ph.D., Department of Ophthalmology, University Eye Clinic, University Hopsital, Zurich, Switzerland; Phone: 1-255-3905; FAX: 1-255-4385; email: email@example.com
Purpose: High levels of visible light induce apoptotic cell death of photoreceptors, a process depending on the activation of the transcription factor AP-1. This suggests that regulation of gene expression might be important for light-induced photoreceptor cell death. We measured expression of AP-1 family members and of several apoptosis-related genes to test their potential involvement in photoreceptor apoptosis.
Methods: Wildtype and c-fos-/- mice were exposed to low (roomlight) or high levels of visible light for up to two hours. Total RNA was prepared from isolated retinas during and after light exposure. Relative mRNA levels were determined semiquantitatively using either competitive or exponential RT-PCR.
Results: Expression of c-fos-/- was upregulated by intense light as early as 15 min after lights on. Highest levels (6-fold induction) were detected at 2 h after lights off declining thereafter to basal levels 20 h after the end of exposure. c-jun mRNA was induced at 30 min after lights on and high expression levels (fourfold induction) persisted at least for 8 h. Similarly, expression of caspase-1 was six to 9-fold increased at 6 to 8 h after light exposure in wildtype but not in c-fos knockout mice. The latter mice are protected against light-induced photoreceptor apoptosis. Expression of other apoptosis-related genes (bcl-2, bcl-XL, bax, bad, caspase-3) was not affected by light exposure or the lack of c-Fos in knockout mice.
Conclusions: Expression of c-fos and c-jun mRNA is transiently induced by exposure to damaging light. Induced expression of c-jun persists longer than expression of c-fos. Among the apoptosis-related genes, only caspase-1 expression was upregulated by light exposure and Caspase-1 might therefore be involved in light-induced retinal degeneration.
Exposure to high levels of visible light induces apoptotic cell death of photoreceptors [1-3]. Photons of the damaging light are absorbed by the visual pigment rhodopsin [1,4] creating an intracellular death signal that leads to activation of the transcription factor AP-1. AP-1 DNA binding activity increases as soon as 15 min after the start of light exposure, reaching a maximum at about 6 h after a 2 h illumination . Activation of AP-1 is essential for light-induced photoreceptor apoptosis since transrepression of AP-1 by activated glucocorticoid receptor protects photoreceptors from light damage (A. Wenzel, personal communication). AP-1 is a complex that consists either of heterodimers of members of the Fos (c-Fos, FosB, Fra-1, Fra-2) and the Jun (c-Jun, JunB, JunD) family of proteins or of homodimers of members of the Jun family of proteins [6,7]. Light-induced complexes are mainly composed of c-Fos, c-Jun and JunD proteins . Whereas JunD is not essential for light-induced photoreceptor apoptosis , the lack of c-Fos completely protects the mouse retina against light damage .
Considerable evidence suggests a role for altered gene expression during apoptosis. Inhibition of both RNA and protein synthesis blocks the onset of apoptosis in a wide variety of systems [11,12] suggesting that specific genes need to be induced and controlled by transcription factors like AP-1. On the other hand, several cell types can express the cell death machinery constitutively at all times. Upon removal of survival signals that seem to suppress the intrinsic death program, such cells die by apoptotic mechanisms without de novo gene expression .
Execution of apoptosis frequently depends on the Bcl-2 family of proteins. Both death antagonists (e.g., Bcl-2, Bcl-XL, Bcl-w, Bfl-1, Bag-1, Mcl-1, A1) and agonists (e.g., Bax, Bak, Bcl-XS, Bad, Bid, Bik, Hrk) belong to the Bcl-2 family of proteins. Most of these proteins contain a transmembrane domain which localizes them predominantly to the outer mitochondrial membrane  where they might be involved in the regulation of the transmembrane potential controlling the release of pro-apoptotic factors like cytochrome c into the cytoplasm.
In the retina, several of these pro- and anti-apoptotic genes are expressed [15,16] and may affect retinal degeneration. Overexpression of Bcl-2 delayed photoreceptor apoptosis in the retinal degeneration slow (rds) mouse  and in the homozygous Pdegtm1 mouse  but not in a mouse carrying a dominant opsin mutation (K296E) [19,20]. Retinal degeneration induced by another rhodopsin mutation (S334ter), however, was delayed by the ectopic expression of Bcl-2 , an effect that was ameliorated by the coexpression of Bag with the Bcl-2 transgene . Mixed results were reported for rescue of retinal degeneration in the retinal degeneration (rd) mouse: opsin driven overexpression of Bcl-2 did not affect photoreceptor apoptosis in a transgenic animal, whereas Bcl-2 delayed the degenerative process when delivered by adenovirus mediated transfer in a gene therapy approach . Furthermore, Bcl-2 overexpression delayed apoptosis induced by constant light  or short term exposure to high intensity green light .
In a variety of tissues, execution of apoptosis frequently relies on the activation of cysteine proteases (caspases) [24,25]. In the retina, several different caspases, mostly including Caspase-3, are activated during apoptosis induced by a variety of stimuli including ischemia, excitotoxicity, treatment with antibodies to heat shock protein 27 , lead and calcium overload , mutations in the opsin gene  or during the degenerative process in the Royal College of Surgeon (RCS) rat . However, retinal degeneration involving oxidative stress could not be prevented by inhibitors of caspase activity  suggesting that both caspase-dependent and caspase-independent apoptosis can occur in the retina. Here, we tested activation of several apoptosis-related genes during light-induced degeneration of photoreceptors in wildtype mice. Besides c-fos and c-jun, caspase-1 was the only apoptosis-related gene upregulated upon light exposure. Gene expression was compared to c-fos-/- mice which are protected against light-induced photoreceptor apoptosis. These mice lack functional c-Fos and may therefore have an AP-1 composition different from wildtype mice. This might affect expression of AP-1 target genes. Therefore, we tested whether any of the common apoptotic genes would be differentially expressed in the protected knockout mice. We show that this was not the case. Except for c-fos, all genes tested were similarly expressed in both wildtype and c-fos-/- mice. This suggests that the protection of c-fos-/- mice against light-induced photoreceptor apoptosis was not due to a generally altered gene expression. Furthermore, our results also suggest that light-induced photoreceptor apoptosis involves upregulation of caspase-1 but not activation of other common pro- or anti-apoptotic genes tested.
All experiments conformed to the ARVO statement for care and use of animals in research and to the guidelines of the Veterinary Authority of Zurich. Wildtype (129SV/Bl6(N2), pigmented; BALB/c, albino) or c-fos-/- mice (genetic background: 129SV/Bl6(N2), pigmented) were raised in cyclic light (12:12 h; 60 lux at cage level) for at least 10 days. The 129SV/Bl6(N2) mice have a mixed 129SV and C57/Bl6 background. They were bred on this background for more than 10 generations.
Light exposure and retinal morphology
Six to 10 week old mice were dark adapted overnight (16 h) and pupils of pigmented mice were dilated under dim red light with 1% Cyclogyl and 5% Phenylephrine 45 min prior to exposure (start at 10 am) to diffuse, white fluorescent light (TLD36 W/965 tubes, Philips; ultraviolet-impermeable diffuser) in cages with reflective interior. After light exposure, mice were either kept in darkness until retinal morphology was analyzed or until retinas were prepared for RNA isolation. For morphological analysis of retinal tissue, enucleated eyes were fixed in 2.5% glutaraldehyde and embedded in Epon 812. Sections were analyzed from both the superior and the inferior central regions of the retina. Shown in Figure 1 are only the inferior central regions, the most affected area in our light damage system.
RNA isolation, cDNA synthesis and PCR
Retinas were removed through a slit in the cornea and immediately frozen in liquid nitrogen. Retinas were stored at -70 °C until RNA preparation. Total retinal RNA was prepared using the RNeasy RNA isolation kit (Qiagen, Hilden, Germany). Reverse transcription was performed on 400 ng of total retinal RNA using oligo(dT) and M-MLV reverse transcriptase (Promega, Madison, USA). cDNAs corresponding to 10 ng of total RNA were amplified with primers specific for b-actin (see below). Amplification products were quantified on a PhosphorImager (Fuji, Tokyo, Japan) for standardization. Standardized cDNAs corresponding to 10 to 20 ng of total RNA were amplified by PCR using the following primer pairs and cycle numbers (linear range of amplification was determined for each amplified fragment in pre-experiments, data not shown): b-actin: 24 cycles; up: 5'-CAA CGG CTC CGG CAT GTG C-3'; down: 5'-CTC TTG CTC TGG GCC TCG-3'. Caspase-3: 30 cycles; up: 5'-AGT CAG TGG ACT CTG GGA TC-3'; down: 5'-GTA CAG TTC TTT CGT GAG CA-3'. Bad: 32 cycles; up: 5'-AGA GTA TGT TCC AGA TCC CAG-3'; down: 5'-GTC CTC GAA AAG GGC TAA GC-3'. Bax: 29 cycles; up: 5'-GCT CTG AAC AGA TCA TGA AG-3'; down: 5'-GAT GGT CAC TGT CTG CCA TG-3'. Bcl-2: 30 cycles; up: 5'-TTG TGG CCT TCT TTG AGT TCG-3'; down: 5'-ATT TCT ACT GCT TTA GTG AAC C-3'. Bcl-XL: 30 cycles; up: 5'-GAC TTT CTC TCC TAC AAG C-3'; down: 5'-CGA AAG AGT TCA TTC ACT AC-3'. Caspase-1: 34 cycles; up: 5'-GAG AAG AGA GTG CTG AAT CAG-3'; down: 5'-CAA GAC GTG TAC GAG TGG TTG-3'. c-Jun: 28 cycles; up: 5'-GCA ATG GGC ACAT CAC CAC-3'; down: 5'-GAA GTT GCT GAG GTT GGC G-3'. c-Fos: 25 cycles; up: 5'-CAA CGC CGA CTA CGA GGC GTC AT-3'; down: 5'-GTG GAG ATG GCT GTC ACC G-3'. Semiquantitative PCR amplification of a 189 bp fragment of c-fos cDNA was done in reactions containing decreasing amounts (5-fold dilutions per step) of a 219 bp long competitor (mimic) DNA. Amplification was done during 30 cycles using the primer pair described above. Downstream primers in all amplification reactions were 32P-end labeled. Amplification products were resolved on a 6% polyacrylamide gel and stained with ethidium bromide. Products were quantified on a PhosphorImager (Fuji).
Expression of c-fos and c-jun
Exposure to high levels of white light induces apoptotic cell death of photoreceptors in wildtype but not in c-fos knockout mice (Figure 1; ). Execution of cell death depends on activation of AP-1 [5,8]. Main components of activated AP-1 are c-Fos, c-Jun and JunD [5,8]. Retinal expression of immediate early genes like c-fos can be regulated by a variety of signals including stress  and light [32,33]. Accordingly, levels of c-fos mRNA increased 3.5- to 4.5-fold at 15 min after dark-adapted mice were exposed to normal roomlight (60 lux; Figure 2A, grey bars) or to damaging light of 13,000 lux (Figure 2A, white bars). In mice exposed to 60 lux, elevated c-fos levels persisted for at least 30 min after lights on, but declined to control levels after 2 h. In contrast, retinal c-fos mRNA levels remained elevated throughout the exposure time of two hours and at least until 2 additional hours after lights off. At 8 h after lights off, retinal c-fos mRNA levels of mice exposed to 13,000 lux declined to levels 2.5-fold above control. After 20 h in darkness, c-fos mRNA levels returned to control levels.
Surprisingly, c-fos mRNA levels of mice not exposed to light also increased transiently (Figure 2A, black bars). Although these mice remained in darkness during the time when experimental mice were exposed, they received low doses (ca. 10 lux) of red light (above 600 nm) during the dilation of their pupils. To test, whether the increase of c-fos mRNA levels in the dark control mice could have been due to handling, pupil dilation and red light illumination, we measured c-fos mRNAs by competitive RT-PCR (i) in retinas of eyes with non-dilated pupils isolated from mice that were dark adapted in separate cages for 16 h, (ii) in retinas of dark adapted mice that were exposed for 30 min to red light (10 lux) and (iii) in retinas of dark adapted mice that had dilated pupils and that were exposed to 30 min of red light (10 lux). Relative c-fos expression in control animals was set as 1 (n=3). Exposure to red light resulted in a 2.3-fold elevation of the RNA levels (n=3). Pupil dilation prior to red light exposure further increased the relative c-fos mRNA levels to a factor of 5.7 (n=3) as compared to the controls.
c-jun mRNA levels were determined by exponential PCR. In contrast to c-fos, c-jun RNA levels were not increased by handling of the animals (data not shown) and only marginally by exposure to room light (Figure 2B, grey bars). c-jun mRNA levels of dark-maintained animals were somewhat elevated during the course of the experiment. The reason for this is not clear but it might be that the control levels (D) were slightly underestimated. Exposure to 13,000 lux resulted in a 3- to 4.5-fold increase of c-jun mRNA levels starting after 30 min of illumination and persisting for more than 8 h post-illumination. Even at 20 h after illumination, c-jun mRNA levels were elevated 3-fold (Figure 2B).
Expression of apoptosis-related genes
Retinal mRNAs of the two anti-apoptotic genes, bcl-2 (Figure 3A) and bcl-XL (Figure 3B), and of the three pro-apoptotic genes bax (Figure 3C), bad (Figure 3D) and caspase-3 (Figure 3E) were expressed at similar levels, independent of the light intensity and duration of exposure. Similarly, RNA levels of dark-maintained animals measured at different timepoints throughout the experiment were not different from control levels (not shown). However, mRNA of caspase-1 was induced more than 9-fold at eight hours after a two hour exposure to 13,000 lux (Figure 3F, white bars). Exposure to 60 lux light did not induce caspase-1 expression (grey bars) and RNA levels of dark-maintained animals remained unchanged throughout the experiment (not shown).
The generality of the induction of caspase-1 expression by damaging light was verified in additional independent experiments using a different mouse strain (BALB/c) and shorter illumination periods (1 h illumination at 13,000 lux instead of 2 h). Although to a lesser extent than in 129SV/Bl6 mice, mRNA levels of caspase-1 were again strongly induced at 6 h after illumination (Figure 4A,B). After the peak of activation, mRNA levels declined steadily and reached almost dark levels at 12 to 14 h after illumination. At 24 h after illumination, however, caspase-1 mRNA levels increased again. This effect was observed also in the experiment shown in Figure 3. In contrast to caspase-1, levels of b-actin mRNA were comparable in all RNA samples (Figure 4C) demonstrating that the induction of caspase-1 was not due to differences in quantity or quality of the RNA preparations.
Lack of c-Fos does not generally alter expression of pro-and anti-apoptotic genes
Since c-Fos is part of the transcription factor AP-1, lack of c-Fos could severely alter expression of pro- and anti-apoptotic genes. This could lead to the observed protection against damaging light. However, with the exception of c-fos (Figure 5A), all genes tested, including caspase-1, were similarly expressed in the retina of dark adapted wildtype and of dark adapted c-fos-/- mice (Figure 5B-H). Immediately after the illumination to 13,000 lux for 2 h, levels of c-fos and c-jun mRNAs were induced in wildtype animals 4- and 2-fold, respectively (Figure 5A,B). At this timepoint, c-jun mRNA was similarly induced (about 2-fold) also in c-fos-/- mice. At 8 h after light exposure, retinal mRNA levels in wildtype mice were elevated 2-fold for c-fos, 4-fold for c-jun and 8-fold for caspase-1; in accordance with the results of the experiments shown in Figure 2. In c-fos-/- mice, however, none of the RNAs tested were elevated above control levels at this timepoint.
Light doses above threshold induce photoreceptor apoptosis in the vertebrate retina. Here we show that exposure to damaging light but not to physiological light levels induces mRNAs of the proto-oncogenes, c-fos and c-jun, and of the cysteine protease caspase-1. Other apoptosis-related genes tested (Bcl-2, Bcl-XL, Bad, Bax, caspase-3) were neither up- nor downregulated by light exposure. Furthermore, all genes tested (except for c-fos) were similarly expressed in dark-adapted wildtype and dark-adapted c-fos-/- mice excluding the possibility that the lack of c-Fos in the knockout mice generally prevents the transcription of apoptosis relevant genes.
Expression of c-fos and c-jun
Activation of c-Fos containing AP-1 is a prerequisite for the induction of the apoptotic program by excessive light [5,8,10]. Induction of the DNA binding activity of AP-1 only occurs in response to damaging light but not after exposure to physiological levels of light . In contrast, c-fos gene expression is also induced by exposure to low levels of light. However, elevated c-fos mRNA levels persist only after exposure to damaging light. When the light pulse was of low intensity, c-fos RNA levels declined rapidly after the initial peak. Similar observations have been made during the exploitation of the diurnal expression of c-fos: when animals received a light pulse during the dark period, c-fos mRNA levels increased transiently for a period of 30 to 60 min .
Retinal c-fos mRNA expression increased even without illumination in our system (Figure 2A). Such a moderate increase in the c-fos mRNA levels could best be explained by stress induced gene expression as has also been observed in animals that were faced, for example, with novelty . Our data of c-fos mRNA levels after handling of the animals, pupil dilation, and exposure to red light, support this hypothesis. In contrast to c-fos, c-jun mRNA varied slightly or not at all following low levels of illumination with white light. When exposed to damaging doses of light, however, c-jun mRNA levels were induced 3 to 5-fold and persisted at elevated levels for at least 20 h (Figure 2B). Induction of c-fos and c-jun gene expression preceded the light-induced increase of AP-1 DNA binding activity which peaks 6 h after the end of light exposure . This suggests that newly made c-Fos and c-Jun proteins contribute to the increase of AP-1 activity. In c-fos-/- mice, c-jun was slightly activated upon exposure to high levels of light (Figure 5B). However, activation did not persist as long as in wildtype animals suggesting that c-Fos containing AP-1 complexes might be involved in the transcriptional regulation of c-jun after light insult. This is supported by the finding that DNA binding of AP-1 is induced as early as 15 min after the onset of light exposure  but elevated c-jun mRNA levels could not be detected before 30 min of light exposure (Figure 2D).
Expression of pro- and anti-apoptotic genes
Apoptosis frequently involves modulation of the transcription of genes encoding proteins involved in the response to apoptotic stimuli. Expression of anti-apoptotic genes like bcl-2 and bcl-XL, for example, have been found to be downregulated after induction of apoptosis by several stimuli in various cell systems [36-41]. This is in contrast to expression of apoptosis promoting genes like bax [42-44], bad , and the cysteine proteases (caspases), which may be upregulated following a pro-apoptotic insult [46-48]. Overexpression of bcl-2 has protective effects on retinal apoptosis induced by a variety of stimuli [17,18,20-23,49]. A major function of Bcl-2 is the regulation of cytochrome c release from mitochondria [50-52] which may induce the apoptotic execution cascade by activating caspase-3 [53,54] a central executioner in many apoptotic systems.
In light-induced photoreceptor degeneration, however, we were unable to detect an alteration in the expression of bcl-2 or caspase-3 (Figure 3 and Figure 5). Similarly, an induction of the enzymatic activity of caspase-3 or the cytoplasmic appearance of cytochrome c could not be detected after light exposure (unpublished results). Therefore, regulation of apoptosis by bcl-2 and/or caspase-3 might play a minor role in light-induced retinal degeneration. The lack of regulation of bcl-XL, another protein thought to be involved in the control of the mitochondrial membrane integrity, and therefore of cytochrome c release  supports this hypothesis.
The role of Caspase-1
In the system of light-induced apoptosis of photoreceptors, caspase-1 was the only gene that was differentially regulated by light exposure. The gene was strongly induced at 6 to 8 h after the end of illumination in two different mouse strains. At present, we do not know why activation of caspase-1 was less strong in BALB/c mice than in 129SV/Bl6 mice. It might be possible, though, that the difference in gene activation reflects strain differences in the regulation of the apoptotic response to damaging light. Caspase-1 (ICE) is an enzyme that might be involved in neuronal cell death. Dorsal root ganglion neurons undergo apoptosis upon withdrawal of nerve growth factor. However, they are protected by the cytokine response modifier crmA, a serpin that specifically inhibits Caspase-1 . Moreover, mice deficient for Caspase-1 [57,58] and mice expressing a dominant negative mutant of this protease  are more resistant to ischemic insult than control animals [60-62]. The main substrate of Caspase-1 is pro-interleukin-1 beta (IL-1b). Cleavage of pro-IL-1b results in the release of the active and pro-inflammatory form of IL-1b [63,64] which has been suggested to have pro-apoptotic effects . A recent report suggests that Caspase-1 can also cleave the inhibitor of caspase-activated deoxyribonuclease (ICAD) leading to the activation of the endonuclease and to the fragmentation of genomic DNA . In the vertebrate retina (rat), caspase-1 expression has been predominantly found in the outer nuclear layer (ONL) . Injection of a specific inhibitor of Caspase-1 decreased the number of apoptotic cells in the ONL in an ischemia-reperfusion model . Increased Caspase-1 activity was also detected during retinal degeneration in RCS rats and inhibitors of Caspase-1 delayed this degenerative process at least partially . Interestingly, light seems to accelerate retinal degeneration in RCS rats . Furthermore, light exposure induced oxidative stress in cultured retinal cells and decreased the anti-apoptotic activity of the transcription factor nuclear factor-kappaB (NF-kappaB) in a Caspase-1 dependent manner . The strong induction of caspase-1 in our model of light-induced retinal degeneration suggests that Caspase-1 might be involved in mediation of apoptotic cell death of photoreceptors after light insult. It is of importance that Caspase-1 was induced only in wildtype mice that were exposed to damaging levels of light but not in mice exposed to physiological light levels or in mutant mice protected against light-induced apoptosis (c-fos-/-). This shows that the induction was not due to the experimental procedure (handling, stress, etc.), and suggests that Caspase-1 might act downstream of c-Fos/AP-1 in the cascade of light-induced photoreceptor apoptosis.
In future experiments, we will test the specific roles of Caspase-1 and of IL-1b in our model of light-induced retinal degeneration and we will test the hypothesis that Caspase-1 plays a central role in the degenerative process of inherited retinal degenerations that are enhanced by light.
We thank D. Greuter, G. Hoegger and C. Imsand for excellent technical assistance in the preparation of histological sections and T. Seiler for continuous support. This work was support by the E & B Grimmke Foundation, Germany, the Bruppacher Foundation, Switzerland, and the Swiss National Science Fondation, Switzerland.
1. Noell WK, Walker VS, Kang BS, Berman S. Retinal damage by light in rats. Invest Ophthalmol 1966; 5:450-73.
2. Reme CE, Grimm C, Hafezi F, Marti A, Wenzel A. Apoptotic cell death in retinal degenerations. Prog Retin Eye Res 1998; 17:443-64.
3. Organisciak DT, Winkler BS. Retinal light damage: practical and theoretical considerations. Prog Retin Eye Res 1994; 13:1-29.
4. Grimm C, Wenzel A, Hafezi F, Yu S, Redmond TM, Reme CE. Protection of Rpe65-deficient mice identifies rhodopsin as mediator of light-induced retinal degeneration. Nat Genet 2000; 25:63-6.
5. Wenzel A, Grimm C, Marti A, Kueng-Hitz N, Hafezi F, Niemeyer G, Reme CE. c-fos controls the "private pathway" of light-induced apoptosis of retinal photoreceptors. J Neurosci 2000; 20:81-8.
6. Curran T, Franza BR Jr. Fos and Jun: the AP-1 connection. Cell 1988; 55:395-7.
7. Hai T, Curran T. Cross-family dimerization of transcription factors Fos/Jun and ATF/CREB alters DNA binding specificity. Proc Natl Acad Sci U S A 1991; 88:3720-4.
8. Hafezi F, Marti A, Grimm C, Wenzel A, Reme CE. Differential DNA binding activities of the transcription factors AP-1 and Oct-1 during light-induced apoptosis of photoreceptors. Vision Res 1999; 39:2511-8.
9. Hafezi F, Grimm C, Wenzel A, Abegg M, Yaniv M, Reme CE. Retinal photoreceptors are apoptosis-competent in the absence of JunD/AP-1. Cell Death Differ 1999; 6:934-6.
10. Hafezi F, Steinbach JP, Marti A, Munz K, Wang ZQ, Wagner EF, Aguzzi A, Reme CE. The absence of c-fos prevents light-induced apoptotic cell death of photoreceptors in retinal degeneration in vivo. Nat Med 1997; 3:346-9.
11. Naora H, Nishida T, Shindo Y, Adachi M, Naora H. Association of nbl gene expression and glucocorticoid-induced apoptosis in mouse thymus in vivo. Immunology 1995; 85:63-8.
12. Yonish-Rouach E, Deguin V, Zaitchouk T, Breugnot C, Mishal Z, Jenkins JR, May E. Transcriptional activation plays a role in the induction of apoptosis by transiently transfected wild-type p53. Oncogene 1995; 11:2197-205.
13. Raff MC, Barres BA, Burne JF, Coles HS, Ishizaki Y, Jacobson MD. Programmed cell death and the control of cell survival: lessons from the nervous system. Science 1993; 262:695-700.
14. Kroemer G, Zamzami N, Susin SA. Mitochondrial control of apoptosis. Immunol Today 1997; 18:44-51.
15. Levin LA, Schlamp CL, Spieldoch RL, Geszvain KM, Nickells RW. Identification of the bcl-2 family of genes in the rat retina. Invest Ophthalmol Vis Sci 1997; 38:2545-53.
16. Shin DH, Lee HY, Lee HW, Kim HJ, Lee E, Cho SS, Baik SH, Lee KH. In situ localization of p53, bcl-2 and bax mRNAs in rat ocular tissue. Neuroreport 1999; 10:2165-7.
17. Nir I, Kedzierski W, Chen J, Travis GH. Expression of Bcl-2 protects against photoreceptor degeneration in retinal degeneration slow (rds) mice. J Neurosci 2000; 20:2150-4.
18. Tsang SH, Chen J, Kjeldbye H, Li WS, Simon MI, Gouras P, Goff SP. Retarding photoreceptor degeneration in Pdegtm1/Pdegtml mice by an apoptosis suppressor gene. Invest Ophthalmol Vis Sci 1997; 38:943-50.
19. Li T, Franson WK, Gordon JW, Berson EL, Dryja TP. Constitutive activation of phototransduction by K296E opsin is not a cause of photoreceptor degeneration. Proc Natl Acad Sci U S A 1995; 92:3551-5.
20. Joseph RM, Li T. Overexpression of Bcl-2 or Bcl-XL transgenes and photoreceptor degeneration. Invest Ophthalmol Vis Sci 1996; 37:2434-46.
21. Chen J, Flannery JG, LaVail MM, Steinberg RH, Xu J, Simon MI. bcl-2 overexpression reduces apoptotic photoreceptor cell death in three different retinal degenerations. Proc Natl Acad Sci U S A 1996; 93:7042-7.
22. Eversole-Cire P, Concepcion FA, Simon MI, Takayama S, Reed JC, Chen J. Synergistic effect of Bcl-2 and BAG-1 on the prevention of photoreceptor cell death. Invest Ophthalmol Vis Sci 2000; 41:1953-61.
23. Bennett J, Zeng Y, Bajwa R, Klatt L, Li Y, Maguire AM. Adenovirus-mediated delivery of rhodopsin-promoted bcl-2 results in a delay in photoreceptor cell death in the rd/rd mouse. Gene Ther 1998; 5:1156-64.
24. Alnemri ES. Mammalian cell death proteases: a family of highly conserved aspartate specific cysteine proteases. J Cell Biochem 1997; 64:33-42.
25. White E. Life, death, and the pursuit of apoptosis. Genes Dev 1996; 10:1-15.
26. Tezel G, Wax MB. Inhibition of caspase activity in retinal cell apoptosis induced by various stimuli in vitro. Invest Ophthalmol Vis Sci 1999; 40:2660-7.
27. He L, Poblenz AT, Medrano CJ, Fox DA. Lead and calcium produce rod photoreceptor cell apoptosis by opening the mitochondrial permeability transition pore. J Biol Chem 2000; 275:12175-84.
28. Liu C, Li Y, Peng M, Laties AM, Wen R. Activation of caspase-3 in the retina of transgenic rats with the rhodopsin mutation s334ter during photoreceptor degeneration. J Neurosci 1999; 19:4778-85.
29. Katai N, Kikuchi T, Shibuki H, Kuroiwa S, Arai J, Kurokawa T, Yoshimura N. Caspaselike proteases activated in apoptotic photoreceptors of Royal College of Surgeons rats. Invest Ophthalmol Vis Sci 1999; 40:1802-7.
30. Carmody RJ, Cotter TG. Oxidative stress induces caspase-independent retinal apoptosis in vitro. Cell Death Differ 2000; 7:282-91.
31. Senba E, Ueyama T. Stress-induced expression of immediate early genes in the brain and peripheral organs of the rat. Neurosci Res 1997; 29:183-207.
32. Imaki J, Yamashita K, Yamakawa A, Yoshida K. Expression of jun family genes in rat retinal cells: regulation by light/dark cycle. Brain Res Mol Brain Res 1995; 30:48-52.
33. Yoshida K, Kawamura K, Imaki J. Differential expression of c-fos mRNA in rat retinal cells: regulation by light/dark cycle. Neuron 1993; 10:1049-54.
34. Nir I, Agarwal N. Diurnal expression of c-fos in the mouse retina. Brain Res Mol Brain Res 1993; 19:47-54.
35. Emmert MH, Herman JP. Differential forebrain c-fos mRNA induction by ether inhalation and novelty: evidence for distinctive stress pathways. Brain Res 1999; 845:60-7.
36. Kihara-Negishi F, Yamada T, Kubota Y, Kondoh N, Yamamoto H, Abe M, Shirai T, Hashimoto Y, Oikawa T. Down-regulation of c-myc and bcl-2 gene expression in PU.1-induced apoptosis in murine erythroleukemia cells. Int J Cancer 1998; 76:523-30.
37. Siegel DS, Zhang X, Feinman R, Teitz T, Zelenetz A, Richon VM, Rifkind RA, Marks PA, Michaeli J. Hexamethylene bisacetamide induces programmed cell death (apoptosis) and down-regulates BCL-2 expression in human myeloma cells. Proc Natl Acad Sci U S A 1998; 95:162-6.
38. Riordan FA, Foroni L, Hoffbrand AV, Mehta AB, Wickremasinghe RG. Okadaic acid-induced apoptosis of HL60 leukemia cells is preceded by destabilization of bcl-2 mRNA and downregulation of bcl-2 protein. FEBS Lett 1998; 435:195-8.
39. Spanaus KS, Schlapbach R, Fontana A. TNF-alpha and IFN-gamma render microglia sensitive to Fas ligand-induced apoptosis by induction of Fas expression and down-regulation of Bcl-2 and Bcl-xL. Eur J Immunol 1998; 28:4398-408.
40. Lin RH, Hwang YW, Yang BC, Lin CS. TNF receptor-2-triggered apoptosis is associated with the down-regulation of Bcl-xL on activated T cells and can be prevented by CD28 costimulation. J Immunol 1997; 158:598-603.
41. Han SS, Chung ST, Robertson DA, Ranjan D, Bondada S. Curcumin causes the growth arrest and apoptosis of B cell lymphoma by downregulation of egr-1, c-myc, bcl-XL, NF-kappa B, and p53. Clin Immunol 1999; 93:152-61.
42. Gong B, Chen Q, Endlich B, Mazumder S, Almasan A. Ionizing radiation-induced, Bax-mediated cell death is dependent on activation of cysteine and serine proteases. Cell Growth Differ 1999; 10:491-502.
43. Perlman H, Zhang X, Chen MW, Walsh K, Buttyan R. An elevated bax/bcl-2 ratio corresponds with the onset of prostate epithelial cell apoptosis. Cell Death Differ 1999; 6:48-54.
44. Kaneda K, Kashii S, Kurosawa T, Kaneko S, Akaike A, Honda Y, Minami M, Satoh M. Apoptotic DNA fragmentation and upregulation of Bax induced by transient ischemia of the rat retina. Brain Res 1999; 815:11-20.
45. Mok CL, Gil-Gomez G, Williams O, Coles M, Taga S, Tolaini M, Norton T, Kioussis D, Brady HJ. Bad can act as a key regulator of T cell apoptosis and T cell development. J Exp Med 1999; 189:575-86.
46. Chen J, Nagayama T, Jin K, Stetler RA, Zhu RL, Graham SH, Simon RP. Induction of caspase-3-like protease may mediate delayed neuronal death in the hippocampus after transient cerebral ischemia. J Neurosci 1998; 18:4914-28.
47. Earnshaw WC, Martins LM, Kaufmann SH. Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu Rev Biochem 1999; 68:383-424.
48. Ni B, Wu X, Du Y, Su Y, Hamilton-Byrd E, Rockey PK, Rosteck P Jr, Poirier GG, Paul SM. Cloning and expression of a rat brain interleukin-1beta-converting enzyme (ICE)-related protease (IRP) and its possible role in apoptosis of cultured cerebellar granule neurons. J Neurosci 1997; 17:1561-9.
49. Chierzi S, Cenni MC, Maffei L, Pizzorusso T, Porciatti V, Ratto GM, Strettoi E. Protection of retinal ganglion cells and preservation of function after optic nerve lesion in bcl-2 transgenic mice. Vision Res 1998; 38:1537-43.
50. Green DR, Reed JC. Mitochondria and apoptosis. Science 1998; 281:1309-12.
51. Kluck RM, Bossy-Wetzel E, Green DR, Newmeyer DD. The release of cytochrome c from mitochondria: a primary site of Bcl-2 regulation of apoptosis. Science 1997; 275:1132-6.
52. Yang J, Liu X, Bhalla K, Kim CN, Ibrado AM, Cai J, Peng TI, Jones DP, Wang X. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 1997; 275:1129-32.
53. Zou H, Henzel WJ, Liu X, Lutschg A, Wang X. Apaf-1, a human protien homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell 1997; 90:405-13.
54. Kluck RM, Martin SJ, Hoffman BM, Zhou JS, Green DR, Newmeyer DD. Cytochrome c activation of CPP32-like proteolysis plays a critical role in a Xenopus cell-free apoptosis system. EMBO J 1997; 16:4639-49.
55. Kharbanda S, Pandey P, Schofield L, Israels S, Roncinske R, Yoshida K, Bharti A, Yuan AM, Saxena S, Weichselbaum R, Nalin C, Kufe D. Role for Bcl-xL as an inhibitor of cytosolic cytochrome C accumulation in DNA damage-induced apoptosis. Proc Natl Acad Sci U S A 1997; 94:6939-42.
56. Gagliardini V, Fernandez PA, Lee RK, Drexler HC, Rotello RJ, Fishman MC, Yuan J. Prevention of vertebrate neuronal death by the crmA gene [published erratum appears in Science 1994; 264:1388]. Science 1994; 263:826-8.
57. Kuida K, Lippke JA, Ku G, Harding MW, Livingston DJ, Su MS, Flavell RA. Altered cytokine export and apoptosis in mice deficient in interleukin-1 beta converting enzyme. Science 1995; 267:2000-3.
58. Li P, Allen H, Banerjee S, Franklin S, Herzog L, Johnston C, McDowell J, Paskind M, Rodman L, Salfeld J, et al. Mice deficient in IL-1 beta-converting enzyme are defective in production of mature IL-1 beta and resistant to endotoxic shock. Cell 1995; 80:401-11.
59. Friedlander RM, Gagliardini V, Hara H, Fink KB, Li W, MacDonald G, Fishman MC, Greenberg AH, Moskowitz MA, Yuan J. Expression of a dominant negative mutant of interleukin-1 beta converting enzyme in transgenic mice prevents neuronal cell death induced by trophic factor withdrawal and ischemic brain injury. J Exp Med 1997; 185:933-40.
60. Schielke GP, Yang GY, Shivers BD, Betz AL. Reduced ischemic brain injury in interleukin-1 beta converting enzyme-deficient mice. J Cereb Blood Flow Metab 1998; 18:180-5.
61. Hara H, Friedlander RM, Gagliardini V, Ayata C, Fink K, Huang Z, Shimizu-Sasamata M, Yuan J, Moskowitz MA. Inhibition of interleukin 1beta converting enzyme family proteases reduces ischemic and excitotoxic neuronal damage. Proc Natl Acad Sci U S A 1997; 94:2007-12.
62. Rabuffetti M, Sciorati C, Tarozzo G, Clementi E, Manfredi AA, Beltramo M. Inhibition of caspase-1-like activity by Ac-Tyr-Val-Ala-Asp-chloromethyl ketone induces long-lasting neuroprotection in cerebral ischemia through apoptosis reduction and decrease of proinflammatory cytokines. J Neurosci 2000; 20:4398-404.
63. Thornberry NA, Bull HG, Calaycay JR, Chapman KT, Howard AD, Kostura MJ, Miller DK, Molineaux SM, Weidner JR, Aunins J, et al. A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes. Nature 1992; 356:768-74.
64. Cerretti DP, Kozlosky CJ, Mosley B, Nelson N, Van Ness K, Greenstreet TA, March CJ, Kronheim SR, Druck T, Cannizzaro LA, et al. Molecular cloning of the interleukin-1 beta converting enzyme. Science 1992; 256:97-100.
65. Friedlander RM, Gagliardini V, Rotello RJ, Yuan J. Functional role of interleukin 1 beta (IL-1 beta) in IL-1 beta-converting enzyme-mediated apoptosis. J Exp Med 1996; 184:717-24.
66. Zhou X, Gordon SA, Kim YM, Hoffman RA, Chen Y, Zhang XR, Simmons RL, Ford HR. Nitric oxide induces thymocyte apoptosis via a caspase-1-dependent mechanism. J Immunol 2000; 165:1252-8.
67. Katai N, Yoshimura N. Apoptotic retinal neuronal death by ischemia-reperfusion is executed by two distinct caspase family proteases. Invest Ophthalmol Vis Sci 1999; 40:2697-705.
68. Organisciak DT, Li M, Darrow RM, Farber DB. Photoreceptor cell damage by light in young Royal College of Surgeons rats. Curr Eye Res 1999; 19:188-96.
69. Krishnamoorthy RR, Crawford MJ, Chaturvedi MM, Jain SK, Aggarwal BB, Al-Ubaidi MR, Agarwal N. Photo-oxidative stress down-modulates the activity of nuclear factor-kappaB via involvement of caspase-1, leading to apoptosis of photoreceptor cells. J Biol Chem 1999; 274:3734-43.