Targeting Bcl-2-IP3 receptor interaction to reverse Bcl-2's inhibition of apoptotic calcium signals.

The antiapoptotic protein Bcl-2 inhibits Ca2+ release from the endoplasmic reticulum (ER). One proposed mechanism involves an interaction of Bcl-2 with the inositol 1,4,5-trisphosphate receptor (IP3R) Ca2+ channel localized with Bcl-2 on the ER. Here we document Bcl-2-IP3R interaction within cells by FRET and identify a Bcl-2 interacting region in the regulatory and coupling domain of the IP3R. A peptide based on this IP3R sequence displaced Bcl-2 from the IP3R and reversed Bcl-2-mediated inhibition of IP3R channel activity in vitro, IP3-induced ER Ca2+ release in permeabilized cells, and cell-permeable IP3 ester-induced Ca2+ elevation in intact cells. This peptide also reversed Bcl-2's inhibition of T cell receptor-induced Ca2+ elevation and apoptosis. Thus, the interaction of Bcl-2 with IP3Rs contributes to the regulation of proapoptotic Ca2+ signals by Bcl-2, suggesting the Bcl-2-IP3R interaction as a potential therapeutic target in diseases associated with Bcl-2's inhibition of cell death.

All-trans-retinal induces Bax activation via DNA damage to mediate retinal cell apoptosis.

The current study investigates the cellular events which trigger activation of proapoptotic Bcl-2-associated × protein (Bax) in retinal cell death induced by all-trans-retinal (atRAL). Cellular events which activate Bax, such as DNA damage by oxidative stress and phosphorylation of p53, were evaluated by immunochemical and biochemical methods using ARPE-19 cells, 661 W cells, cultured neural retinas and a retinal degeneration model, Abca4(-/-)Rdh8(-/-) mice. atRAL-induced Bax activation in cultured neural retinas was examined by pharmacological and genetic methods. Other Bax-related cellular events were also evaluated by pharmacological and biochemical methods. Production of 8-OHdG, a DNA damage indicator, and the phosphorylation of p53 at Ser46 were detected prior to Bax activation in ARPE-19 cells incubated with atRAL. Light exposure to Abca4(-/-)Rdh8(-/-) mice also caused the above mentioned events in conditions of short term intense light exposure and regular room lighting conditions. Incubation with Bax inhibiting peptide and deletion of the Bax gene partially protected retinal cells from atRAL toxicity in cultured neural retina. Necrosis was demonstrated not to be the main pathway in atRAL mediated cell death. Bcl-2-interacting mediator and Bcl-2 expression levels were not altered by atRAL in vitro. atRAL-induced oxidative stress results in DNA damage leading to the activation of Bax by phosphorylated p53. This cascade is closely associated with an apoptotic cell death mechanism rather than necrosis.

Cytoprotective Small Compound M109S Attenuated Retinal Ganglion Cell Degeneration Induced by Optic Nerve Crush in Mice.

BAX plays an essential role in retinal ganglion cell (RGC) death induced by optic nerve injury. Recently, we developed M109S, an orally bioactive and cytoprotective small compound (CPSC) that inhibits BAX-mediated cell death. We examined whether M109S can protect RGC from optic nerve crush (ONC)-induced apoptosis. M109S was administered starting 5 h after ONC for 7 days. M109S was orally administered in two groups (5 mg/kg twice a day or 7.5 mg/kg once a day). The retina was stained with anti-BRN3A and cleaved Caspase-3 (active Caspase-3) that are the markers of RGC and apoptotic cells, respectively. ONC decreased the number of BRN3A-positive RGC and increased the number of active Caspase-3-expressing apoptotic cells. In ONC-treated retina, there were cells that were double stained with anti-BRN3A and ant-cleaved Caspase-3, indicating that apoptosis in BRN3A-positive RGCs occurred. M109S inhibited the decrease of BRN3A-positive cells whereas it inhibited the increase of active Caspase-3-positive cells in the retina of ONC-treated mice, suggesting that M109S inhibited apoptosis in RGCs. M109S did not induce detectable histological damage to the lungs or kidneys in mice, suggesting that M109S did not show toxicities in the lung or kidneys when the therapeutic dose was used. The present study suggests that M109S is effective in rescuing damaged RGCs. Since M109S is an orally bioactive small compound, M109S may become the basis for a portable patient-friendly medicine that can be used to prevent blindness by rescuing damaged optic nerve cells from death.

Unconventional activation of PRKDC by TNF-α: deciphering its crucial role in Th1-mediated inflammation beyond DNA repair as part of the DNA-PK complex.

BACKGROUND: The DNA-dependent protein kinase (DNA-PK) complex comprises a catalytic (PRKDC) and two requisite DNA-binding (Ku70/Ku80) subunits. The role of the complex in repairing double-stranded DNA breaks (DSBs) is established, but its role in inflammation, as a complex or individual subunits, remains elusive. While only ~ 1% of PRKDC is necessary for DNA repair, we reported that partial inhibition blocks asthma in mice without causing SCID. METHODS: We investigated the central role of PRKDC in inflammation and its potential association with DNA repair. We also elucidated the relationship between inflammatory cytokines (e.g., TNF-α) and PRKDC by analyzing its connections to inflammatory kinases. Human cell lines, primary human endothelial cells, and mouse fibroblasts were used to conduct the in vitro studies. For animal studies, LPS- and oxazolone-induced mouse models of acute lung injury (ALI) and delayed-type hypersensitivity (DHT) were used. Wild-type, PRKDC+/-, or Ku70+/- mice used in this study. RESULTS: A ~ 50% reduction in PRKDC markedly blocked TNF-α-induced expression of inflammatory factors (e.g., ICAM-1/VCAM-1). PRKDC regulates Th1-mediated inflammation, such as DHT and ALI, and its role is highly sensitive to inhibition achieved by gene heterozygosity or pharmacologically. In endothelial or epithelial cells, TNF-α promoted rapid PRKDC phosphorylation in a fashion resembling that induced by, but independent of, DSBs. Ku70 heterozygosity exerted little to no effect on ALI in mice, and whatever effect it had was associated with a specific increase in MCP-1 in the lungs and systemically. While Ku70 knockout blocked VP-16-induced PRKDC phosphorylation, it did not prevent TNF-α - induced phosphorylation of the kinase, suggesting Ku70 dispensability. Immunoprecipitation studies revealed that PRKDC transiently interacts with p38MAPK. Inhibition of p38MAPK blocked TNF-α-induced PRKDC phosphorylation. Direct phosphorylation of PRKDC by p38MAPK was demonstrated using a cell-free system. CONCLUSIONS: This study presents compelling evidence that PRKDC functions independently of the DNA-PK complex, emphasizing its central role in Th1-mediated inflammation. The distinct functionality of PRKDC as an individual enzyme, its remarkable sensitivity to inhibition, and its phosphorylation by p38MAPK offer promising therapeutic opportunities to mitigate inflammation while sparing DNA repair processes. These findings expand our understanding of PRKDC biology and open new avenues for targeted anti-inflammatory interventions.

Epigenetic aging of human blood cells is influenced by the age of the host body.

Allogenic hematopoietic stem cell transplantation is a therapeutic procedure performed over a wide range of donor and recipient age combinations, representing natural experiments of how the age of the recipient affects aging in transplanted donor cells in vivo. We measured DNA methylation and epigenetic aging in donors and recipients and found that biological epigenetic clocks are accelerated in cells transplanted into an older body and decelerated in a younger body. This is the first evidence that the age of the circulating environment influences human epigenetic aging in vivo.

Induction of Ca²+-driven apoptosis in chronic lymphocytic leukemia cells by peptide-mediated disruption of Bcl-2-IP3 receptor interaction.

Bcl-2 contributes to the pathophysiology and therapeutic resistance of chronic lymphocytic leukemia (CLL). Therefore, developing inhibitors of this protein based on a thorough understanding of its mechanism of action is an active and promising area of inquiry. One approach centers on agents (eg, ABT-737) that compete with proapoptotic members of the Bcl-2 protein family for binding in the hydrophobic groove formed by the BH1-BH3 domains of Bcl-2. Another region of Bcl-2, the BH4 domain, also contributes to the antiapoptotic activity of Bcl-2 by binding to the inositol 1,4,5-trisphosphate receptor (IP3R) Ca²(+) channel, inhibiting IP(3)-dependent Ca²(+) release from the endoplasmic reticulum. We report that a novel synthetic peptide, modeled after the Bcl-2-interacting site on the IP3R, binds to the BH4 domain of Bcl-2 and functions as a competitive inhibitor of the Bcl-2-IP3R interaction. By disrupting the Bcl-2-IP3R interaction, this peptide induces an IP3R-dependent Ca²(+) elevation in lymphoma and leukemia cell lines and in primary CLL cells. The Ca²(+) elevation evoked by this peptide induces apoptosis in CLL cells, but not in normal peripheral blood lymphocytes, suggesting the involvement of the Bcl-2-IP3R interaction in the molecular mechanism of CLL and indicating the potential merit of targeting this interaction therapeutically.

Development of novel cytoprotective small compounds inhibiting mitochondria-dependent cell death.

We identified cytoprotective small molecules (CSMs) by a cell-based high-throughput screening of Bax inhibitors. Through a medicinal chemistry program, M109S was developed, which is orally bioactive and penetrates the blood-brain/retina barriers. M109S protected retinal cells in ocular disease mouse models. M109S directly interacted with Bax and inhibited the conformational change and mitochondrial translocation of Bax. M109S inhibited ABT-737-induced apoptosis both in Bax-only and Bak-only mouse embryonic fibroblasts. M109S also inhibited apoptosis induced by staurosporine, etoposide, and obatoclax. M109S decreased maximal mitochondrial oxygen consumption rate and reactive oxygen species production, whereas it increased glycolysis. These effects on cellular metabolism may contribute to the cytoprotective activity of M109S. M109S is a novel small molecule protecting cells from mitochondria-dependent apoptosis both in vitro and in vivo. M109S has the potential to become a research tool for studying cell death mechanisms and to develop therapeutics targeting mitochondria-dependent cell death pathway.

Glucocorticoid elevation of dexamethasone-induced gene 2 (Dig2/RTP801/REDD1) protein mediates autophagy in lymphocytes.

Glucocorticoid hormones, including dexamethasone, induce apoptosis in lymphocytes and consequently are used clinically as chemotherapeutic agents in many hematologic malignancies. Dexamethasone also induces autophagy in lymphocytes, although the mechanism is not fully elucidated. Through gene expression analysis, we found that dexamethasone induces the expression of a gene encoding a stress response protein variously referred to as Dig2, RTP801, or REDD1. This protein is reported to inhibit mammalian target of rapamycin (mTOR) signaling. Because autophagy is one outcome of mTOR inhibition, we investigated the hypothesis that Dig2/RTP801/REDD1 elevation contributes to autophagy induction in dexamethasone-treated lymphocytes. In support of this hypothesis, RNAi-mediated suppression of Dig2/RTP801/REDD1 reduces mTOR inhibition and autophagy in glucocorticoid-treated lymphocytes. We observed similar results in Dig2/Rtp801/Redd1 knock-out murine thymocytes treated with dexamethasone. Dig2/RTP801/REDD1 knockdown also leads to increased levels of dexamethasone-induced cell death, suggesting that Dig2/RTP801/REDD1-mediated autophagy promotes cell survival. Collectively, these findings demonstrate for the first time that elevation of Dig2/RTP801/REDD1 contributes to the induction of autophagy.

CRM1 protein-mediated regulation of nuclear clusterin (nCLU), an ionizing radiation-stimulated, Bax-dependent pro-death factor.

Expression of the clusterin (CLU) gene results in the synthesis of a conventional secretory isoform set (pre- and mature secretory clusterin proteins, psCLU/sCLU), as well as another set of intracellular isoforms, appearing in the cytoplasm (pre-nuclear CLU, pnCLU) and in the nucleus as an ∼55-kDa mature nuclear clusterin (nCLU) form. These two isoform sets have opposing cell functions: pro-survival and pro-death, respectively. Although much is known about the regulation and function of sCLU as a pro-survival factor, the regulation and function of endogenous nCLU in cell death are relatively unexplored. Here, we show that depletion of endogenous nCLU protein using siRNA specific to its truncated mRNA increased clonogenic survival of ionizing radiation (IR)-exposed cells. nCLU-mediated apoptosis was Bax-dependent, and lethality correlated with accumulation of mature nCLU protein. nCLU accumulation was regulated by CRM1 because binding between CRM1 and nCLU proteins was significantly diminished by leptomycin B (LMB), and nuclear levels of nCLU protein were significantly enhanced by LMB and IR co-treatment. Moreover, LMB treatment significantly enhanced IR-induced nCLU-mediated cell death responses. Importantly, bax(-/-) and bax(-/-)/bak(-/-) double knock-out cells were resistant to nCLU-mediated cell death, whereas bak(-/-) or wild-type bax(+/+)/bak(+/+) cells were hypersensitive. The regulation of nCLU by CRM1 nuclear export/import may explain recent clinical results showing that highly malignant tumors have lost the ability to accumulate nCLU levels, thereby avoiding growth inhibition and cell death.

Cytoprotective membrane-permeable peptides designed from the Bax-binding domain of Ku70.

Bax is a pro-apoptotic member of Bcl-2 family proteins and is central to mitochondria-dependent apoptosis. Bax resides in the cytosol as a quiescent protein and translocates into mitochondria after apoptotic stimuli. Ku70 is a 70K subunit of the Ku complex, which has an important role in DNA double-strand break (DSB) repair in the nucleus. In another article in this issue, we reported that Ku70 interacts with pro-apoptotic protein Bax in the cytosol and prevents its mitochondrial translocation, suggesting that Ku70 suppresses Bax-mediated apoptosis. Here, we describe the development of a new membrane-permeable peptide, Bax-inhibiting peptide (BIP) that inhibits Bax-mediated apoptosis, on the basis of the previous finding that showed an interaction between Ku70 and Bax. BIP is comprised of five amino acids designed from the Bax-binding domain of Ku70, and suppresses the mitochondrial translocation of Bax. BIP inhibited Bax-mediated apoptosis induced by staurosporine, UVC irradiation and anti-cancer drugs in several types of cells. BIP may provide valuable information in the development of therapeutics that control apoptosis-related diseases.

Ku70 suppresses the apoptotic translocation of Bax to mitochondria.

Bax induces mitochondrial-dependent cell death signals in mammalian cells. However, the mechanism of how Bax is kept inactive has remained unclear. Yeast-based functional screening of Bax inhibitors from mammalian cDNA libraries identified Ku70 as a new Bax suppressor. Bax-mediated apoptosis was suppressed by overexpression of Ku70 in mammalian cells, but enhanced by downregulation of Ku70. We found that Ku70 interacts with Bax, and that the carboxyl terminus of Ku70 and the amino terminus of Bax are required for this interaction. Bax is known to translocate from the cytosol to mitochondria when cells receive apoptotic stimuli. We found that Ku70 blocks the mitochondrial translocation of Bax. These results suggest that in addition to its previously recognized DNA repair activity in the nucleus, Ku70 has a cytoprotective function in the cytosol that controls the localization of Bax.

Securinine induces p73-dependent apoptosis preferentially in p53-deficient colon cancer cells.

The identification of agents that preferentially kill cancer cells while protecting normal cells offers the potential to overcome toxicities found in many existing chemotherapeutic agents. Because p53 is frequently inactivated in cancer, agents that preferentially kill p53-null cells and protect wild-type p53-expressing cells are highly desirable chemotherapeutic agents. By using pairs of isogenic colon cancer cell lines that differ only in p53 expression (RKO and HCT116), securinine was found to exhibit these properties. Securinine (30 microM) induces apoptosis in 73% of p53-null HCT116 cells (LD(50) 17.5 microM) as opposed to 17.6% of HCT116 parental cells (LD(50) 50 microM) at 72 h after treatment. The mechanism of securinine-mediated death in p53-deficient cells involves the induction of the p53 family member, p73. Interestingly, the proapoptotic protein p73 is down-regulated in colon cancer cells expressing p53. This differential regulation of p73 in a p53-dependent fashion reveals a novel pathway for preferentially targeting cancer cells. In contrast to p53-deficient cells, cells expressing p53 are protected from cell death through the p53-mediated up-regulation of p21. These studies reveal a novel approach to specifically target colon cancer cells lacking p53 as well as identify a novel clinically relevant pathway to selectively induce p73 in p53-null cells.

Analysis of epigenetic aging in vivo and in vitro: Factors controlling the speed and direction.

IMPACT STATEMENT: Aging is associated with DNA methylation (DNAm) changes. Recent advancement of the whole-genome DNAm analysis technology allowed scientists to develop DNAm-based age estimators. A majority of these estimators use DNAm data from a single tissue type such as blood. In 2013, a multi-tissue age estimator using DNAm pattern of 353 CpGs was developed by Steve Horvath. This estimator was named "epigenetic clock", and the improved version using DNAm pattern of 391 CpGs was developed in 2018. The estimated age by epigenetic clock is named DNAmAge. DNAmAge can be used as a biomarker of aging predicting the risk of age-associated diseases and mortality. Although the DNAm-based age estimators were developed, the mechanism of epigenetic aging is still enigmatic. The biological significance of epigenetic aging is not well understood, either. This minireview discusses the current understanding of the mechanism of epigenetic aging and the future direction of aging research.

Histone deacetylase inhibitors sensitize prostate cancer cells to agents that produce DNA double-strand breaks by targeting Ku70 acetylation.

This study reports a histone deacetylation-independent mechanism whereby histone deacetylase (HDAC) inhibitors sensitize prostate cancer cells to DNA-damaging agents by targeting Ku70 acetylation. Ku70 represents a crucial component of the nonhomologous end joining repair machinery for DNA double-strand breaks (DSB). Our data indicate that pretreatment of prostate cancer cells with HDAC inhibitors (trichostatin A, suberoylanilide hydroxamic acid, MS-275, and OSU-HDAC42) led to increased Ku70 acetylation accompanied by reduced DNA-binding affinity without disrupting the Ku70/Ku80 heterodimer formation. As evidenced by increased Ser(139)-phosphorylated histone H2AX (gammaH2AX), impaired Ku70 function diminished cellular capability to repair DNA DSBs induced by bleomycin, doxorubicin, and etoposide, thereby enhancing their cell-killing effect. This sensitizing effect was most prominent when cells were treated with HDAC inhibitors and DNA-damaging agents sequentially. Mimicking acetylation was done by replacing K282, K317, K331, K338, K539, or K542 with glutamine via site-directed mutagenesis, which combined with computer docking analysis was used to analyze the role of these lysine residues in the interactions of Ku70 with DNA broken ends. Mutagenesis of K282, K338, K539, or K542 suppressed the activity of Ku70 to bind DNA, whereas mutagenesis of K317 or K331 with glutamine had no significant effect. Moreover, overexpression of K282Q or K338Q rendered DU-145 cells more susceptible to the effect of DNA-damaging agents on gammaH2AX formation and cell killing. Overall, the ability of HDAC inhibitors to regulate cellular ability to repair DNA damage by targeting Ku70 acetylation underlies the viability of their combination with DNA-damaging agents as a therapeutic strategy for prostate cancer.

Epigenetic clock analysis of human fibroblasts in vitro: effects of hypoxia, donor age, and expression of hTERT and SV40 largeT.

Aging is associated with a genome-wide change of DNA methylation (DNAm). "DNAm age" is defined as the predicted chronological age by the age estimator based on DNAm. The estimator is called the epigenetic clock. The molecular mechanism underlining the epigenetic clock is still unknown. Here, we evaluated the effects of hypoxia and two immortalization factors, hTERT and SV40-LargeT (LT), on the DNAm age of human fibroblasts in vitro. We detected the cell division-associated progression of DNAm age after >10 population doublings. Moreover, the progression of DNAm age was slower under hypoxia (1% oxygen) compared to normoxia (21% oxygen), suggesting that oxygen levels determine the speed of the epigenetic aging. We show that the speed of cell division-associated DNAm age progression depends on the chronological age of the cell donor. hTERT expression did not arrest cell division-associated progression of DNAm age in most cells. SV40LT expression produced inconsistent effects, including rejuvenation of DNAm age. Our results show that a) oxygen and the targets of SV40LT (e.g. p53) modulate epigenetic aging rates and b) the chronological age of donor cells determines the speed of mitosis-associated DNAm age progression in daughter cells.

Pharmacological inhibition of Bax-induced cell death: Bax-inhibiting peptides and small compounds inhibiting Bax.

IMPACT STATEMENT: Bax induces mitochondria-dependent programed cell death. While cytotoxic drugs activating Bax have been developed for cancer treatment, clinically effective therapeutics suppressing Bax-induced cell death rescuing essential cells have not been developed. This mini-review will summarize previously reported Bax inhibitors including peptides, small compounds, and antibodies. We will discuss potential applications and the future direction of these Bax inhibitors.

Epigenetic age is a cell-intrinsic property in transplanted human hematopoietic cells.

The age of tissues and cells can be accurately estimated by DNA methylation analysis. The multitissue DNA methylation (DNAm) age predictor combines the DNAm levels of 353 CpG dinucleotides to arrive at an age estimate referred to as DNAm age. Recent studies based on short-term observations showed that the DNAm age of reconstituted blood following allogeneic hematopoietic stem cell transplantation (HSCT) reflects the age of the donor. However, it is not known whether the DNAm age of donor blood remains independent of the recipient's age over the long term. Importantly, long-term studies including child recipients have the potential to clearly reveal whether DNAm age is cell-intrinsic or whether it is modulated by extracellular cues in vivo. Here, we address this question by analyzing blood methylation data from HSCT donor and recipient pairs who greatly differed in chronological age (age differences between 1 and 49 years). We found that the DNAm age of the reconstituted blood was not influenced by the recipient's age, even 17 years after HSCT, in individuals without relapse of their hematologic disorder. However, the DNAm age of recipients with relapse of leukemia was unstable. These data are consistent with our previous findings concerning the abnormal DNAm age of cancer cells, and it can potentially be exploited to monitor the health of HSCT recipients. Our data demonstrate that transplanted human hematopoietic stem cells have an intrinsic DNAm age that is unaffected by the environment in a recipient of a different age.

Ku70 is stabilized by increased cellular SUMO.

Ku70 is a protein that finds itself at the heart of several important cellular processes. It is essential to the non-homologous end joining pathway as a part of the DNA-end binding complex, required for proper maintenance of telomeres and contributes to DNA damage recognition and regulation of apoptosis. Forces that regulate Ku70 are therefore likely to have large consequences on the physiologic state of the cell. We report here that transient expression of the small protein SUMO resulted in a dramatic increase in the abundance of Ku70. Surprisingly, the direct SUMOylation of Ku70 does not appear to be required for this effect. Rather, Ku70 appears to be stabilized through indirect effects on the rate of degradation. The same outcome was obtained by raising the expression of enzymes that promote SUMOylation. It is likely that many other proteins will be similarly regulated, providing a general control of cellular state.

Epigenetic clock for skin and blood cells applied to Hutchinson Gilford Progeria Syndrome and ex vivo studies.

DNA methylation (DNAm)-based biomarkers of aging have been developed for many tissues and organs. However, these biomarkers have sub-optimal accuracy in fibroblasts and other cell types used in ex vivo studies. To address this challenge, we developed a novel and highly robust DNAm age estimator (based on 391 CpGs) for human fibroblasts, keratinocytes, buccal cells, endothelial cells, lymphoblastoid cells, skin, blood, and saliva samples. High age correlations can also be observed in sorted neurons, glia, brain, liver, and even bone samples. Gestational age correlates with DNAm age in cord blood. When used on fibroblasts from Hutchinson Gilford Progeria Syndrome patients, this age estimator (referred to as the skin & blood clock) uncovered an epigenetic age acceleration with a magnitude that is below the sensitivity levels of other DNAm-based biomarkers. Furthermore, this highly sensitive age estimator accurately tracked the dynamic aging of cells cultured ex vivo and revealed that their proliferation is accompanied by a steady increase in epigenetic age. The skin & blood clock predicts lifespan and it relates to many age-related conditions. Overall, this biomarker is expected to become useful for forensic applications (e.g. blood or buccal swabs) and for a quantitative ex vivo human cell aging assay.

Hypoxia-induced retinal ganglion cell death and the neuroprotective effects of beta-adrenergic antagonists.

Hypoxia-induced retinal ganglion cell (RGC) death has been implicated in glaucomatous optic neuropathy. However, the precise mechanism of death signaling and how neuroprotective agents affect it are still unclear. The aim of this study is to characterize the mechanisms of hypoxia-induced apoptosis of cultured purified RGCs and to study the neuroprotective effects of beta-adrenergic antagonists. Rat RGCs were purified utilizing a modified two-step immuno-panning procedure. First, the extent of apoptosis in RGCs under hypoxia was quantified. Next, the effects of glutamate-channel antagonists (MK801 or DNQX), Bax inhibiting peptide (BIP), and beta-adrenergic antagonists (betaxolol, nipradilol, timolol or carteolol) on hypoxia-induced RGC death were investigated by the cell viability assay. Third, the effects of beta-adrenergic antagonists on hypoxia-induced increase of intracellular calcium concentrations ([Ca(2+)](i)) and the additional effect of NO scavenger to nipradilol were evaluated. Apoptotic RGC percentages under hypoxia were significantly increased compared to the control. The viability of RGCs under hypoxia was not affected by MK801 or DNQX, whereas it was increased in a dose-dependent manner with exposure to BIP, and to betaxolol, nipradilol, timolol, but not to carteolol. These effective beta-adrenergic antagonists showed no significant change in hypoxia-induced [Ca(2+)](i) levels. The NO scavenger alleviated neuroprotective effect by nipradilol. In conclusion, purified RGC damage induced by hypoxia involves Bax-dependent apoptotic pathway, but mostly independent of glutamate receptor-mediated excitotoxicity. Betaxolol, timolol and nipradilol showed a protective effect against hypoxia-induced RGC death, which was thought to be irrelevant either to calcium channel or beta-adrenoceptor blocking effects.