Formation of caspase-3 complexes and fragmentation of caspase-12 during anisomycin-induced apoptosis in AKR-2B cells without aggregation of Apaf-1

Treatment of AKR-2B fibroblasts with anisomycin (10 microM) led to a rapid disintegration of the cells (t1/2 = 5 h) which was complete after 24 h. Cell death was associated with typical hallmarks of apoptosis like membrane blebbing, exposure of phophatidylserine on the cell surface, nuclear condensation and specific cleavage of rRNA. However, there was no dissipation of the mitochondrial potential and no intranucleosomal fragmentation. By affinity labeling with YVK(-bio)D.aomk in combination with immunostaining against activated caspase-3 analyzed by 2-D gel electrophoresis it was shown that caspase-3 is the dominant executioner caspase. Gel filtration experiments of cytosolic extract analyzed by Western blotting revealed the formation of high-molecular-weight complexes of caspase-3 (600 kDa and 250 kDa, respectively), but there was no complex formation of Apaf-1. Anisomycin treatment led to a strong activation of the stress kinases p38 kinases and the jun kinases, that was not sufficient for the activation of caspase-3 which required much higher concentrations. By using the selective inhibitors SB 203580 for p38 kinases and SP 600125 for c-jun kinases, respectively, it is shown that activation of these kinases is not necessary for cell death induced by anisomycin in AKR-2B cells. Furthermore, we disclose the activation of caspase-12 in AKR-2B cells following the addition of anisomycin. Caspase-12 zymogen present as a cytosolic complex (> 600 kDa) is activated by anisomycin leading to an uncomplexed cleaved enzyme. Since anisomycin treatment did neither lead to stress of the endoplasmic reticulum nor to a breakdown of intracellular Ca(2+)-stores, alternative pathways involved in the activation of caspases are discussed.     

Ribotoxic stress sensitizes glioblastoma cells to death receptor induced apoptosis: requirements for c-Jun NH2-terminal kinase and Bim

A prominent feature of glioblastoma is its resistance to death receptor-mediated apoptosis. In this study, we explored the possibility of modulating death receptor-induced cell death with the c-Jun-NH2-terminal kinase (JNK) activator anisomycin. Anisomycin activates JNK by inactivating the ribosome and inducing "ribotoxic stress." We found that anisomycin and death receptor ligand anti-Fas antibody CH-11 or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) synergistically induce apoptosis in multiple human glioblastoma cell lines. For example, in U87 cells, anisomycin reduced the IC50 of CH-11 by more than 20-fold (from 500 to 25 ng/mL). Cell viability in response to anisomycin, CH-11, and their combination was 79%, 91%, and 28% (P<0.001), respectively. Anisomycin and TRAIL were found to be similarly synergistic in glioblastoma cells maintained as tumor xenografts. The potentiation of death receptor-dependent cell death by anisomycin was specific because emetine, another ribosome inhibitor that does not induce ribotoxic stress or activate JNK, did not have a similar effect. Synergistic cell death was predominantly apoptotic involving both extrinsic and intrinsic pathways. Expression of Fas, FasL, FLIP, and Fas-associated death domain (FADD) was not changed following treatment with anisomycin+CH-11. JNK was activated 10- to 22-fold by anisomycin+CH-11 in U87 cells. Inhibiting JNK activation with pharmacologic inhibitors of JNKK and JNK or with dominant negative mitogen-activated protein kinase (MAPK) kinase kinase 2 (MEKK2) significantly prevented cell death induced by the combination of anisomycin+CH-11. We further found that anisomycin+CH-11 up-regulated the proapoptotic protein Bim by approximately 14-fold. Simultaneously inhibiting Bim expression and JNK activation additively desensitized U87 cells to anisomycin+CH-11. These findings show that anisomycin-induced ribotoxic stress sensitizes glioblastoma cells to death receptor-induced apoptosis via a specific mechanism requiring both JNK activation and Bim induction.     

Analysis of apoptotic cells using Beadlyte suspension arrays

Here we describe a new approach to study apoptosis pathways using multiplex suspension arrays. Apoptosis was induced in Jurkat T cells using the protein synthesis inhibitor, anisomycin. Cells grown in 96-well plates were treated with anisomycin for up to 7 h, washed, and lysed in their respective wells. Samples of each lysate were analyzed using Beadlyte suspension arrays consisting of total Akt/PKB, phosphorylated Akt/PKB, active caspase-3, and single-stranded DNA (ssDNA)-specific Beadmate microspheres and quantified with the X-MAP system. We found that phosphorylated Akt levels dropped dramatically with 2 h or more of anisomycin treatment, whereas active caspase-3 levels rose sharply with 2 h of treatment, signifying the onset of apoptosis. Longer incubation with anisomycin showed increases in ssDNA, which is consistent with the characteristic degradation of DNA that occurs in late-stage apoptosis. This approach demonstrates how apoptosis pathways can be studied from a small amount of sample without the use of more lengthy techniques such as immunoprecipitation or Western blotting. The suspension array is being expanded to measure many other intracellular proteins including posttranslational modifications and should prove to be extremely useful for studying apoptosis and other important cellular pathways.     

Activation of p38 and JNK MAPK pathways abrogates requirement for new protein synthesis for phorbol ester mediated induction of select MMP and TIMP genes.

The human matrix metalloproteinase (MMP) gene family includes 24 genes whose regulated expression, together with that of four tissue inhibitors of metalloproteinases (TIMPs), is essential in tissue remodelling and cell signalling. Quantitative real-time-PCR (qPCR) analysis was used to evaluate the shared and unique patterns of control of these two gene families in human MRC-5 and WI-38 fibroblasts in response to the protein kinase C (PKC) activator phorbol-12-myristate-13-acetate (PMA). The requirement for ongoing translation was analysed using three protein synthesis inhibitors, anisomycin, cycloheximide and emetine. PMA induced MMP1, 3, 8, 9, 10, 12, 13, 14 and TIMP1 and TIMP3 RNAs after 4-8 h, and induction of all except MMP9 and TIMP3 was blocked by all protein synthesis inhibitors. However, even though all inhibitors effectively blocked translation, PMA-induction of MMP9 and TIMP3 was blocked by emetine but was insensitive to cycloheximide and anisomycin. Anisomycin alone induced MMP9 and TIMP3, along with MMP25 and MMP19. The extracellular signal-regulated kinases (ERKs)-1/2 were strongly activated by PMA, while anisomycin activated the c-Jun N-terminal kinase (JNK) and p38 pathways, and cycloheximide activated p38, but emetine had no effect on the stress-activated mitogen-activated protein kinase (MAPK) pathways. The involvement of the p38 and JNK pathways in the selective effects of anisomycin and cycloheximide on MMP/TIMP expression was supported by use of pharmacological inhibitors. These data confirm that most inducible MMPs and TIMP1 behave as "late" activated, protein synthesis-dependent genes in fibroblasts. However, the requirement of protein synthesis for PMA-induction of MMPs and TIMPs is not universal, since it is abrogated for MMP9 and TIMP3 by stimulation of the stress-activated MAPK pathways. The definition of clusters of co-regulated genes among the two gene families will aid in bioinformatic dissection of control mechanisms.     

Anisomycin affects both pro- and antiapoptotic mechanisms in PC12 cells

Survival and differentiation of PC12 cells depend on the proper balance between the activities of several mitogen-activated protein kinase (MAPK) pathways. We have previously shown that low, nontoxic doses of anisomycin stimulated these MAPKs as well as the expression of several early-response genes and inhibited NGF-induced neurite formation. In the present work we show that protein synthesis-inhibiting concentrations of anisomycin, in contrast, cause apoptosis of PC12 cells. To try to characterize the apoptosis-inducing mechanisms of anisomycin we compared the signaling effects of subinhibitory and inhibitory drug concentrations. Anisomycin in a nontoxic dosis activates the same MAPK pathways and early-response genes as in protein synthesis inhibiting concentrations. In contrast, while the subinhibitory anisomycin treatment stimulates Akt and induces Bcl-2, two antiapoptotic proteins, the translation-inhibiting concentration of the drug prevents these survival-promoting biochemical events. Anisomycin thus triggers both pro- and antiapoptotic processes in PC12 cells; stimulation of stress-responsive MAPK cascades is not sufficient to mediate apoptotic signaling: the inhibition of key antiapoptotic proteins appears to be more important for PC12 cell death by anisomycin treatment.     

Cellular, biochemical, and genetic analysis of mechanism of small molecule IAP inhibitors

XIAP is member of the IAP family of anti-apoptotic proteins and is known for its ability to bind and suppress caspase family cell death proteases. A phenylurea series of chemical inhibitors of XIAP was recently generated by our laboratories (Schimmer, A. D., Welsh, K., Pinilla, C., Bonneau, M., Wang, Z., Pedersen, I. M., Scott, F. L., Glinsky, G. V., Scudiero, D. A., Sausville, E., Salvesen, G., Nefzi, A., Ostresh, J. M., Houghten, R. A., and Reed, J. C. (2004) Cancer Cell 5, 25-35). We examined the mechanisms of action of these chemical compounds using biochemical, molecular biological, and genetic methods. Active phenylurea-based compounds dissociated effector protease caspase-3 but not initiator protease caspase-9 from XIAP in vitro and restored caspase-3 but not caspase-9 enzymatic activity. When applied to tumor cell lines in culture, active phenylurea-based compounds induced apoptosis in a rapid, concentration-dependent manner, associated with activation of cellular caspases. Apoptosis induced by active phenylurea-based compounds was blocked by chemical inhibitors of caspases, with inhibitors of downstream effector caspases displaying more effective suppression than inhibitors of upstream initiator caspases. Phenylurea-based XIAP antagonists induced apoptosis (defined by annexin V staining) prior to mitochondrial membrane depolarization, in contrast to cytotoxic anticancer drugs. Consistent with these findings, apoptosis induced by phenylurea-based compounds was not altered by genetic alterations in the expression of Bcl-2 family proteins that control mitochondria-dependent cell death pathways, including over-expression of anti-apoptotic proteins Bcl-2 or Bcl-X(L) and genetic ablation of pro-apoptotic proteins Bax and Bak. Conversely, conditional over-expression of an active fragment of XIAP or genetic ablation of XIAP expression altered the apoptosis dose-response of the compounds. Altogether, these findings indicate that phenylurea-based XIAP antagonists block interaction of downstream effector caspases with XIAP, thus inducing apoptosis of tumor cell lines through a caspase-dependent, Bcl-2/Bax-independent mechanism.     

Comparison of the apoptosis-inducing abilities of various protein synthesis inhibitors in U937 cells

We compared the abilities of ricin, diphtheria toxin, cycloheximide, and anisomycin to induce apoptosis, using human myeloid leukemia U937 cells at the concentration of each toxin at which almost complete protein synthesis inhibition was attained within 3 h. Among these toxins, anisomycin was found to be the most potent apoptosis inducer. After a 6-h exposure to anisomycin (1 microg/ml), nearly 95% of the cells had apoptotic nuclear morphological changes, while 53%, 30%, and 10% of the cells showed apoptotic changes after exposure to ricin (0.1 microg/ml), diphtheria toxin (10 microg/ml), and cycloheximide (10 microg/ml), respectively. Furthermore, a rapid increase in caspase-3-like activity was observed in anisomycin-treated cells. A similar increase in caspase-3-like activity was also observed in ricin-treated cells on a slower time schedule. However, only a slight increase in the protease activity was induced by diphtheria toxin or cycloheximide even after 6 h of incubation. Since both ricin and anisomycin are known to act on 28S ribosomal RNA, our results suggest that this action mechanism may be responsible for their potent apoptosis induction, and protein synthesis inhibition alone is not sufficient to induce apoptosis.     

Jolkinolide B from <Emphasis Type="Italic">Euphorbia fischeriana Steud</Emphasis> induces apoptosis in human leukemic U937 cells through PI3K/Akt and XIAP pathways

Jolkinolide B, a bioactive diterpene isolated from the roots of Euphorbia fischeriana Steud, is known to induce apoptosis in cancer cells. However, the molecular mechanism of its anti-cancer activity has not been fully elucidated. In the present study, we found that Jolkinolide B reduced cell viability and induced apoptosis in a dose- and timedependent manner in human leukemic U937. The induction of apoptosis was also accompanied by the downregulation of PI3K/Akt and the inhibitor of apoptosis protein (IAP) family proteins. Moreover, we observed that Jolkinolide B treatment resulted in activation of caspase-3 and -9, which may partly explain the anti-cancer activity of Jolkinolide B. Taken together, our study for the first time suggest that Jolkinolide B is able to enhance apoptosis of U937 cells, at least in part, through downregulation of PI3K/Akt and IAP family proteins. Moreover, triggering of caspase-3 and -9 activation mediated apoptotic induction.     

Inhibition of protein synthesis and JNK activation are not required for cell death induced by anisomycin and anisomycin analogues

Anisomycin was identified in a screen of clinical compounds as a drug that kills breast cancer cells (MDA16 cells, derived from the triple negative breast cancer cell line, MDA-MB-468) that express high levels of an efflux pump, ABCB1. We show the MDA16 cells died by a caspase-independent mechanism, while MDA-MB-468 cells died by apoptosis. There was no correlation between cell death and either protein synthesis or JNK activation, which had previously been implicated in anisomycin-induced cell death. In addition, anisomycin analogues that did not inhibit protein synthesis or activate JNK retained the ability to induce cell death. These data suggest that either a ribosome-ANS complex is a death signal in the absence of JNK activation or ANS kills cells by binding to an as yet unidentified target.     

Translation inhibition in apoptosis: caspase-dependent PKR activation and eIF2-alpha phosphorylation

The protein kinase PKR is a major player in the cellular antiviral response, acting mainly by phosphorylation of the alpha-subunit of the eukaryotic translation initiation factor 2 (eIF2-alpha) to block de novo protein synthesis. PKR activation requires binding of double-stranded RNA or PACT/RAX proteins to its regulatory domain. Since several reports have demonstrated that translation is inhibited in apoptosis, we investigated whether PKR and eIF2-alpha phosphorylation contribute to this process. We show that PKR is proteolysed and that eIF2-alpha is phosphorylated at the early stages of apoptosis induced by various stimuli. Both events coincide with the onset of caspase activity and are prevented by caspase inhibitors. Using site-directed mutagenesis we show that PKR is specifically proteolysed at Asp(251) during cellular apoptosis. This site is cleaved in vitro by recombinant caspase-3, caspase-7, and caspase-8 and not by the proinflammatory caspase-1 and caspase-11. The released kinase domain efficiently phosphorylates eIF2-alpha at the cognate Ser(51) residue, and its overexpression in mammalian cells impairs the translation of its own mRNA and of reporter mRNAs. Our results demonstrate a new and caspase-dependent activation mode for PKR, leading to eIF2-alpha phosphorylation and translation inhibition in apoptosis.     

Anisomycin protects against sepsis by attenuating IκB kinase-dependent NF-κB activation and inflammatory gene expression

Anisomycin is known to inhibit eukaryotic protein synthesis and has been established as an antibiotic and anticancer drug. However, the molecular targets of anisomycin and its mechanism of action have not been explained in macrophages. Here, we demonstrated the anti-inflammatory effects of anisomycin both in vivo and in vitro. We found that anisomycin decreased the mortality rate of macrophages in cecal ligation and puncture (CLP)- and lipopolysaccharide (LPS)-induced acute sepsis. It also declined the gene expression of proinflammatory mediators such as inducible nitric oxide synthase, tumor necrosis factor-α, and interleukin-1β as well as the nitric oxide and proinflammatory cytokines production in macrophages subjected to LPS-induced acute sepsis. Furthermore, anisomycin attenuated nuclear factor (NF)-κB activation in LPS-induced macrophages, which correlated with the inhibition of phosphorylation of NF-κB-inducing kinase and IκB kinase, phosphorylation and IκBα proteolytic degradation, and NF-κB p65 subunit nuclear translocation. These results suggest that anisomycin prevented acute inflammation by inhibiting NF-κB-related inflammatory gene expression and could be a potential therapeutic candidate for sepsis.     

Induction of interleukin-2 receptor-alpha gene expression is regulated by post-translational activation of kappa B specific DNA binding proteins

T cell mitogens induce the expression of specific trans-acting DNA binding proteins that in turn regulate the expression of the interleukin-2 receptor-alpha (IL-2R alpha) gene. To investigate whether de novo protein synthesis is required for the activation of these transacting factors and the induced expression of this receptor gene, Jurkat T cells were incubated with various inhibitors of protein synthesis prior to stimulation with phytohemagglutinin and phorbol 12-myristate 13-acetate (PMA). Despite the presence of cycloheximide or anisomycin at concentrations sufficient to block greater than 97% of cellular protein synthesis, phytohemagglutinin and phorbol 12-myristate 13-acetate effectively induced the expression of the IL-2R alpha gene as measured at the mRNA level. Similarly, gel retardation, DNA footprinting, and DNA-protein cross-linking studies revealed that these mitogens induced the activation of two predominant DNA binding proteins (50-55 and 80-90 kDa) in the presence or absence of cycloheximide and anisomycin. Both of these proteins specifically interacted with a kappa B-like binding site present in the IL-2R alpha promoter (-267 to -256) that is requisite for mitogen-induced expression of this receptor gene. These findings support a post-translational mechanism of induction of pre-existing, but inactive, DNA binding proteins which in turn bind to and activate the IL-2R alpha gene.     

Hypoxia induces apoptosis of HUVECs in an in vitro capillary model by activating proapoptotic signal p38 through suppression of ERK1/2

We recently reported that hypoxia induces chromatin condensation and cell nuclear fragmentation, morphological markers of apoptosis, to tube-forming HUVECs in an in vitro blood vessel model by activating p38 MAPK. In this report, we further examined what role p38 plays and how it is activated during hypoxia-induced apoptosis. First, in order to confirm that p38 can indeed induce apoptosis, the cells were treated with anisomycin, a p38 activator, during normoxia. The activator treatment induced apoptosis and activation of p38 and caspase-3 in a very short time, which indicated that p38 activation alone was sufficient to trigger apoptosis in tube-forming HUVECs. We then observed hypoxia-induced changes in intracellular signals, ERK1/2 and Akt. ERK1/2 inactivation was shown to occur prior to p38 activation and caspase-3 cleavage during hypoxia. On the other hand, anisomycin had no inhibitory effect on ERK1/2 activation during normoxia. It was also shown that the amount of Akt protein slightly decreased by either hypoxia or anisomycin treatment. We then investigated how these two survival signals, ERK1/2 and Akt, are involved in p38 activation by using MEK inhibitor U0126 and PI3K inhibitor LY294002. When tube-forming HUVECs were treated with U0126 or LY294002 during normoxia, the two inhibitors were able to induce apoptosis and activation of p38 and caspase-3 in a relatively short time. U0126 was able to inhibit ERK1/2 activation, but had almost no effect on Akt activation. In contrast, LY294002 was able to inhibit Akt activation, but had very little effect on ERK1/2 activation. These results indicate that ERK1/2 inactivation, rather than Akt decrease, is responsible for hypoxia-induced p38 activation. Taken together, our results strongly suggest that hypoxia-induced apoptosis is regulated through signal transduction in which inactivation of ERK1/2 leads to activation of p38, which then triggers caspase cascade as an execution mechanism of apoptosis.     

XIAP regulates DNA damage-induced apoptosis downstream of caspase-9 cleavage

The IAP (inhibitor of apoptosis) family of anti-apoptotic proteins regulates programmed cell death. Of the six known human IAP-related proteins, XIAP is the most potent inhibitor. To study the mechanistic effects of XIAP on DNA damage-induced apoptosis, we prepared U-937 cells that stably overexpress XIAP. The results demonstrate that XIAP inhibits apoptosis induced by 1-[beta-d-arabinofuranosyl]cytosine (ara-C) and other genotoxic agents. XIAP had no detectable effect on ara-C-induced release of mitochondrial cytochrome c and attenuated cleavage of procaspase-9. In addition, we show that ara-C induces the association of XIAP with the cleaved fragments of caspase-9 and thereby inhibition of caspase-9 activity. The results also demonstrate that ara-C induces cleavage of procaspase-3 by a caspase-8-dependent mechanism and that XIAP inhibits caspase-3 activity. These results demonstrate that XIAP functions downstream of procaspase-9 cleavage as an inhibitor of both proteolytically processed caspase-9 and -3 in the cellular response to genotoxic stress.     

Cytotoxic mechanism of 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (ECyd).

The molecular mechanism of cell death induced by 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (ECyd: Figure 1), a potent inhibitor of RNA synthesis, was performed using mouse mammary tumor FM3A cells and human fibrosarcoma HT1080 cells. ECyd induced the characteristics of apoptosis on these cells, such as morphological changes, DNA fragmentations (Figure 2), and caspase-3-like protease activation. General caspases inhibitor (Z-Asp-CH2-DCB) inhibited these changes and cell death. We also found that ECyd induced DNA and 28S ribosomal RNA (rRNA) fragmentations. Though the mechanisms of rRNA fragmentations haven't revealed, it suggests that translational function of the treated cells should be disturbed. These results indicate that antitumor mechanism of ECyd are characteristics of apoptosis on the cells and rRNA fragmentations is one of the death events resulted inhibition of RNA synthesis.     

Antibiotic anisomycin induces cell cycle arrest and apoptosis through inhibiting mitochondrial biogenesis in osteosarcoma

The anti-cancer activities of antibiotic anisomycin have been demonstrated in kidney, colon and ovarian cancers whereas its underlying mechanisms are not well elucidated. In this work, we investigated whether anisomycin is effective in sensitizes osteosarcoma cell response to chemotherapy. We show that anisomycin inhibits proliferation via inducing osteosarcoma cell arrest at G2/M phase, accompanied by the increased levels of mitotic marker cyclin B and the decreased levels of Rb and E2F-1. Anisomycin also induces apoptosis in a caspase-dependent manner in osteosarcoma cells. Importantly, anisomycin is less effective in normal control NIH3T3 cells compared to osteosarcoma cells. In addition, anisomycin inhibits osteosarcoma growth in xenograft mouse model and enhances the inhibitory effects of doxorubicin in osteosarcoma in vitro and in vivo. Mechanistically, anisomycin targets mitochondrial biogenesis in osteosarcoma as shown by the decreased mitochondrial membrane potential, suppressed mitochondrial respiration via decreasing complex I activity, reduced ATP production. Furthermore, mitochondrial biogenesis stimulator acetyl-L-Carnitine (ALCAR) significantly rescues the inhibitory effects of anisomycin in osteosarcoma cells. Our work demonstrates that anisomycin is active against osteosarcoma cells and the molecular mechanism of its action is the inhibition of mitochondrial biogenesis.     

Protein kinase C inhibition induces DNA fragmentation in COLO 205 cells which is blocked by cysteine protease inhibition but not mediated through caspase-3

Enhancing apoptosis to remove abnormal cells has potential in reversing cancerous processes. Caspase-3 activation generally accompanies apoptosis and its substrates include enzymes responsible for DNA fragmentation and isozymes of protein kinase C (PKC). Recent data, however, question its obligatory role in apoptosis. We have examined whether modulation of PKC activity induces apoptosis in COLO 205 cells and the role of caspase-3. Proliferation ([3H]thymidine) and apoptosis (DNA fragmentation and FACS) of COLO 205 cells were measured in response to PKC activation and inhibition. Caspase-3 activity was assayed and the effects of its inhibition with Ac-DEVD-cmk, and the effect of other protease inhibitors, on apoptosis were determined. PKC activation and inhibition both reduced DNA synthesis and induced DNA fragmentation. As PKC inhibitors induced DNA fragmentation more rapidly than PKC activators and failed to block activator effects, we conclude that it is PKC down-regulation (i.e., inhibition) after activator exposure that mediates apoptosis. Increases in caspase-3 activity occurred during apoptosis but apoptosis was not blocked by caspase inhibition. By contrast, the cysteine protease inhibitor, E-64d, blocked apoptosis. Cysteine proteases not of the caspase family may either act more closely to the apoptotic process than caspases or lie on an alternative, more active pathway.     

Nitric oxide fully protects against UVA-induced apoptosis in tight correlation with Bcl-2 up-regulation

A variety of toxic and modulating events induced by UVA exposure are described to cause cell death via apoptosis. Recently, we found that UV irradiation of human skin leads to inducible nitric-oxide synthase (iNOS) expression in keratinocytes and endothelial cells (ECs). We have now searched for the role of iNOS expression and nitric oxide (NO) synthesis in UVA-induced apoptosis as detected by DNA-specific fluorochrome labeling and in DNA fragmentation visualized by in situ nick translation in ECs. Activation with proinflammatory cytokines 24 h before UVA exposure leading to iNOS expression and endogenous NO synthesis fully protects ECs from the onset of apoptosis. This protection was completely abolished in the presence of the iNOS inhibitor L-N5-(1-iminoethyl)-ornithine (0.25 mM). Additionally, preincubation of cells with the NO donor (Z)-1-[N(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-i um-1, 2-diolate at concentrations from 10 to 1000 microM as an exogenous NO-generating source before UVA irradiation led to a dose-dependent inhibition of both DNA strand breaks and apoptosis. In search of the molecular mechanism responsible for the protective effect, we find that protection from UVA-induced apoptosis is tightly correlated with NO-mediated increases in Bcl-2 expression and a concomitant inhibition of UVA-induced overexpression of Bax protein. In conclusion, we present evidence for a protective role of iNOS-derived NO in skin biology, because NO either endogenously produced or exogenously applied fully protects against UVA-induced cell damage and death. We also show that the NO-mediated expression modulation of proteins of the Bcl-2 family, an event upstream of caspase activation, appears to be the molecular mechanism underlying this protection.