Bortezomib sensitises TRAIL-resistant HPV-positive head and neck cancer cells to TRAIL through a caspase-dependent, E6-independent mechanism.

Human papillomavirus (HPV) is causative for a new and increasing form of head and neck squamous cell carcinomas (HNSCCs). Although localised HPV-positive cancers have a favourable response to radio-chemotherapy (RT/CT), the impact of HPV in advanced or metastatic HNSCC remains to be defined and targeted therapeutics need to be tested for cancers resistant to RT/CT. To this end, we investigated the sensitivity of HPV-positive and -negative HNSCC cell lines to TRAIL (tumour necrosis factor-related apoptosis-inducing ligand), which induces tumour cell-specific apoptosis in various cancer types. A clear correlation was observed between HPV positivity and resistance to TRAIL compared with HPV-negative head and neck cancer cell lines. All TRAIL-resistant HPV-positive cell lines tested were sensitised to TRAIL-induced cell death by treatment with bortezomib, a clinically approved proteasome inhibitor. Bortezomib-mediated sensitisation to TRAIL was associated with enhanced activation of caspase-8, -9 and -3, elevated membrane expression levels of TRAIL-R2, cytochrome c release and G2/M arrest. Knockdown of caspase-8 significantly blocked cell death induced by the combination therapy, whereas the BH3-only protein Bid was not required for induction of apoptosis. XIAP depletion increased the sensitivity of both HPV-positive and -negative cells to TRAIL alone or in combination with bortezomib. In contrast, restoration of p53 following E6 knockdown in HPV-positive cells had no effect on their sensitivity to either single or combination therapy, suggesting a p53-independent pathway for the observed response. In summary, bortezomib-mediated proteasome inhibition sensitises previously resistant HPV-positive HNSCC cells to TRAIL-induced cell death through a mechanism involving both the extrinsic and intrinsic pathways of apoptosis. The cooperative effect of these two targeted anticancer agents therefore represents a promising treatment strategy for RT/CT-resistant HPV-associated head and neck cancers.

Differential response of head and neck cancer cell lines to TRAIL or Smac mimetics is associated with the cellular levels and activity of caspase-8 and caspase-10.

BACKGROUND: Current treatment strategies for head and neck cancer are associated with significant morbidity and up to 50% of patients relapse, highlighting the need for more specific and effective therapeutics. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and Smac mimetics (SMs) are promising anticancer agents, but their effect on head and neck squamous cell carcinoma (HNSCC) remains unknown. METHODS: We examined the response of a panel of nine HNSCC cell lines to TRAIL and SMs and investigated the mechanism of cell type-specific response by functional analysis. RESULTS: Head and neck cancer cell lines revealed a converse response pattern with three cell lines being highly sensitive to Smac-164 (SM) but resistant to TRAIL, whereas the other six were sensitive to TRAIL but resistant to SM. Distinct protein expression and activation patterns were found to be associated with susceptibility of HNSCC cell lines to TRAIL and SM. Tumour necrosis factor-related apoptosis-inducing ligand sensitivity was associated with high caspase-8 and Bid protein levels, and TRAIL-sensitive cell lines were killed via the type II extrinsic apoptotic pathway. Smac mimetic-sensitive cells expressed low levels of caspase-8 and Bid but had high TNF-α expression. Smac mimetic-induced cell death was associated with caspase-10 activation, suggesting that in the absence of caspase-8, caspase-10 mediates response to SM. Cotreatment with TNF-α sensitised the resistant cells to SM, demonstrating a decisive role for TNF-α-driven feedback loop in SM sensitivity. CONCLUSIONS: Tumour necrosis factor-related apoptosis-inducing ligand and SMs effectively kill HNSCC cell lines and therefore represent potential targeted therapeutics for head and neck cancer. Distinct molecular mechanisms determine the sensitivity to each agent, with levels of TNF-α, caspase-8, Bid and caspase-10 providing important predictive biomarkers of response to these agents.

Development of a human three-dimensional organotypic skin-melanoma spheroid model for in vitro drug testing.

Despite remarkable efforts, metastatic melanoma (MM) still presents with significant mortality. Recently, mono-chemotherapies are increasingly replenished by more cancer-specific combination therapies involving death ligands and drugs interfering with cell signaling. Still, MM remains a fatal disease because tumors rapidly develop resistance to novel therapies thereby regaining tumorigenic capacity. Although genetically engineered mouse models for MM have been developed, at present no model is available that reliably mimics the human disease and is suitable for studying mechanisms of therapeutic obstacles including cell death resistance. To improve the increasing requests on new therapeutic alternatives, reliable human screening models are demanded that translate the findings from basic cellular research into clinical applications. By developing an organotypic full skin equivalent, harboring melanoma tumor spheroids of defined sizes we have invented a cell-based model that recapitulates both the 3D organization and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention. By extending our previous findings on melanoma cell sensitization toward TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) by co-application of sublethal doses of ultraviolet-B radiation (UVB) or cisplatin, we show significant differences in the therapeutical outcome to exist between regular two-dimensional (2D) and complex in vivo-like 3D models. Of note, while both treatment combinations killed the same cancer cell lines in 2D culture, skin equivalent-embedded melanoma spheroids are potently killed by TRAIL+cisplatin treatment but remain almost unaffected by the TRAIL+UVB combination. Consequently, we have established an organotypic human skin-melanoma model that will facilitate efforts to improve therapeutic outcomes for malignant melanoma by providing a platform for the investigation of cytotoxic treatments and tailored therapies in a more physiological setting.

Caspase-3 cleaves XIAP in a positive feedback loop to sensitize melanoma cells to TRAIL-induced apoptosis.

Successful treatment of melanoma is still challenging, because metastasis remain chemoresistant and radioresistant. Accordingly, combinational treatments involving death ligands are mandatory. In a recent study from our lab, the majority out of 18 melanoma cell lines remained resistant against treatment with the death ligand TRAIL (tumor necrosis factor related apoptosis inducing ligand). Resistance was shown to be mainly due to incomplete processing of caspase-3 into catalytically inactive p21 by binding of the anti-apoptotic protein X-linked inhibitor of apoptosis protein (XIAP). Co-irradiation with sub-lethal ultraviolet (UV) B caused depletion of XIAP resulting in synergistic sensitization of all but two melanoma cell lines to TRAIL. We show here the XIAP depletion to essentially require initial caspase-mediated cleavage, which promotes proteasomal degradation of XIAP. Utilizing specific caspase inhibitors and small interfering RNA-mediated knockdown, we further identified caspase-3 to be responsible for performing the initial cleavage of XIAP after UVB treatment. Additional evidence suggests an accelerated mitochondrial outer membrane permeabilization in response to co-treatment with TRAIL and UVB, which directs the release of XIAP antagonizing factors including Smac. Distraction of XIAP consequently liberates caspase-3 to autocatalytically process into active p17. Activated caspase-3 cleaves XIAP and further enhances its activation in a positive regulatory feedback loop. The molecular mechanism discovered here appears to have broader implications, because cleavage of XIAP was also shown to accompany cisplatin-induced sensitization of melanoma cells to TRAIL.

Sensitization of melanoma cells to TRAIL by UVB-induced and NF-kappaB-mediated downregulation of xIAP.

Effective treatment of malignant melanoma with the tumor-selective death ligand tumor necrosis-related apoptosis-inducing ligand (TRAIL) is curtailed by the fact that many melanoma cell lines are a priori resistant against TRAIL-induced apoptosis. By investigating 18 melanoma cell lines, we show that TRAIL susceptibility is completely independent of the tumor progression stage but can be positively stimulated by co-exposure to a sublethal ultraviolet B light (UVB) dose, providing an excellent tool to study the mechanism underlying TRAIL resistance. TRAIL resistance could be linked to the ratio of x-linked inhibitor of apoptosis proteins (xIAP) and caspase-3 levels within the cell. UVB-induced sensitization coincides with enhanced xIAP degradation, allowing full caspase-3 processing and activation. It is also accompanied by concomitant IkappaBalpha degradation, resulting in nuclear factor-kappaB (NF-kappaB)-dependent transcriptional repression of xIAP. Loss of xIAP in turn was reduced upon overexpression of an IkappaBalpha super-repressor, thus NF-kappaB activation seems to be responsible for differential regulation of xIAP and consequently determines TRAIL susceptibility. As xIAP-mediated blockade of apoptosis seems to be the dominant cause of TRAIL resistance of all melanoma cell lines investigated here, our data suggest that direct chemical xIAP inhibition or combination treatment with DNA-damaging agents may offer new therapeutic strategies to generally sensitize melanoma toward TRAIL-induced apoptosis.

Identification of PP2A as a crucial regulator of the NF-kappaB feedback loop: its inhibition by UVB turns NF-kappaB into a pro-apoptotic factor.

Although nuclear factor-kappaB (NF-kappaB) usually exerts anti-apoptotic activity, upon activation by interleukin-1 (IL-1) it enhances ultraviolet-B radiation (UVB)-induced apoptosis. This paradoxical effect is associated with NF-kappaB-dependent pronounced secretion of tumour necrosis factor-alpha (TNF) which activates TNF-R1 in an autocrine fashion to enhance UVB-induced apoptosis. We demonstrate that sustained TNF transcription in UVB+IL-1-treated cells involves complete abrogation of the negative feedback loop of NF-kappaB preventing IkappaBalpha resynthesis, hence allowing uncontrolled NF-kappaB activity. We show that IkappaBalpha is not transcriptionally inhibited but resynthesized protein is immediately marked for degradation due to persistent inhibitor of kappaB kinasebeta (IKKbeta) activity. Continuous IKKbeta phosphorylation and activation is caused by UVB-mediated inhibition of the phosphatase PP2A. This study demonstrates that the cellular response to different NF-kappaB activators may be converted to the opposite reaction when both stimuli act in concert. Our data shed new light on the significance of negative feedback regulation of NF-kappaB and identifies PP2A as the key regulator of this process.

Combined targeting of MAPK and AKT signalling pathways is a promising strategy for melanoma treatment.

BACKGROUND: In melanoma, several signalling pathways are constitutively activated. Among them, the RAS/RAF/MEK/ERK (MAPK) and PI3K/AKT (AKT) signalling pathways are activated through multiple mechanisms and appear to play a major role in melanoma development and progression. OBJECTIVES: In this study, we examined whether targeting the MAPK and/or AKT signalling pathways would have therapeutic effects against melanoma. METHODS: Using a panel of pharmacological inhibitors (BAY 43-9006, PD98059, U0126, wortmannin, LY294002) we inhibited the MAPK and AKT signalling pathways at different levels and evaluated the effects on growth, survival and invasion of melanoma cells in monolayer and organotypic skin culture. RESULTS: Antiproliferative and proapoptotic effects of inhibitors alone in monolayer culture were disappointing and varied among the different cell lines. In contrast, combined targeting of the MAPK and AKT signalling pathways significantly inhibited growth and enhanced apoptosis in monolayer culture. To verify our data in a more physiological context we incorporated melanoma cells into regenerated human skin mimicking the microenvironment of human melanoma. Combinations of MAPK and AKT inhibitors completely suppressed invasive tumour growth of melanoma cells in regenerated human skin. CONCLUSIONS: Combined targeting of MAPK and AKT signalling pathways is a promising strategy for melanoma treatment and should encourage further in-depth investigations.

Differential effects of NF-kappaB on apoptosis induced by DNA-damaging agents: the type of DNA damage determines the final outcome.

The transcription factor nuclear factor kappa-B (NF-kappaB) is generally regarded as an antiapoptotic factor. Accordingly, NF-kappaB activation inhibits death ligand-induced apoptosis. In contrast, ultraviolet light B (UVB)-induced apoptosis is not inhibited but even enhanced upon NF-kappaB activation by interleukin-1 (IL-1). This study was performed to identify the molecular mechanisms underlying this switch of NF-kappaB. Enhancement of UVB-induced apoptosis was always associated with increased release of tumour necrosis factor-alpha (TNF-alpha), which was dependent on NF-kappaB activation. The same was observed when UVA and cisplatin were used, which like UVB induce base modifications. In contrast, apoptosis caused by DNA strand breaks was not enhanced by IL-1, indicating that the type of DNA damage is critical for switching the effect of NF-kappaB on apoptosis. Surprisingly, activated NF-kappaB induced TNF-alpha mRNA expression in the presence of all DNA damage-inducing agents. However, in the presence of DNA strand breaks, there was no release of the TNF-alpha protein, which is so crucial for enhancing apoptosis. Together, this indicates that induction of DNA damage may have a significant impact on biological effects but it is the type of DNA damage that determines the final outcome. This may have implications for the role of NF-kappaB in carcinogenesis and for the application of NF-kappaB inhibitors in anticancer therapy.

Apoptosis induced by disruption of the actin cytoskeleton is mediated via activation of CD95 (Fas/APO-1).

Activation of the death receptor CD95 by its ligand or by UV radiation is associated with receptor clustering. The mechanism underlying this clustering is mostly unclear. Here we show that although disruption of the actin cytoskeleton by cytochalasin B (CyB) itself induces moderate apoptosis, it enhances apoptosis in HeLa cells induced either by UV radiation or an agonistic anti-CD95 antibody. CyB augments UV-induced apoptosis independently of UV-mediated DNA damage, since induction of DNA repair by exogenous DNA repair enzymes did not alter its enhancing effect. Inhibition of caspase-8, the most upstream caspase in CD95 signaling, blocked the apoptotic effect of CyB and the enhancing effect on UV- and CD95-induced apoptosis. Confocal laser scanning microscopy revealed that (i) CyB induces CD95 clustering, (ii) enhances UV-induced CD95 clustering, and (iii) CD95 clusters colocalize with disrupted actin filaments, suggesting a link between receptor clustering and actin rearrangement. Disruption of CD95 signaling by a dominant negative mutant of the signaling protein FADD protected from CyB-induced apoptosis and prevented the UV-enhancing effect. Accordingly, both the apoptotic and the enhancing effect of CyB was reduced in epidermal cells obtained from CD95 deficient mice (lpr) when compared to wild-type mice. These data suggest that disruption of the cytoskeleton causes apoptosis via activation of CD95 and enhances UV-induced apoptosis, possibly via aiding receptor clustering.

The molecular determinants of sunburn cell formation.

Sunburn cell (SBC) formation in the epidermis is a characteristic consequence of ultraviolet radiation (UVR) exposure at doses around or above the minimum erythema dose. SBC have been identified morphologically and biologically as keratinocytes undergoing apoptosis. There is evidence that SBC formation is a protective mechanism to eliminate cells at risk of malignant transformation. The level of DNA photodamage is a major determinant of SBC induction by a process controlled by the tumor suppressor gene p53. However, extra-nuclear events also contribute to SBC formation, such as the activation of death receptors including CD95/Fas. UVR triggers death receptors either by direct activation of these surface molecules or by inducing the release of their ligands such as CD95 ligand or tumor necrosis factor. Oxidative stress also appears to be involved, probably via mitochondrial pathways, resulting in the release of cytochrome C. Pathways which modify SBC formation are now extensively studied given the importance of apoptosis in eliminating irreparably damaged cells. A greater understanding of the mechanisms that induce and prevent UVR-induced apoptosis will contribute to our understanding of mechanisms relevant in genomic integrity.

Ultraviolet radiation inhibits interleukin-2-induced tyrosine phosphorylation and the activation of STAT5 in T lymphocytes.

UV radiation was recently found to hinder interferon-gamma from exerting its biological effects by inhibiting the phosphorylation of signal transducer and activator of transcription (STAT)-1, a crucial signal transducing protein in the interferon-gamma pathway. Because this activity by UV may contribute to its immunosuppressive properties we studied whether this is specific for STAT1 or whether UV also affects other members of the STAT family. STAT5 is crucially involved in signaling of interleukin (IL)-2, enabling up-regulation of the IL-2 receptor alpha chain, an essential component of the high affinity IL-2 receptor. Exposure of the murine T cell line CTLL to IL-2 caused tyrosine phosphorylation of STAT5 that was remarkably reduced when cells were exposed to UV. Accordingly, STAT5 binding activity was significantly impaired in UV-exposed cells. In contrast, IL-2-induced tyrosine phosphorylation of the kinases Jak1 and Jak3 located upstream of STAT5 was not affected by UV. The effect of UV on STAT5 phosphorylation was antagonized by orthovanadate, implying involvement of a phosphatase in this process. Accordingly, up-regulation of the IL-2 receptor alpha chain was reduced in cells that were treated with IL-2 plus UV. Because STAT5-mediated IL-2 effects are vital for normal immune functions, inhibition of STAT5 signaling by UV may contribute to its well known immunosuppressive properties.

Molecular mechanisms of UV-induced apoptosis.

Sunburn cells, single standing cells with typical morphologic features occurring in UV-exposed skin, have been recognized as keratinocytes undergoing apoptosis following UV irradiation. Induction of apoptosis following UV exposure appears to be a protective mechanism, getting rid off severely damaged cells that bear the risk of malignant transformation. UV-mediated apoptosis is a highly complex process in which different molecular pathways are involved. These include DNA damage, activation of the tumor suppressor gene p53, triggering of cell death receptors either directly by UV or by autocrine release of death ligands, mitochondrial damage and cytochrome C release. Detailed knowledge about the interplay between these pathways will increase our understanding of photocarcinogenesis. This review briefly discusses recent findings concerning the molecular mechanisms underlying UV-induced apoptosis.

Ultraviolet radiation-induced interleukin 6 release in HeLa cells is mediated via membrane events in a DNA damage-independent way.

Evidence exists that ultraviolet radiation (UV) affects molecular targets in the nucleus or at the cell membrane. UV-induced apoptosis was found to be mediated via DNA damage and activation of death receptors, suggesting that nuclear and membrane effects are not mutually exclusive. To determine whether participation of nuclear and membrane components is also essential for other UV responses, we studied the induction of interleukin-6 (IL-6) by UV. Exposing HeLa cells to UV at 4 degrees C, which inhibits activation of surface receptors, almost completely prevented IL-6 release. Enhanced repair of UV-mediated DNA damage by addition of the DNA repair enzyme photolyase did not affect UV-induced IL-6 production, suggesting that in this case membrane events predominant over nuclear effects. UV-induced IL-6 release is mediated via NFkappaB since the NFkappaB inhibitor MG132 or transfection of cells with a super-repressor form of the NFkappaB inhibitor IkappaB reduced IL-6 release. Transfection with a dominant negative mutant of the signaling protein TRAF-2 reduced IL-6 release upon exposure to UV, indicating that UV-induced IL-6 release is mediated by activation of the tumor necrosis factor receptor-1. These data demonstrate that UV can exert biological effects mainly by affecting cell surface receptors and that this is independent of its ability to induce nuclear DNA damage.

Interleukin-1 protects transformed keratinocytes from tumor necrosis factor-related apoptosis-inducing ligand- and CD95-induced apoptosis but not from ultraviolet radiation-induced apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a new member of the tumor necrosis factor (TNF) family, induces apoptosis primarily of transformed cells. Interleukin-1 was previously found to protect the keratinocyte cell line KB from TRAIL-induced apoptosis, thus we studied whether interleukin-1 also protects from other apoptotic stimuli (ultraviolet radiation (UV), CD95-ligand). Interleukin-1 rescued KB cells from TRAIL- and CD95-induced apoptosis, which was critically dependent on nuclear factor kappaB, because cells transfected with a super-repressor form of the nuclear factor kappaB inhibitor IkappaB were less protected. In contrast, UV-mediated apoptosis was not only not prevented by interleukin-1 but even enhanced. This opposite effect of interleukin-1 was also observed for the expression of the inhibitor of apoptosis proteins (IAP). Whereas TRAIL- and CD95-mediated suppression of IAP expression was partially reversed by interleukin-1, UV-mediated down-regulation of IAPs was not reversed but even further enhanced. Increased apoptosis induced by interleukin-1 plus UV was accompanied by excessive TNFalpha release, implying that enhanced cytotoxicity is due to the additive effect of these two apoptotic stimuli. Accordingly, enhanced apoptosis was reduced by blocking the TNF receptor-1. The opposite effects of interleukin-1 indicate that different mechanisms are involved in UV-induced apoptosis compared with CD95- and TRAIL-mediated apoptosis. Furthermore, the data suggest that whether a signal acts in an antiapoptotic way or not does not only depend on the signal itself but also on the stimulus causing apoptosis.

Nuclear and cell membrane effects contribute independently to the induction of apoptosis in human cells exposed to UVB radiation.

UVB-induced DNA damage is a crucial event in UVB-mediated apoptosis. On the other hand, UVB directly activates death receptors on the cell surface including CD95, implying that UVB-induced apoptosis can be initiated at the cell membrane through death receptor clustering. This study was performed to measure the relative contribution of nuclear and membrane effects in UVB-induced apoptosis of the human epithelial cell line HeLa. UVB-mediated DNA damage can be reduced by treating cells with liposomes containing the repair enzyme photolyase followed by exposure to photoreactivating light. Addition of photolyase followed by photoreactivation after UVB reduced the apoptosis rate significantly, whereas empty liposomes had no effect. Likewise, photoreactivating treatment did not affect apoptosis induced by the ligand of CD95, CD95L. UVB exposure at 4 degrees C, which prevents CD95 clustering, also reduced the apoptosis rate, but to a lesser extent. When cells were exposed to UVB at 4 degrees C and treated with photolyase plus photoreactivating light, UVB-induced apoptosis was almost completely prevented. Inhibition of caspase-3, a downstream protease in the CD95 signaling pathway, blocked both CD95L and UVB-induced apoptosis, whereas blockage of caspase-8, the most proximal caspase, inhibited CD95L-mediated apoptosis completely, but UVB-induced apoptosis only partially. Although according to these data nuclear effects seem to be slightly more effective in mediating UVB-induced apoptosis than membrane events, both are necessary for the complete apoptotic response. Thus, this study shows that nuclear and membrane effects are not mutually exclusive and that both components contribute independently to a complete response to UVB.

Interleukin-1 protects transformed keratinocytes from tumor necrosis factor-related apoptosis-inducing ligand.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor family. It induces apoptosis primarily of transformed but not of normal cells and may therefore be a promising anti-cancer drug. Studying the role of TRAIL in apoptosis of keratinocytes, we detected TRAIL transcripts and protein in both normal human keratinocytes and transformed keratinocyte cell lines HaCaT and KB. Although normal keratinocytes were resistant to TRAIL, HaCaT and KB cells underwent apoptosis following TRAIL exposure. When HaCaT and KB cells were pretreated with the pro-inflammatory cytokine interleukin-1 (IL-1), cells became resistant to TRAIL-induced apoptosis. IL-1 significantly induced activation of the transcription factor NFkappaB in transformed keratinocytes. Moreover, the proteasome inhibitor MG132, which inhibits IL-1-induced NFkappaB activation, completely prevented the protective effect of IL-1. Thus, IL-1 appears to protect transformed keratinocytes from the cytotoxic effect of TRAIL via activation of NFkappaB. These data suggest that NFkappaB activation may protect cells from TRAIL-induced apoptosis and indicate a TRAIL receptor-independent pathway, which allows cells to escape the cytotoxic effect of TRAIL. Because IL-1 is secreted by a variety of tumor cells and is also released by inflammatory cells participating in the tumor-host immune response, tumors under these conditions could become resistant to TRAIL.

Ultraviolet light induces apoptosis via direct activation of CD95 (Fas/APO-1) independently of its ligand CD95L.

Induction of apoptosis in keratinocytes by UV light is a critical event in photocarcinogenesis. Although p53 is of importance in this process, evidence exists that other pathways play a role as well. Therefore, we studied whether the apoptosis-related surface molecule CD95 (Fas/APO-1) is involved. The human keratinocyte cell line HaCaT expresses CD95 and undergoes apoptosis after treatment with UV light or with the ligand of CD95 (CD95L). Incubation with a neutralizing CD95 antibody completely prevented CD95L-induced apoptosis but not UV-induced apoptosis, initially suggesting that the CD95 pathway may not be involved. However, the protease CPP32, a downstream molecule of the CD95 pathway, was activated in UV-exposed HaCaT cells, and UV-induced apoptosis was blocked by the ICE protease inhibitor zVAD, implying that at least similar downstream events are involved in CD95- and UV-induced apoptosis. Activation of CD95 results in recruitment of the Fas-associated protein with death domain (FADD) that activates ICE proteases. Immunoprecipitation of UV-exposed HaCaT cells revealed that UV light also induces recruitment of FADD to CD95. Since neutralizing anti-CD95 antibodies failed to prevent UV-induced apoptosis, this suggested that UV light directly activates CD95 independently of the ligand CD95L. Confocal laser scanning microscopy showed that UV light induced clustering of CD95 in the same fashion as CD95L. Prevention of UV-induced CD95 clustering by irradiating cells at 10 degrees C was associated with a significantly reduced death rate. Together, these data indicate that UV light directly stimulates CD95 and thereby activates the CD95 pathway to induce apoptosis independently of the natural ligand CD95L. These findings further support the concept that UV light can affect targets at the plasma membrane, thereby even inducing apoptosis.

Down-regulation of interferon gamma-activated STAT1 by UV light.

STAT1 is a cytoplasmic transcription factor that is phosphorylated by Janus kinases (Jak) in response to interferon-gamma (IFNgamma). Phosphorylated STAT1 translocates to the nucleus, where it turns on specific sets of IFNgamma-inducible genes. Here, we show that UV light interferes with tyrosine phosphorylation of STAT1, thereby hindering IFNgamma from exerting its biological effects. This effect is not due to a down-regulation of the IFNgamma receptor because phosphorylation of upstream-located Jak1 and Jak2 was not suppressed by UV light. In contrast, UV light had no effect on the phosphorylation of STAT3, which is activated by the proinflammatory cytokine interleukin 6. The UV light effect on STAT1 phosphorylation could be antagonized by vanadate, indicating at least partial involvement of a protein tyrosine phosphatase. Therefore, this study indicates a mechanism by which UV light can inhibit gene activation and suggests STAT1 as a new extranuclear UV target closely located to the membrane.

Overproduction of Sac7d and Sac7e reveals only Sac7e to be a DNA-binding protein with ribonuclease activity from the extremophilic archaeon Sulfolobus acidocaldarius.

Genomic DNA from Sulfolobus acidocaldarius was screened using a degenerate oligodeoxyribonucleotide, derived from the sequence of 16 N-terminal amino acids from SaRD protein. SaRD protein was previously isolated in our laboratory and identified as a protein from S. acidocaldarius exhibiting ribonuclease activity as well as DNA-binding properties. On the basis of Southern hybridization analysis two genes from S. acidocaldarius have been cloned, sequenced and overproduced in Escherichia coli. The deduced amino acid sequences revealed that one gene encodes Sac7d and the other one Sac7e; two small, previously described basic proteins from S. acidocaldarius, and furthermore the N-termini of Sac7e and SaRD are identical. Northern blot analysis demonstrated that the genes are transcribed separately. After expression of sac7d and sac7e genes in E. coli it was shown that only recombinant Sac7e protein exhibits RNase activity and is catalytically indistinguishable from SaRD protein. Western blot analysis using a polyclonal antiserum raised against purified SaRD protein further confirmed that Sac7e and SaRD are identical proteins endowed with RNase activity and DNA-binding properties. A new RNA cleavage mechanism has to be postulated for Sac7e since, in contrast to common RNases (e.g. RNase A and T1), no histidines are present in the amino acid sequence. Differences between the very closely related 7 kDa proteins from two Sulfolobus strains converting DNA-binding proteins into RNases are pointed out and discussed, whereas substitutions of Glu by Gln (S. solfataricus) or by Lys (S. acidocaldarius) seem to be crucial.

SaRD, a new protein isolated from the extremophile archaeon Sulfolobus acidocaldarius, is a thermostable ribonuclease with DNA-binding properties.

We have isolated the thermostable 9 kDa SaRD-protein from Sulfolobus acidocaldarius which exhibit RNase activity as well as DNA-binding properties (SaRD). The amino acid composition and the sequence of the 16 N-terminal amino acids show similarities to different RNases as well as to DNA-binding proteins from thermophilic archea. The RNase activity was demonstrated by 5S rRNA degradation, thin layer chromatography and a zymogram. The temperature optimum for the RNase activity is 65 degrees C. The pH optimum ranges from 6.5-7.0. DNA-binding properties were shown by gel-shift assays on agarose gels. In a similar way SaRD mediated protection of DNA against DNase I digestion and Sau3A I restriction could be demonstrated. The melting point (Tm) of genomic DNA was raised from 68 degrees C to 90 degrees C by addition of the SaRD-protein. CD spectroscopy indicated that SaRD is very stable near neutral pH and can neither be unfolded by temperatures up to 85% C nor by addition of 8 M urea.