Original Paper | Published:

DNA damage, death receptor activation and reactive oxygen species contribute to ultraviolet radiation-induced apoptosis in an essential and independent way

Oncogene volume 21, pages 58445851 (29 August 2002) | Download Citation

Subjects

Abstract

Nuclear DNA damage and death receptor (CD95) activation by ultraviolet-B radiation (UVB) play a major role in UVB-induced apoptosis. Removal of DNA damage combined with inhibition of death receptor activation resulted in pronounced but not complete suppression of apoptosis, indicating that a third independent pathway is involved. Since reactive oxygen species (ROS) cause apoptosis and are induced by UVB, the radical scavenger pyrrolidene-dithiocarbamate (PDTC) was used. PDTC prevented UVB-induced apoptosis partially, H2O2-induced cell death largely, but not CD95-mediated apoptosis. The same was observed for cytochrome c release from mitochondria, another important event during apoptosis. The proapoptotic protein Bid was cleaved upon exposure to UVB or to agonistic anti-CD95-antibodies, but not to H2O2, indicating that H2O2 uses a different pathway. The fact that PDTC neither inhibited CD95-mediated apoptosis nor affected UV-induced DNA damage indicated that ROS generated during UVB irradiation may directly trigger mitochondrial cytochrome c release, thereby contributing to apoptosis. Accordingly, complete inhibition of apoptosis was observed when in addition to DNA damage removal via photoreactivation and blockade of CD95 signaling by caspase-8 inhibitor zIETD, PDTC was added before UVB exposure. This indicates that DNA damage, death receptor activation and ROS formation contribute to UVB-induced apoptosis in an essential and independent way.

Introduction

One of the major biological features of ultraviolet radiation (UV) in the middle wave length range (290–320 nm, UVB) is the induction of apoptotic cell death of keratinocytes, which in vivo appear as sunburn cells within the epidermis (Murphy et al., 2001). The observation that the tumor suppressor gene p53 is closely linked to UVB-induced apoptosis suggested that formation of sunburn cells is a controlled mechanism which limits the survival of cells with irreparable UVB-induced DNA damage and thus avoids malignant transformation (Ziegler et al., 1994). While in previous years UVB-mediated apoptosis of keratinocytes was just regarded as a marker for the severity of UVB damage, it is now accepted to be a protective mechanism preventing the generation of UVB-induced skin cancer, the most common malignancy in Caucasians in Western Europe and in the United States (Gloster and Brodland, 1996). Due to its antiapoptotic and therefore putatively procarcinogenic function, dysregulations in UVB-induced apoptosis may have profound impacts on photocarcinogenesis. Therefore, detailed knowledge about the signaling pathways involved in UVB-induced apoptosis is of practical relevance.

UVB-induced apoptosis has been recognized as a complex mechanism in which a variety of signaling pathways are involved (Kulms and Schwarz, 2000). For years, it was proposed that UVB-induced DNA damage is the only mediator of UVB-induced cell death. UVB induces two types of lesions in chromosomal DNA, <6–4> photoproducts and cyclobutane pyrimidine dimers (CPD), the latter being the predominant ones (Patrick, 1977). The majority of DNA lesions are removed by the nucleotide excision repair (de Laat et al., 1999). Accordingly, patients suffering from xeroderma pigmentosum, a disease which is based on genetic defects in various components of the nucletoide excision repair (Kraemer et al., 1994) reveal an increased number of sunburn cells following UVB exposure (Petit-Frere et al., 2000). The crucial role of DNA damage in UVB-induced apoptosis has been further confirmed by in vivo studies showing that enhancement of DNA repair by the topical application of repair enzymes in liposomes reduces sunburn cell formation (Stege et al., 2000; Wolf et al., 1995). In addition, detection of sunburn cells in skin treated with other DNA damaging irradiation procedures (e.g. psoralen and UVA) supports the view that DNA is the relevant chromophore in the formation of sunburn cells (Woodcock and Magnus, 1976).

Rosette and Karin demonstrated that UVB is able to directly activate cell surface receptors by inducing receptor clustering without the need of the respective ligand (Rosette and Karin, 1996). Accordingly, we and others showed that this applies also for the death receptor CD95/Fas (Aragane et al., 1998; Rehemtulla et al., 1997). Using confocal laser scanning microscopy we observed that UVB induces clustering of the CD95 receptor. CD95 clustering by UVB is functionally relevant since inhibition of CD95 clustering by keeping cells at low temperature (4–10°C) during UVB exposure was associated with a partial reduction of UVB-induced apoptosis (Aragane et al., 1998). Furthermore, transfection of cells with a dominant negative mutant against FADD (Fas-associated protein with death domain), an important member of the CD95 signaling pathway (Boldin et al., 1995; Chinnaiyan et al., 1996), resulted in reduction of UVB-induced apoptosis (Aragane et al., 1998), indicating that death receptor activation by UVB does play a role in UVB-induced apoptosis.

The above mentioned findings clearly demonstrated that UVB-induced apoptosis can be initiated both at the cell membrane and in the nucleus. To determine the relative contribution of nuclear and membrane effects to UVB-induced apoptosis, Kulms et al. (1999) used the approach of enhancing DNA repair by addition of the exogenous DNA repair enzyme photolyase encapsulated into liposomes (Yarosh and Klein, 1994). The photolyase binds to an UVB-induced CPD in DNA and catalyzes its splitting by electron transfer from absorbing wavelengths above 320 nm, a process called photoreactivation (Eker et al., 1990). Accordingly, photoreactivation reduced both CPD and UVB-induced apoptosis remarkably but did not protect cells completely. Exposure of HeLa cells to UVB at 4°C which prevents death receptor clustering also reduced apoptosis but to a lesser extent than photoreactivation. When cells were exposed to UVB at 4°C and subsequently photoreactivated, UVB-induced apoptosis was reduced in an additive way, indicating that nuclear and membrane effects are not mutually exclusive and that both components contribute independently to the apoptotic UVB response (Kulms et al., 1999). However, even by blocking death receptor activation and reducing DNA damage, complete inhibition of apoptosis was not achieved, some residual apoptotic activity was still left. This indicates that at least a third independent signaling pathway should be involved as well.

UV is known to be an inducer of reactive oxygen species (ROS), including superoxide radical (O2−), hydrogen peroxide (H2O2), singulett oxygen (1O2) and hydroxyl radical (OH) (Farber, 1994; Masaki et al., 1995; Peus et al., 1999). These products have been shown themselves to initiate cellular damage and apoptosis (Tyrrell, 1995). Therefore, ROS have been implicated in cutaneous aging as well as in the pathogenesis of inflammatory skin diseases and of skin cancer (Black, 1987; Black and Lambert, 2001; Tyrrell, 1994). The cytotoxic potential of ROS involves lipid peroxidation which causes changes in the structure of plasma membranes (Bongarzone et al., 1995; de Kok et al., 1994) and damaging of the inner mitochondrial membrane resulting in loss of the membrane potential Δψm and consequently in cytochrome c release into the cytoplasm (Farber, 1994). Additional genotoxic effects derive from an increased level of ROS, especially DNA strand breaks, formation of altered bases like 8-hydroxyguanine and modification of the DNA binding sites of transcription factors (Floyd, 1990).

Therefore, we tested whether generation of ROS by UVB is involved in UVB-induced apoptosis and may represent the additional pathway involved. Here, we show that in addition to DNA damage and death receptor activation by UVB, ROS contribute to UVB-induced apoptosis in an essential and independent way.

Results

The radical scavenger PDTC reduces UVB- and H2O2-induced apoptosis but not CD95-mediated cell death

To determine whether ROS are involved in UVB-induced apoptosis, the radical scavenger PDTC was used. Addition of PDTC to HeLa cells 1 h before UVB exposure resulted in partial inhibition of UVB-mediated cell death as determined by a cell death detection ELISA (Figure 1a). Similar findings were obtained when annexin V staining was used as a read-out system for apoptosis (data not shown). H2O2-mediated apoptosis was almost completely suppressed by PDTC, while apoptosis induced by an agonistic anti-CD95-Ab was not affected at all by PDTC (Figure 1a).

Figure 1
Figure 1

PDTC reduces UVB-, H2O2-, but not CD95-induced apoptosis and cytochrome c release. (a), After preincubation with PDTC (50 μM) for 1 h, HeLa cells were exposed to UVB (400 J/m2), to H2O2 (500 μM) or to an agonistic anti-CD95-Ab (1 μg/ml). After incubation for 16 h, apoptosis was evaluated using a cell death detection ELISA. Rate of apoptosis is reflected by the enrichment of nucleosomes in the cytoplasm, shown on the y axis (mean±s.d. of three independently performed experiments). (b), After 16 h cytoplasmic protein extracts were obtained and subjected to Western blot analysis with an Ab directed against cytochrome c. Equal loading was monitored with an Ab directed against α-tubulin

Activation of the CD95 receptor either by UVB or by an anti-CD95-Ab leads to activation of the upstream caspase-8 subsequently resulting in direct cleavage of the downstream effector caspase-3 as well as of the proapoptotic Bid protein (Li et al., 1998; Luo et al., 1998). Activated Bid then interacts with the antiapoptotic proteins of the Bcl-2 family at the mitochondrial membrane, finally causing cytochrome c release into the cytoplasm (Desagher et al., 1999). Cytochrome c interacts with Apaf-1 and ATP to activate caspase-9, resulting in further cleavage of caspase-3 (Hu et al., 1999; Zou et al., 1999). Western blot analysis demonstrated that activation of CD95 induced cytochrome c release into the cytoplasm, which was not influenced by PDTC (Figure 1b). In contrast, UVB-induced cytochrome c release was partially reduced by PDTC. H2O2 induced pronounced mitochondrial cytochrome c release, which was almost completely inhibited upon PDTC treatment. These data indicate that ROS are involved in UVB-mediated apoptosis but do not play a role in death receptor/CD95-mediated cell death.

To exclude that PDTC may inhibit UVB-induced apoptosis by affecting other pathways than blocking ROS, other radical scavengers were utilized. For that purpose N-acetyl-cysteine (NAC) and butylated hydroxyanisole (BHA), respectively, were used. As observed with PDTC, UVB-induced apoptosis was partially inhibited by NAC or BHA. Both substances inhibited H2O2-mediated apoptosis which was very pronounced, while apoptosis induced by an agonistic anti-CD95-Ab remained unaffected (Figure 2).

Figure 2
Figure 2

Radical scavengers BHA and NAC reduce UVB, and H2O2-, but not CD95-induced apoptosis. After preincubation with BHA (50 μM) or NAC (10 mM) for 1 h, HeLa cells were exposed to UVB (400 J/m2), to H2O2 (500 μM) or to an agonistic anti-CD95-Ab (1 μg/ml). After incubation for 16 h, apoptosis was evaluated using a cell death detection ELISA. Rate of apoptosis is reflected by the enrichment of nucleosomes in the cytoplasm, shown on the y axis. Data show one representative of three independently performed experiments

PDTC does not affect UVB-induced DNA damage

UVB-induced apoptosis is a complex process in which DNA damage and CD95 activation are critically involved (Kulms and Schwarz, 2000). Since the data displayed in Figures 1 and 2 suggest that activation of CD95 may be independent of the formation of ROS we next determined whether ROS are involved in the mediation of UVB-induced DNA damage. PDTC was applied to cells 1 h prior to UVB-irradiation and Southwestern dot-blot analysis was performed using an Ab directed against CPD, the main lesions in genomic DNA induced by UVB. As a positive control for a reduction of DNA damage, DNA repair was induced by photoreactivation through addition of Photosomes®. As documented in Figure 3, cells subjected to photoreactivation following UVB irradiation showed a significant reduction of CPD compared to DNA extracts obtained from cells which were only UVB exposed. In contrast, addition of PDTC did not reduce the level of UVB-induced CPD. This indicates that ROS are not involved in the generation of UVB-induced CPDs.

Figure 3
Figure 3

PDTC does not affect the formation of UVB-induced CPD. HeLa cells were subjected to UVB (400 J/m2). PDTC (50 μM) was added 1 h before UVB exposure. Photosomes (Photo) were added after UV exposure, cells were incubated for 1 h at 37°C in the dark followed by photoreactivation by irradiating cells with photoreactivating light. One hour after photoreactivation, genomic DNA was extracted and Southwestern dot-blot analysis was performed using an Ab directed against CPD

In constrast to UVB- and CD95-induced apoptosis, H2O2-mediated cell death is independent of Bid cleavage

To further elucidate the differences in the signaling pathways, kinetic analysis of cytochrome c release was performed. Cells were exposed either to UVB, H2O2 or to an agonistic anti-CD95-Ab. Cytoplasmic protein extracts were obtained after 2, 4, 6, 10 or 16 h and subjected to Western blot analysis. Release of cytochrome c from mitochondria into the cytoplasm occurred 4 h after exposure to UVB or to H2O2, whereas cytochrome c release started only after 6 h upon anti-CD95-treatment (Figure 4).

Figure 4
Figure 4

H2O2-mediated cell death is independent of Bid cleavage. HeLa cells were exposed to either UVB (400 J/m2), H2O2 (500 μM) or to an agonistic anti-CD95-Ab (1 μg/ml). At different time points, cytoplasmic protein extracts were obtained and subjected to Western blot analysis using Abs directed against α-tubulin, Bid, and cytochrome c, respectively

CD95-mediated cytochrome c release correlated with Bid cleavage (Figure 4). The same was observed upon UVB exposure which is in accordance with previous findings that CD95 triggering is involved in UV-induced apoptosis (Aragane et al., 1998; Kulms et al., 1999; Rehemtulla et al., 1997). In contrast, Bid was not cleaved when apoptosis was induced by H2O2, indicating that ROS-mediated apoptosis is independent of Bid cleavage, and consequently that ROS do not influence death receptor activation.

Only early application of PDTC inhibits cytochrome c release during UVB-induced apoptosis

Dysfunction of mitochondria is a common feature of the apoptotic scenario and results in an enhanced appearance of ROS within the cell (Zamzami et al., 1996). The reducing effect of PDTC on UVB-induced apoptosis can be due to scavenging radicals which are either directly induced by UVB or are generated as a consequence of damage to the mitochondria. To determine whether ROS formation during UVB-induced apoptosis occurs prior to or coincides with cytochrome c release, PDTC was added at different time points before and after UVB exposure. Sixteen hours after UVB irradiation, cells were harvested and in parallel analysed in a cell death detection ELISA (Figure 5a) and subjected to Western blot analysis with an Ab directed against cytochrome c (Figure 5b). The maximum reducing effect on both UVB-induced cell death and cytochrome c release was observed when PDTC was applied either 1 h before or up to 1 h after UVB exposure. Addition of PDTC at later timepoints ranging from 2 to 8 h after UVB irradiation had no or only marginal effects on cytochrome c release (Figure 5b), while inhibition of apoptosis was still observed upon addition of PDTC 2 h after UVB exposure (Figure 5A). The inhibitory effects of PDTC on cytochrome c release were more pronounced than those on apoptosis, suggesting that UVB-induced apoptosis is partially, but cytochrome c release almost exclusively, mediated via ROS. Furthermore, these data support the concept that ROS formation is an early event in UVB-induced apoptosis and is rather the cause for, but not the consequence of cytochrome c release from mitochondria.

Figure 5
Figure 5

UVB-induced apoptosis can only be inhibited by early application of PDTC. (a), Cells were exposed to UVB (400 J/m2). PDTC was added either 1 h prior to irradiation, immediately after irradiation or 1, 2, 4 or 8 h after irradiation. Sixteen hours after UVB exposure apoptosis was measured using a cell death detection ELISA. Rate of apoptosis is reflected by the enrichment of nucleosomes in the cytoplasm, shown on the y axis (mean±s.d. of three independently performed experiments). (b), Cells were subjected to the identical treatment as in (a). Sixteen hours after UVB exposure cytoplasmic proteins were extracted and Western blot analysis was performed with an Ab directed against cytochrome c. Equal loading was monitored by reprobing the membrane with an Ab directed against α-tubulin

Complete inhibition of UVB-induced apoptosis is achieved by the combination of DNA repair, inhibition of death receptor signaling and scavenging of ROS

To determine the relative contribution of ROS formation, DNA damage and death receptor activation to UVB-induced apoptosis, it was tested whether inhibition of all three pathways resulted in complete inhibition of UVB-mediated cell death. DNA damage was reduced via induction of photoreactivation through application of photolyase incorporated into liposomes. Death receptor signaling was inhibited by the tetrapeptide z-IETD which inhibits caspase-8, the most upstream located caspase in CD95 signaling. As demonstrated previously (Kulms et al., 1999), the zIETD concentration applied inhibits CD95-mediated apoptosis induced by an agonistic CD95 antibody completely. Formation of ROS was inhibited by PDTC.

Cells were analysed both for apoptosis and cytochrome c release. Removal of DNA damage via photoreactivation resulted in pronounced reduction of apoptosis and of cytochrome c release. z-IETD also reduced apoptosis and cytochrome c release but to a lesser extent, confirming previous data (Kulms et al., 1999). In accordance with previous observations, the combination of zIETD and photoreactivation resulted in a further but not complete reduction of both apoptosis and cytochrome c release. When PDTC was added to this combination, UVB-induced apoptosis was completely inhibited (Figure 6a). Accordingly, cytochrome c release was almost completely inhibited as well, when UV-exposed cells were photoreactivated and treated with zIETD plus PDTC (Figure 6b). The fact that complete inhibition of UVB-induced apoptosis was only observed after addition of PDTC indicates that besides DNA damage and death receptor activation, UVB-mediated ROS formation is the third independent pathway being involved in UVB-induced cell death. Taken together, these data suggest that DNA damage, death receptor activation and ROS induction by UVB contribute in an essential way to UVB-mediated apoptosis.

Figure 6
Figure 6

Complete inhibition of UVB-induced apoptosis by the combination of DNA repair, inhibition of death receptor signaling and ROS formation. (a), Cells were exposed to 400 J/m2 UVB. PDTC (50 μM) was added 1 h before irradiation. z-IETD (20 μM) was added 1 h before UVB exposure. Photoreactivation was induced by adding Photosomes immediately after UVB exposure and keeping cells in the dark for 1 h followed by exposure to photoreactivating light. Cells were exposed to the different combinations of treatments as indicated. Sixteen hours after UVB exposure the apoptosis rate was determined using a cell death detection ELISA and is reflected by the enrichment of nucleosomes in the cytoplasm, shown on the y axis (mean±s.d. of three independently performed experiments). (b), Sixteen hours after UVB exposure cytoplasmic proteins were extracted and analysed for the amounts of cytochrome c using a quantitative human cytochrome c ELISA

Discussion

UVB-induced apoptosis has been recognized as a complex process in which a variety of pathways appear to be involved. UVB-induced DNA damage is certainly the main trigger of UVB-induced cell death, since accelerated removal of DNA damage by enhanced DNA repair via application of exogenous repair enzymes (T4N5 endonuclease, photolyase) most effectively reduces the formation of sunburn cells (Stege et al., 2000; Wolf et al., 1995). There is also recent evidence that induction of nucleotide excision repair by the cytokine interleukin-12 not only reduces UVB-induced CPD but also significantly suppresses UVB-mediated cell death both in vitro and in vivo (Schwarz et al., 2001).

Nevertheless, DNA damage does not appear to be the only mediator causing UVB-induced apopotosis. There is convincing evidence that UVB directly activates death receptors including CD95 and the TNF receptor 1 (Aragane et al., 1998; Rehemtulla et al., 1997; Sheikh et al., 1998). Inhibition of death receptor activation by exposing cells at a low temperature (4–10°C) to UVB is associated with a partial reduction in UVB-induced apoptosis. A similar reduction is observed when CD95 signaling is interrupted by adding zIETD, a tetrapeptide which inhibits caspase-8, the most upstream located caspase.

These observations indicated that UVB-induced apoptosis can be initiated both at the membrane and in the nucleus. Kulms et al. (1999) demonstrated that both pathways are not mutually exclusive but that both contribute independently to UVB-induced cell death. When DNA damage was repaired and UVB-induced death receptor activation was blocked, an additive effect with regard to inhibition of apoptosis was observed. Nevertheless, inhibition of apoptosis was not complete. The fact that there was still some residual apoptotic activity left under these conditions indicated that at least a third independent pathway mediating UVB-induced apoptosis may exist.

Here we provide evidence that ROS induced by UVB may be the missing link. To determine the role of ROS in UVB-induced apoptosis, we utilized the radical scavenger PDTC. Addition of PDTC partially inhibited UVB-induced apoptosis, while H2O2-mediated cell death, included as a positive control, was almost completely inhibited as determined by a cell death detection ELISA. A similar pattern was observed when cytochrome c release into the cytoplasm was investigated by Western blot analysis. Cell death induced by an agonistic anti-CD95-Ab was not affected by PDTC, indicating that CD95 triggering and signaling is independent of ROS. This is also supported by the observation that the proapoptotic protein Bid was cleaved upon triggering of CD95, while addition of H2O2 induced apoptosis without affecting Bid cleavage. The molecular mechanisms underlying CD95 clustering still remain to be determined, however, the present findings imply that ROS do not appear to be involved. To exclude that PDTC may inhibit UVB-induced apoptosis by affecting other pathways than blocking ROS, other radical scavengers were utilized. When using N-acetyl-cysteine (NAC) or butylated hydroxyanisole (BHA) similar effects were observed (Figure 2).

PDTC did not affect the formation of CPD, the major DNA photoproduct induced by UVB. This observation is in accordance with previous findings that UVB-induced DNA damage is mostly independent of ROS formation (Kuluncsics et al., 1999). In contrast, addition of the repair enzyme photolyase, as a positive control, significantly reduced CPD, confirming previous observations (Kulms et al., 1999; Stege et al., 2000). ROS are known to induce other DNA lesions than CPD, mostly 8-hydroxyguanine. However, 8-hydroxyguanine lesions do not appear to be relevant in our system since the UVB dose applied in this study only minimally induced 8-hydroxyguanine lesions which were almost below the detection limit (data not shown). In addition, CPDs appear to mediate apoptosis independently of ROS since otherwise the application of PDTC should be more or at least equally potent in reducing apoptosis than the application of photolyase.

The fact that PDTC did not affect CPD formation and did not interfere with CD95 signaling but reduced UVB-induced apoptosis indicated that ROS may independently contribute to UVB-mediated cell death. This appears to be the case, since complete inhibition of UVB-induced apoptosis was only observed when, in addition to removal of DNA damage and inhibition of CD95 signaling, PDTC was added. These data also suggest that ROS may be the missing additional pathway being responsible for the residual apoptotic activity which was still detectable when CD95 signaling was blocked and DNA damage removed (Figure 6).

Mitochondria play an important role in the pathogenesis of apoptosis (Kroemer and Reed, 2000). Triggering of the death receptor CD95 induces cleavage of the proapoptotic protein Bid (Li et al., 1998; Luo et al., 1998). Cleaved Bid translocates to the outer mitochondrial membrane and induces the release of cytochrome c from the mitochondria into the cytoplasm. Cytochrome c in association with Apaf-1 and ATP forms the apoptosome which induces cleavage of caspase-9. Activated caspase-9 cleaves the executioner caspase-3 which ultimately induces apoptosis by further substrate cleavage (Hu et al., 1999; Zou et al., 1999). Mitochondria have been recognized not only to be a target for ROS but may also function as a source for ROS (Cadenas and Davies, 2000). In the late phase of UV-induced apoptosis, mitochondrial dysfunction leads to enrichment of ROS in the cell where they exert their cytotoxic effects e.g. lipid peroxidation (Buttke and Sandstrom, 1995). To determine whether ROS formation precedes mitochondrial cytochrome c release or whether ROS formation simply coincides with cytochrome c release as a consequence of mitochondrial dysfunction during UVB-induced apoptosis, we performed experiments elucidating the time dependency of the PDTC effect. UVB-induced apoptosis as well as cytochrome c release could only be reduced if PDTC was added at early timepoints. Addition of PDTC at later time points did not affect cytochrome c release, suggesting that cytochrome c release may be a direct consequence of free radical formation. In contrast, PDTC was still able to reduce the apoptosis rate moderately when added even 2 h after UVB, and marginally after 4 h. This may be due to the fact that PDTC added at these later time points may still mitigate the cytotoxic effect caused by the release of ROS as a consequence of mitochondrial dysfunction. Together, these data suggest that ROS formation during UVB-induced apoptosis cannot only be the consequence of the loss of the mitochondrial membrane potential (Δψm) but may also be directly induced by UVB and cause cytochrome c release at early timepoints during UVB-mediated cell death (Figure 7).

Figure 7
Figure 7

Signaling pathways involved in UVB-induced apoptosis. UVB damages nuclear DNA via induction of cyclobutane pyrimidine dimers (1). In addition, UVB directly activates death receptors like CD95, inducing receptor trimerization and clustering (2). Furthermore, UVB induces formation of ROS which induce cytochrome c release (3). All these events induce the apoptosis program in an essential and independent way. This graph represents a simplified cartoon, since for the sake of clarity many details have been omitted

UVB-induced apoptosis is a protective mechanism since it eliminates cells with damaged DNA which are prone to malignant transformation (Kraemer, 1997). Therefore, inhibition of UVB-induced cell death may enhance the carcinogenic risk, unless it is due to enhanced removal of DNA damage. This can be achieved either by exogenous repair enzymes (Stege et al., 2000; Wolf et al., 1995) or as demonstrated very recently by inducing the nucleotide excision repair, e.g. by interleukin-12 (Schwarz et al., 2002). Accordingly, topical application of T4N5 endonuclease was not only associated with a reduction in sunburn cells but also with a decreased incidence of skin tumors in chronically UV-exposed mice (Yarosh et al., 1992). In contrast, other strategies reducing UVB-induced apoptosis, e.g. overexpression of antiapoptotic proteins or inhibition of death receptor signaling, may enhance the carcinogenic risk since they enable survival of cells with damaged DNA. According to our findings, free radical scavengers and antioxidants may be added to this list.

There is convincing evidence that ROS are crucially involved in premature skin aging and in connective tissue destruction (Wenk et al., 2001). Therefore, it is suggested that the topical application of radical scavengers and antioxidants like vitamin E, vitamin C and their derivatives may prevent premature skin aging and indeed these substances are widely used (Dreher and Maibach, 2001). Our findings that antioxidants inhibit UVB-induced apoptosis and thus enable survival of damaged keratinocytes would imply that the chronic application of these substances may enhance the risk for skin cancer. Unless long term studies will have clarified this issue, caution with these substances may be warranted.

Materials and methods

Cells and reagents

The human epithelial carcinoma cell line HeLa (American Tissue Culture Collection, Rockville, MD, USA) was cultured in RPMI 1640 with 5% FCS. Before stimulation cells were kept in serum free medium for 24 h. Cells were exposed to UV through colorless medium without FCS. For UV irradiation a bank of 6 TL12 fluorescent bulbs (Philips, Eindhoven, The Netherlands) was used which emit most of their energy within the UVB range (290–320 nm) with an emission peak at 313 nm. Throughout this study a dose of 400 J/m2 was used. Control cells were subjected to the identical procedure without being exposed to UV. To induce CD95-mediated apoptosis, an agonistic mouse IgM Ab directed against human CD95 (CH-11, Immunotech, Luminy, France) was added to the cell medium at a final concentration of 1 μg/ml. H2O2 (Sigma, Munich, Germany) was used in a final concentration of 500 μM to induce apoptosis. The radical scavenger pyrrolidene-dithiocarbamate (PDTC, Sigma) was used in a final concentration of 50 μM, N-acetly-cysteine (NAC, Sigma) at 10 mM and butylated hydroxyanisole (BHA, Sigma) at 50 μM. Activation of caspase-8 was blocked by the specific oligopeptide z-Ile-Glu-Thr-Asp-CH2F (z-IETD, Enzyme Systems Products, Livermore, CA, USA) added at a final concentration of 20 μM.

Induction of DNA repair via photoreactivation

Exogenous induction of DNA repair via photoreactivation was performed as described previously in detail (Kulms et al., 1999). Briefly, photolyase was encapsulated into liposomes (Photosomes®, AGI Dermatics, Freeport, NY, USA) at a concentration of 1.2 mg/ml. Liposomes consisted of the lipids egg phosphatidylcholine, egg phosphatidyl trans-ethanolamine, oleic acid and the membrane stabilizer cholesterol hemisuccinate. For photoreactivation, HeLa cells were irradiated as described above and photosomes (40 μl/ml) were added. Cells were incubated at 37°C for 1 h in the dark followed by illumination with photoreactivating light. As a light source for photoreactivating light, UVA fluorescent bulbs (TL09, Philips) filtered through a 6 mm glass plate with peak emission at 365 nm were used. Cells were exposed for 20 min which corresponds to a photoreactivating light fluence of 12 J/m2. After photoreactivation, cells were supplemented with normal RPMI containing 5% FCS and incubated for 16 h at 37°C.

For Southwestern dot-blot analysis, genomic DNA was extracted from cells according to the protocol from Biozym Diagnostics (Hessisch Oldendorf, Germany) 1 h after stimulation. DNA damage was detected utilizing an Ab directed against thymine dimers (Kamiya Biomedical, Thousand Oaks, CA, USA) as described previously (Kulms et al., 1999).

Detection of cell death

Sixteen hours after stimulation cells were detached from the dishes, and apoptosis analysed by a cell death detection ELISA (Boehringer Mannheim, Germany). The enrichment of mono- and oligonucleosomes released into the cytoplasm of cell lysates is detected by biotinylated anti-histone- and peroxidase-coupled anti-DNA-antibodies and is calculated according to the formula: absorbance of sample cells/absorbance of control cells. Unless otherwise stated, this factor was used as a parameter of apoptosis and is given as the mean±s.d. of three independently performed experiments.

Western blot analysis

To determine cytochrome c release from the mitochondria into the cytoplasm, cytoplasmic protein extracts were obtained by lysing cells in buffer consisting of 250 mM sucrose, 80 mM KCl and 1 μg/ml digitonin (Sigma) followed by passing cells through a 26 gauge needle eight times. After centrifugation, supernatants were collected and the protein content measured by BioRad Protein assay kit (BioRad, Hercules, CA, USA). Thirty μg of the protein samples were subjected to 12% SDS–PAGE, blotted onto nitrocellulose membranes and incubated with Abs directed against cytochrome c (Biosource, Nivelles, Belgium), or Bid (R&D Systems Inc., Minneapolis, MN, USA). Equal loading was monitored by reprobing membranes with an Ab directed against α-tubulin (Calbiochem, San Diego, CA, USA). Signals were detected with an ECL-kit (Amersham, Buckinghamshire, UK).

In addition, cytochrome c release was quantified by using the Mitochondria/Cytosol Fractionation kit from Bio Vision (Mountain View, CA, USA) and the Quantakine human Cytochrome c ELISA (R&D Systems Inc.)

References

  1. , , , , , , . 1998 J. Cell Biol. 140: 171–182

  2. . 1987 Photochem. Photobiol. 46: 213–221

  3. , . 2001 Curr. Probl. Dermatol. 29: 140–156

  4. , , , , , . 1995 J. Biol. Chem. 270: 7795–7798

  5. , , . 1995 J. Neuroscience Res. 41: 213–221

  6. , . 1995 Free Radic. Res. 5: 389–397

  7. , . 2000 Free Radic. Biol. Med. 29: 222–230

  8. , , , , , , , , . 1996 J. Biol. Chem. 271: 4961–4965

  9. , , , , . 1994 Carcinogenesis 15: 1399–1404

  10. , , . 1999 Genes Dev. 13: 768–785

  11. , , , , , , , , . 1999 J. Cell. Biol. 144: 891–901

  12. , . 2001 Curr. Probl. Dermatol. 29: 157–164

  13. , , , . 1990 J. Biol. Chem. 265: 8009–8015

  14. . 1994 Environ. Health Perspect. 102: 10 17–24

  15. . 1990 FASEB J. 4: 2587–2597

  16. , . 1996 Dermatol. Surg. 22: 217–226

  17. , , , . 1999 EMBO J. 18: 3586–3589

  18. , , , . 1994 Arch. Dermatol. 130: 1018–1021

  19. . 1997 Proc. Natl. Acad. Sci., USA 94: 1–14

  20. , . 2000 Nat. Med. 6: 513–519

  21. , , , , , . 1999 Proc. Natl. Acad. Sci. USA 96: 7974–7979

  22. , . 2000 Photodermatol. Photoimmunol. Photomed. 16: 195–201

  23. , , , , . 1999 J. Photochem. Photobiol. B. 49: 71–80

  24. , , , . 1998 Cell 94: 491–501

  25. , , , , . 1998 Cell 94: 481–490

  26. , , . 1995 Biochem. Biophys. Res. Commun. 17: 474–479

  27. , , , , . 2001 Exp. Dermatol. 10: 155–160

  28. . 1977 Photochem. Photobiol. 25: 357–372

  29. , , , , , , , , . 2000 J. Invest. Dermatol. 115: 687–693

  30. , , , , , . 1999 J. Invest. Dermatol. 112: 751–756

  31. , , , . 1997 J. Biol. Chem. 272: 25783–25786

  32. , . 1996 Science 274: 1194–1197

  33. , , , , , , , , . 2002 Nat. Cell Biol. 4: 26–31

  34. , , , . 1998 Oncogene 17: 2555–2563

  35. , , , , , , . 2000 Proc. Natl. Acad. Sci. USA 97: 1790–1795

  36. . 1994 Mol. Aspects Med. 15: 1–77

  37. . 1995 Biochem. Soc. Symp. 61: 547–553

  38. , , , , , , , , , . 2001 Curr. Probl. Dermatol. 29: 83–94

  39. , , , . 1995 J. Invest. Dermatol. 104: 287–292

  40. , . 1976 Br. J. Dermatol. 95: 459–468

  41. , , , , , , , , . 1992 Cancer Res. 52: 4227–4231

  42. , . 1994 Trends Photochem. Photobiol. 3: 175–181

  43. , , , , , , . 1996 J. Exp. Med. 183: 1533–1544

  44. , , , , , , , , . 1994 Nature 372: 773–776

  45. , , , . 1999 J. Biol. Chem. 274: 11549–11556

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Acknowledgements

The authors are grateful to D Yarosh, AGI-Dermatics for generous supply of Photosomes®. This work was supported by grants from the Federal Ministery of Education and Research (07UVB63A/5), the European Community (ENV4-CT97-0556) and the Interdisciplinary Center for Clinical Research (1ZKF, E10).

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  1. Ludwig Boltzmann Institute for Cell Biology and Immunobiology of the Skin, Department of Dermatology, University Münster, D-48149 Münster, Germany

    • Dagmar Kulms
    • , Elke Zeise
    • , Birgit Pöppelmann
    •  & Thomas Schwarz

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Correspondence to Thomas Schwarz.

Glossary

BHA

butylated hydroxyanisole

CPD

cyclobutane pyrimidine dimers

NAC

N-acetyl-cysteine

PDTC

pyrrolidene-dithiocarbamate

ROS

reactive oxygen species

UV

ultraviolet radiation

UVB

ultraviolet-B radiation

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DOI

https://doi.org/10.1038/sj.onc.1205743

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