Dagmar Kulms

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.

Interleukin-12 suppresses ultraviolet radiation-induced apoptosis by inducing DNA repair

Induction of apoptosis of keratinocytes by ultraviolet (UV) radiation is a protective phenomenon relevant in limiting the survival of cells with irreparable DNA damage. Changes in UV-induced apoptosis may therefore have significant impact on photocarcinogenesis. We have found that the immunomodulatory cytokine IL-12 suppresses UV-mediated apoptosis of keratinocytes both in vitro and in vivo. IL-12 caused a remarkable reduction in UV-specific DNA lesions which was due to induction of DNA repair. In accordance with this, IL-12 induced the expression of particular components of the nucleotide-excision repair complex. Our results show that cytokines can protect cells from apoptosis induced by DNA-damaging UV radiation by inducing DNA repair, and that nucleotide-excision repair can be manipulated by cytokines.

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

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.

Independent contribution of three different pathways to ultraviolet-B-induced apoptosis

Ultraviolet-B radiation (UVB) causes a variety of biological effects which include the induction of apoptosis. UVB-induced apoptosis provides a well controlled scavenging mechanism protecting cells from malignant transformation. To induce programmed cell death, UVB uses a variety of cellular signaling pathways. In this context induction of nuclear DNA damage seems to be the predominant pathway, since experimental reduction of DNA damage was associated with a strong suppression of apoptosis. Additionally, UVB has been shown to target cytoplasmatically located or membrane bound components to induce signal transduction. UVB was found to directly activate cell surface death receptors, thereby triggering the apoptotic machinery. Furthermore, UVB-induced intracellular formation of reactive oxygen species (ROS) accompanied by mitochondrial dysfunction and cytochrome c release was demonstrated to be additionally involved in the apoptotic program. The following review will briefly discuss current aspects of the interplay between the different signaling pathways involved in UVB-induced apoptosis.

The molecular determinants of sunburn cell formation

Abstract: 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.

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 NFκB in transformed keratinocytes. Moreover, the proteasome inhibitor MG132, which inhibits IL-1-induced NFκB 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 NFκB. These data suggest that NFκB 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.

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.

Molecular Mechanisms Involved in UV-Induced Apoptotic Cell Death

Ultraviolet radiation (UV) induces a variety of biological effects which include the induction of programmed cell death. UV-induced apoptosis seems to represent a controlled scavenging mechanism which protects cells from malignant transformation in human skin. To exert these effects on a cellular base, UV uses a variety of signaling pathways, involving nuclear DNA damage as a predominant pathway, since experimental reduction of DNA damage is associated with a loss of these effects. On the other hand, UV has been found to utilize extranuclear components located in the cytoplasm or at the cell membrane for signaling. UV can directly activate cell surface death receptors, thereby triggering the apoptotic machinery. Oxidative stress accompanied by mitochondrial changes and cytochrome c release are further involved in UV-mediated apoptosis. The following review will briefly discuss current aspects of the interplay between the different signaling pathways involved in UV-induced apoptosis.

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 κB, because cells transfected with a super-repressor form of the nuclear factor κB inhibitor IκB 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 TNFα 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.

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

The transcription factor nuclear factor kappa-B (NF-κB) is generally regarded as an antiapoptotic factor. Accordingly, NF-κB activation inhibits death ligand-induced apoptosis. In contrast, ultraviolet light B (UVB)-induced apoptosis is not inhibited but even enhanced upon NF-κB activation by interleukin-1 (IL-1). This study was performed to identify the molecular mechanisms underlying this switch of NF-κB. Enhancement of UVB-induced apoptosis was always associated with increased release of tumour necrosis factor-α (TNF-α), which was dependent on NF-κB 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-κB on apoptosis. Surprisingly, activated NF-κB induced TNF-α 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-α 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-κB in carcinogenesis and for the application of NF-κB inhibitors in anticancer therapy.