Marjan Garmyn

Epidermal differentiation does not involve the pro-apoptotic executioner caspases, but is associated with caspase-14 induction and processing

The epidermis is a stratified squamous epithelium in which keratinocytes progressively undergo terminal differentiation towards the skin surface leading to programmed cell death. In this respect we studied the role of caspases. Here, we show that caspase-14 synthesis in the skin is restricted to differentiating keratinocytes and that caspase-14 processing is associated with terminal epidermal differentiation. The pro-apoptotic executioner caspases-3, -6, and -7 are not activated during epidermal differentiation. Caspase-14 does not participate in apoptotic pathways elicited by treatment of differentiated keratinocytes with various death-inducing stimuli, in contrast to caspase-3. In addition, we show that non-cornifying oral keratinocyte epithelium does not express caspase-14 and that the parakeratotic regions of psoriatic skin lesions contain very low levels of caspase-14 as compared to normal stratum corneum. These observations strongly suggest that caspase-14 is involved in the keratinocyte terminal differentiation program leading to normal skin cornification, while the executioner caspases are not implicated. Cell Death and Differentiation (2000) 7, 1218–1224

1,25‐dihydroxyvitamin D3 inhibits ultraviolet B‐induced apoptosis, Jun kinase activation, and interleukin‐6 production in primary human keratinocytes

We investigated the capacity of 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3] to protect human keratinocytes against the hazardous effects of ultraviolet B (UVB)‐irradiation, recognized as the most important etiological factor in the development of skin cancer. Cytoprotective effects of 1,25(OH)2D3 on UVB‐irradiated keratinocytes were seen morphologically and quantified using a colorimetric survival assay. Moreover, 1,25(OH)2D3 suppressed UVB‐induced apoptotic cell death. An ELISA, detecting DNA‐fragmentation, demonstrated that pretreatment of keratinocytes with 1,25(OH)2D3 1 μM for 24 h reduced UVB‐stimulated apoptosis by 55–70%. This suppression required pharmacological concentrations 1,25(OH)2D3 and a preincubation period of several hours. In addition, 1,25(OH)2D3 also inhibited mitochondrial cytochrome c release (90%), a hallmark event of UVB‐induced apoptosis. Furthermore, we demonstrated that 1,25(OH)2D3 reduced two important mediators of the UV‐response, namely, c‐Jun‐NH2‐terminal kinase (JNK) activation and interleukin‐6 (IL‐6) production. As shown by Western blotting, pretreatment of keratinocytes with 1,25(OH)2D3 1 μM diminished UVB‐stimulated JNK activation with more than 30%. 1,25(OH)2D3 treatment (1 μM) reduced UVB‐induced IL‐6 mRNA expression and secretion with 75–90%. Taken together, these findings suggest the existence of a photoprotective effect of active vitamin D3 and create new perspectives for the pharmacological use of active vitamin D compounds in the prevention of UVB‐induced skin damage and carcinogenesis. J. Cell. Biochem. 89: 663–673, 2003.

Oxidative DNA damage induced by visible light in mammalian cells: extent, inhibition by antioxidants and genotoxic effects

The extent of the indirect DNA damage generated in mammalian cells by visible light because of the presence of endogenous photosensitizers was studied by means of repair endonucleases. In immortalized human keratinocytes (HaCaT cells) exposed to low doses of natural sunlight, the yield of oxidative DNA base modifications sensitive to the repair endonuclease formamidopyrimidine-DNA glycosylase (Fpg protein) generated by this indirect mechanism was 10% of that of pyrimidine dimers (generated by direct DNA excitation). A similar yield of Fpg-sensitive modifications, which include 8-hydroxyguanine, was observed in primary keratinocytes. The relative yield of oxidative base modifications decreased at higher light doses, probably as a result of photodecomposition of the endogenous chromophore involved. For the three cell lines tested, viz. HaCaT cells, L1210 mouse leukemia cells and AS52 Chinese hamster cells, the yield of oxidative base modifications generated by a low dose of visible light appeared to be correlated with the basal concentrations of porphyrins in the cells. Induction of cellular porphyrin synthesis by pretreatment with 5-aminolaevulinic acid increased the light-induced oxidative damage in L1210 cells several-fold. In both induced and uninduced cells, the damage was inhibited by more than 50% in the presence of ascorbic acid (100 microM), while alpha-tocopherol and the iron chelator alpha-phenanthroline had no effect and beta-carotene even increased the damage. Even high doses of visible light did not significantly increase the numbers of micronuclei in L1210 cells or of gpt mutations in AS52 cells. The negative outcome can be fully explained by the photobleaching of the endogenous photosensitizers, which prevents the generation of sufficiently high levels of oxidative DNA damage. Therefore, the mutagenic risk arising from the indirectly generated oxidative DNA modifications induced by sunlight may be underestimated when results obtained at high doses are extrapolated to low doses or low dose rates.

Concomitant inhibition of AKT and autophagy is required for efficient cisplatin-induced apoptosis of metastatic skin carcinoma

Cutaneous squamous cell carcinoma (cSCC) is one of the most common cancers in the Caucasian population. Although early stages of skin cancer have a high curability and excellent prognosis, advanced cSCC shows resistance to chemotherapy, including cisplatin. The PI3‐K/AKT pathway is known to have a role in both skin cancer development and resistance to therapeutic drugs. In this study, we used isogenic cell lines representing different stages of malignant transformation of the keratinocytes that were derived from dysplastic forehead skin (PM1), primary cutaneous SCC (MET1) and its lymph node metastasis (MET4) of an immunosuppressed patient. We show that skin tumor progression parallels enhanced AKT activation and increased resistance to cisplatin‐induced apoptosis. Pharmacological AKT inhibition, or specific AKT1 knock down, sensitizes the apoptosis‐resistant MET1 and, to a lesser extent, MET4 cells to cisplatin‐mediated cell death. Concomitantly autophagy induction was observed in MET4, as demonstrated by accumulation of the autophagic protein marker LC3‐II, by analysis of full autophagosome maturation process using tandem mRFP‐GFP fluorescence microscopy and by electron microscopy. Counteracting the autophagic process by 3‐methyladenine or specific ATG5 knock down enhanced cytotoxicity of cisplatin combined with AKT inhibitor, thus revealing a key role for autophagy in chemoresistance. Taken together, these results indicate that concomitant inhibition of autophagy is required to increase the therapeutic benefit of AKT inhibition for combination therapy with the standard chemotherapeutic agent cisplatin in advanced skin carcinoma.

New Strategies of Photoprotection

Adequate photoprotection is essential to control UV‐related disorders, including sunburn, photoaging and photocarcinogenisis. Sun avoidance, protection of skin with clothing, and sunscreens are presently the best way of photoprotection, assuming that they are used properly. However, new strategies, which are based on or make use of the endogenous protective response to UV light, may further improve currently used photoprotective means. The addition of repair enzymes and/or antioxidants has a positive effect on skins recovery from UV‐induced DNA‐damage. Several botanical agents, mainly vitamins and polyphenols, have shown to influence signal transduction pathways leading to photo‐protective effects. Also stimulation of endogenous UV‐response pathways via irradiation with a low UV dose or via simulation of UV‐induced DNA‐damage results in photo‐protective effects. Future research in this field and combination of different photoprotective strategies will hopefully lead to improved photoprotection.

Insulin-like growth factor-1-mediated AKT activation postpones the onset of ultraviolet B-induced apoptosis, providing more time for cyclobutane thymine dimer removal in primary human keratinocytes

Insulin-like growth factor-1 (IGF-1) acts as a potent survival factor in numerous cell lines, primarily through activation of the AKT signaling pathway. Although some targets of this pathway have known anti-apoptotic functions, its relationship with the improved survival of cells after exposure to environmental stresses, including UVB, remains largely unclear. We report that in growth factor-deprived keratinocytes, IGF-1 significantly and consistently delayed the onset of UVB-induced apoptosis by >7 h. This delay allowed IGF-1-supplemented keratinocytes to repair significantly more cyclobutane thymine dimers than their growth factor-deprived counterparts. This increase in cyclobutane thymine removal resulted in enhanced survival if the amount of DNA damage was not too high. To increase cell survival after UVB irradiation, IGF-1 supplementation was required only during this initial time period in which extra repair was executed. Finally, we show that IGF-1 mediated this delay in the onset of UVB-induced apoptosis through activation of the AKT signaling pathway. We therefore believe that the AKT signaling pathway increases cell survival after a genotoxic insult such as UVB irradiation not by inhibiting the apoptotic stimulus, but only by postponing the induction of apoptosis, giving the DNA repair mechanism more time to work.

Influence of Eicosapentaenoic Acid, an Omega-3 Fatty Acid, on Ultraviolet-B Generation of Prostaglandin-E2 and Proinflammatory Cytokines Interleukin-1β, Tumor Necrosis Factor-α, Interleukin-6 and Interleukin-8 in Human Skin In Vivo¶

Abstract Dietary omega-3 polyunsaturated fatty acids (ω-3 PUFA) reduce sunburn, an acute inflammatory response, in humans. We assessed whether this may be mediated by reduced ultraviolet-B (UV-B) induction of proinflammatory mediators tumor necrosis factor–α (TNF-α), interleukin (IL)-1β, IL-6, IL-8 and prostaglandin (PG)E2 in healthy skin. In a double-blind, randomized study, 28 humans received 4 g daily of 95% ethyl esters of eicosapentaenoic acid (EPA) or oleic acid (OA) orally for 3 months. Skin biopsies and suction blister fluid were taken from unexposed and UV-B–exposed skin and examined for mediator expression immunohistochemically and quantitatively by immunoassay; plasma levels were also assayed. The subjects taking EPA, but not OA, showed a significant rise in their minimal erythemal dose (MED) (data reported elsewhere). Before supplementation, irradiation with 3× MED UV-B increased blister fluid TNF-α, IL-6, IL-8 and PGE2 at 16 h (all P < 0.001). No significant change occurred in baseline or UV-B–induced skin levels of cytokines after either supplement, whereas UV-B induction of PGE2 was abolished after EPA but not OA. Immunohistochemical expression of the cytokines at baseline and after UV-B was unaltered by EPA and OA; circulating cytokine and PGE2 levels were also unchanged. Hence, in healthy skin in vivo, there was no evidence that reduction of the sunburn response by EPA is mediated by the proinflammatory cytokines examined; abrogation of UV-B–generated PGE2 may play a role.

Allergic and photoallergic contact dermatitis from ketoprofen: results of (photo) patch testing and follow-up of 42 patients.

Background:  Photoallergic contact dermatitis from topical ketoprofen (KP), a nonsteroidal anti‐inflammatory agent, is a well‐known side effect.

Ultraviolet B radiation-induced apoptosis in human keratinocytes: cytosolic activation of procaspase-8 and the role of Bcl-2

In this study, we show that ultraviolet B radiation (UVB)‐induced apoptosis of human keratinocytes involves mainly cytosolic signals with mitochondria playing a central role. Overexpression of Bcl‐2 inhibited UVB‐induced apoptosis by blocking the early generation of reactive oxygen species, mitochondrial cardiolipin degradation and cytochrome c release, without affecting Fas ligand (FasL)‐induced cell death. It also prevented the subsequent activation of procaspase‐3 and ‐8 as well as Bid cleavage in UVB‐treated cells. Comparative analysis of UVB and FasL death pathways revealed a differential role and mechanism of caspase activation, with the UVB‐induced activation of procaspase‐8 only being a bystander cytosolic event rather than a major initiator mechanism, as is the case for the FasL‐induced cell death. Our results suggest that Bcl‐2 overexpression, by preventing reactive oxygen species production, helps indirectly to maintain the integrity of lysosomal membranes, and therefore inhibits the release of cathepsins, which contribute to the cytosolic activation of procaspase‐8 in UVB‐irradiated keratinocytes.

Pathways involved in sunburn cell formation: deregulation in skin cancer

The incidence of squamous cell carcinoma of the skin is rising worldwide for decades. Chronic exposure to sunlight is the most important environmental risk factor for this type of skin cancer. This is predominantly due to the DNA damaging effect of ultraviolet-B (UVB) in sunlight. UVB induces also sunburn cells, i.e. apoptotic keratinocytes, which is a crucial protective mechanism against the carcinogenic effects of UVB irradiation. This process is regulated by a wide range of molecular determinants involved in the balance between pro- and anti-apoptotic pathways. Growing evidence suggests that the deregulation of this balance by chronic UVB irradiation, contributes to the development of skin cancer. This review gives a brief summary of major known pathways involved in the regulation of keratinocyte survival and cell death upon UVB damage and discusses the contribution of the deregulation of these cascades to photocarcinogenesis.