Up‐Regulation and Redistribution of Bax in Ultraviolet B‐Irradiated Melanocytes

Ultraviolet B (UVB) radiation may activate or deteriorate cultured human epidermal melanocytes, depending on the doses and culture conditions. It is also reported that cultured human epidermal melanocytes derived from different pigmentary phenotypes showed different responses to UVB radiation. In this study, we examined whether apoptosis of melanocytes can be induced by physiologic doses of UVB irradiation using cultured human epidermal melanocytes derived from oriental males of skin types III and IV. Propidium iodide staining for DNA condensation and flow cytometric analyses demonstrated the apoptotic cell death of melanocytes following UVB irradiation (0–30 mJ/cm²). The levels of p53, Bax, and Bcl‐2, determined by immunoblotting, revealed a dose‐dependent increase in p53 and Bax, but the level of Bcl‐2 remained unchanged. Confocal microscopic examination showed that Bax moved from a diffuse to a punctate distribution after UVB irradiation. However, there were no changes in the pattern of distribution of Bcl‐2. These data suggest that the high constitutional level of Bcl‐2 may protect melanocytes from UVB‐induced injury, and that apoptotic death of melanocytes may be induced by the elevation and redistribution of Bax.     

Ciprofloxacin as a potential radio-sensitizer to tumor cells and a radio-protectant for normal cells: differential effects on γ-H2AX formation,p53 phosphorylation,Bcl-2 production,and cell death

Ionizing radiation increases cell mortality in a dose-dependent manner. Increases in DNA double strand breaks, γ-H2AX, p53 phophorylation, and protein levels of p53 and Bax also occur. We investigated the ability of ciprofloxacin (CIP), a widely prescribed antibiotic, to inhibit DNA damage induced by ionizing radiation. Human tumor TK6, NH32 (p53 ^?/? of TK6) cells, and human normal peripheral blood mononuclear cells (PBMCs) were exposed to 2–8 Gy ⁶⁰Co-γ-photon radiation. γ-H2AX (an indicator of DNA strand breaks), phosphorylated p53 (responsible for cell-cycle arrest), Bcl-2 (an apoptotic protein, and cell death were measured. Ionizing irradiation increased γ-H2AX amounts in TK6 cells (p53^+/+) within 1 h in a radiation dose-dependent manner. CIP pretreatment and posttreatment effectively inhibited the increase in γ-H2AX. CIP pretreatment reduced Bcl-2 production but promoted p53 phosphorylation, caspase-3 activation and cell death. In NH32 cells, CIP failed to significantly inhibit the radiation-induced γ-H2AX increase, suggesting that CIP inhibition involves in p53-dependent mechanisms. In normal healthy human PBMCs, CIP failed to block the radiation-induced γ-H2AX increase but effectively increased Bcl-2 production, but blocked the phospho-p53 increase and subsequent cell death. CIP increased Gadd45α, and enhanced p21 protein 24 h postirradiation. Results suggest that CIP exerts its effect in TK6 cells by promoting p53 phosphorylation and inhibiting Bcl-2 production and in PBMCs by inhibiting p53 phosphorylation and increasing Bcl-2 production. Our data are the first to support the view that CIP may be effective to protect normal tissue cells from radiation injury, while enhancing cancer cell death in radiation therapy.     

Potentiation of UVB-induced apoptosis by novel phytosphingosine derivative, tetraacetyl phytosphingosine in HaCaT cell and mouse skin

Inappropriate apoptosis results in the epidermal hyperplasia as in psoriasis and UVB irradiation has been successfully used to treat this kind of skin disorders. Previously, we reported that the novel phytosphingosine derivative, tetraacetyl phytosphingosine (TAPS) induced apoptosis in HaCaT cells. This study examined the effect of UVB irradiation and/or TAPS on the induction of apoptosis in HaCaT. 10 mJ/cm2 of UVB irradiation or 10 microM of TAPS alone exhibited weak cytotoxicity but co-treatment of UVB and TAPS synergistically enhanced the cytotoxicity and apoptosis in HaCaT. The cells treated with UVB and TAPS showed much higher levels of cleaved caspase-3, -8, -9 and Bax than with UVB or TAPS alone, whereas Bcl-2 level was decreased by co-administration of UVB and TAPS. In hairless mice, co-treatment of UVB and TAPS synergistically increased apoptosis, as shown in the HaCaT co-treated with UVB and TAPS. Furthermore, UVB irradiation caused an increase of apoptotic cells in the epidermis and the TAPS-treated mice showed an increase of apoptotic cells in the dermis as well as in the epidermis. These results suggest that the TAPS co-treatment synergistically increases the level of UVB-induced apoptosis via caspase activation by regulating the level of pro-apoptotic Bax and anti-apoptotic Bcl-2.     

Nitric oxide inhibits ultraviolet B-induced murine keratinocyte apoptosis by regulating apoptotic signaling cascades

Cytotoxic effects of nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) are considered to be one of the major causes of inflammatory diseases. On the other hand, protective effects of NO on toxic insults-induced cellular damage/apoptosis have been demonstrated recently. Ultraviolet B (UVB)-induced apoptosis of epidermal keratinocytes leads to skin inflammation and photoageing. However, it has not been elucidated what kind of effects NO has on UVB-induced keratinocyte apoptosis. Thus, in the present study, we investigated the problem and demonstrated that NO from NO donor suppressed UVB-induced apoptosis of murine keratinocytes. In addition, NO significantly suppressed activities of caspase 3, caspase 8 and caspase 9 that had been upregulated by UVB radiation. NO also suppressed p53 expression that had been upregulated by UVB radiation and upregulated Bcl-2 expression that had been downregulated by UVB radiation. These findings suggested that NO might suppress UVB-induced keratinocyte apoptosis by regulating apoptotic signaling cascades in p53, Bcl-2, caspase3, caspase 8 and caspase 9.     

Aldose reductase in keratinocytes attenuates cellular apoptosis and senescence induced by UV radiation

Although aldose reductase (AR) has been implicated in the cellular response to oxidative stress, the role of AR in ultraviolet-B (UVB)-induced cellular injury has not been investigated. Here, we show that an increased expression of AR in human keratinocytes modulates UVB-induced apoptotic cell death and senescence. Overexpression of AR in HaCaT cells significantly attenuated UVB-induced cellular damage and apoptosis, with a decreased generation of reactive oxygen species (ROS) and aldehydes. Ablation of AR with small interfering RNA or inhibition of AR activity abolished these effects. We also show that increased AR activity suppressed UVB-induced activation of the p38 and c-Jun N-terminal kinases, but did not affect the extracellular signal-regulated kinase and phosphatidylinositol 3-kinase pathways. Similarly, UVB-induced translocation of Bax and Bcl-2 to mitochondria and cytosol, respectively, was markedly attenuated in cells overexpressing AR. Knockdown or inhibition of AR activity in primary cultured keratinocytes enhanced UVB-induced cellular senescence and increased the level of a cell-cycle regulatory protein, p53. Finally, cellular apoptosis induced by UVB radiation was significantly reduced in the epidermis of transgenic mice overexpressing human AR. These findings suggest that AR plays an important role in the cellular response to oxidative stress by sequestering ROS and reactive aldehydes generated in keratinocytes.     

The greater lethality of UVB radiation to cultured human cells is associated with the specific activation of a DNA damage-independent signaling pathway

UV radiation causes cell death through the activation of various intracellular signaling molecules in both DNA damage-dependent and -independent manners. The ability of middle-wavelength UV (UVB) radiation to form DNA photoproducts is less than that of short-wavelength UV (UVC) radiation; however, the differences between UVB and UVC radiation in the extent of DNA damage-independent signaling and its contribution to cell death have not been well characterized. When cells were irradiated with UVB or UVC radiation at doses that generated equivalent amounts of DNA photoproducts, UVB radiation induced more clonogenic cell death, apoptotic cells, mitochondrial cytochrome C release, and intracellular oxidative stress. Among the signaling molecules examined, levels of p53 phosphorylated at Ser-392 and p38 were higher in UVB-irradiated cells than in UVC-irradiated cells. Both phosphorylations were reduced by treating cells with an antioxidant. Furthermore, an inhibitor of p38 also blocked the phosphorylation of p53 at Ser-392. These results suggest that UVB radiation activates the p38 pathway through the generation of oxidative stress, which merges with the DNA p53 pathway by phosphorylation of p53 at ser392. This greater contribution of the DNA damage-independent pathway in UVB-irradiated cells may explain the greater lethality of UVB radiation.     

alpha-Melanocyte-stimulating hormone protects from ultraviolet radiation-induced apoptosis and DNA damage

Ultraviolet radiation is a well established epidemiologic risk factor for malignant melanoma. This observation has been linked to the relative resistance of normal melanocytes to ultraviolet B (UVB) radiation-induced apoptosis, which consequently leads to accumulation of UVB radiation-induced DNA lesions in melanocytes. Therefore, identification of physiologic factors regulating UVB radiation-induced apoptosis and DNA damage of melanocytes is of utmost biological importance. We show that the neuropeptide alpha-melanocyte-stimulating hormone (alpha-MSH) blocks UVB radiation-induced apoptosis of normal human melanocytes in vitro. The anti-apoptotic activity of alpha-MSH is not mediated by filtering or by induction of melanin synthesis in melanocytes. alpha-MSH neither leads to changes in the cell cycle distribution nor induces alterations in the expression of the apoptosis-related proteins Bcl(2), Bcl(x), Bax, p53, CD95 (Fas/APO-1), and CD95L (FasL). In contrast, alpha-MSH markedly reduces the formation of UVB radiation-induced DNA damage as demonstrated by reduced amounts of cyclobutane pyrimidine dimers, ultimately leading to reduced apoptosis. The reduction of UV radiation-induced DNA damage by alpha-MSH appears to be related to induction of nucleotide excision repair, because UV radiation-mediated apoptosis was not blocked by alpha-MSH in nucleotide excision repair-deficient fibroblasts. These data, for the first time, demonstrate regulation of UVB radiation-induced apoptosis of human melanocytes by a neuropeptide that is physiologically expressed within the epidermis. Apart from its ability to induce photoprotective melanin synthesis, alpha-MSH appears to exert the capacity to reduce UV radiation-induced DNA damage and, thus, may act as a potent protection factor against the harmful effects of UV radiation on the genomic stability of epidermal cells.     

Granulocyte‐Macrophage Colony‐Stimulating Factor (GM‐CSF) Controls the Proliferation and Differentiation of Mouse Epidermal Melanocytes from Pigmented Spots Induced by Ultraviolet Radiation B

Repeated exposure of ultraviolet radiation B (UVB) on the dorsal skin of hairless mice induces the development of pigmented spots long after its cessation. The proliferation and differentiation of epidermal melanocytes in UVB‐induced pigmented spots are greatly increased, and those effects are regulated by keratinocytes rather than by melanocytes. However, it remains to be resolved what factor(s) derived from keratinocytes are involved in regulating the proliferation and differentiation of epidermal melanocytes. In this study, primary melanoblasts (c. 80%) and melanocytes (c. 20%) derived from epidermal cell suspensions of mouse skin were cultured in a basic fibroblast growth factor‐free medium supplemented with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). GM‐CSF induced the proliferation and differentiation of melanocytes in those keratinocyte‐depleted cultures. Moreover, an antibody to GM‐CSF inhibited the proliferation of melanoblasts and melanocytes from epidermal cell suspensions derived from the pigmented spots of UV‐irradiated mice, but not from control mice. Further, the GM‐CSF antibody inhibited the proliferation and differentiation of melanocytes co‐cultured with keratinocytes derived from UV‐irradiated mice, but not from control mice. The quantity of GM‐CSF secreted from keratinocytes derived from the pigmented spots of UV‐irradiated mice was much greater than that secreted from keratinocytes derived from control mice. Moreover, immunohistochemistry revealed the expression of GM‐CSF in keratinocytes derived from the pigmented spots of skin in UV‐irradiated mice, but not from normal skin in control mice. These results suggest that GM‐CSF is one of the keratinocyte‐derived factors involved in regulating the proliferation and differentiation of mouse epidermal melanocytes from UVB‐induced pigmented spots.     

UVC radiation-induced effect on human primary thyroid cell proliferation and HLA-DR expression

The aim of this study was to examine how UVC irradiation will affect normal human thyroid cell proliferation and HLA-DR expression. Primary human thyroid cells were exposed to UVC (254 nm wavelength) irradiation. In some experiments 0.5 mM buthionine sulfoximine (BSO) was added. Apoptosis was detected measuring annexin V, proteins involved in apoptotic process (p53, Bax, Bcl-2, caspase 3, and 9) by immunoblot analysis and HLA-DR expression by FACS. UVC induced a cell cycle arrest in G0/G1 phase in the first 24 h, accumulation of cells in the S phase 72 h after treatment, and an increase of apoptotic cells. BSO pretreatment showed an earlier appearance and a higher percentage of apoptosis. p53, caspase 3 and 9 were increased, while Bax and Bcl-2 were decreased. We also observed a transient significant increase in HLA-DR expression. UVC inhibited cell proliferation and induced apoptosis in normal human primary thyroid cells. An inhibitor of glutathione synthesis induced an earlier appearance and higher percentage of apoptosis suggesting that oxidative stress may play a role. Apoptotis involved components of the intrinsic mitochondrial pathway. A transient increase in HLA-DR expression after UVC irradiation could play a role in inducing AITD.     

Distinct Melanogenic Response of Human Melanocytes in Mono‐culture,in Co‐Culture with Keratinocytes and in Reconstructed Epidermis,to UV Exposure

Striking differences are observed in the melanogenic response of normal human melanocytes to UVA and UVB irradiation depending on culture conditions and the presence of keratinocytes. Exposure of melanocytes co‐cultured with keratinocytes to UVB irradiation triggered, already at low doses (5 mJ/cm²), an increase in melanin synthesis whereas in melanocyte mono‐cultures, UVB doses up to 50 mJ/cm² had no melanogenic effect. Unlike UVB, UVA exposure caused the same melanogenic response in both mono‐ and co‐cultures. Removing certain keratinocyte growth factors from the co‐culture medium abolished the melanogenic response to UVB, but not to UVA exposure. When integrated into the basal layer of a reconstructed human epidermis, human melanocytes similarly reacted to UVA and UVB irradiation as in vivo by increasing their production and transfer of melanin to the neighboring keratinocytes which resulted in a noticeable tanning of the reconstructed epidermis. The presence of a dense stratum corneum, known to scatter and absorb UV light, is responsible for higher minimal UVB and UVA doses required to trigger a melanogenic response in the reconstructed epidermis compared to keratinocyte–melanocyte co‐cultures. Furthermore, an immediate tanning response was observed in the pigmented epidermis following UVA irradiation. From these results we conclude that: (i) keratinocytes play an important role in mediating UVB‐induced pigmentation, (ii) UVA‐induced pigmentation is the result of a rather direct effect on melanocytes and (iii) reconstructed pigmented epidermis is the most appropriate model to study UV‐induced pigmentation in vitro.     

Keratinocytes Control the Proliferation and Differentiation of Cultured Epidermal Melanocytes from Ultraviolet Radiation B‐Induced Pigmented Spots in the Dorsal Skin of Hairless Mice

Long‐term exposure of ultraviolet radiation B (UVB)‐induced pigmented spots in the dorsal skin of hairless mice of Hos:(HR‐1 X HR//De) F₁. Previous study showed that the proliferative and differentiative activities of cultured epidermal melanoblasts//melanocytes from UVB‐induced pigmented spots increased with the development of the pigmented spots. To determine whether the increase in the proliferative and differentiative activities of epidermal melanoblasts//melanocytes was brought about by direct changes in melanocytes, or by indirect changes in surrounding keratinocytes, pure cultured melanoblasts//melanocytes and keratinocytes were prepared and co‐cultured in combination with control and irradiated mice in a serum‐free culture medium. Keratinocytes from irradiated mice stimulated the proliferation and differentiation of both neonatal and adult non‐irradiated melanoblasts//melanocytes more greatly than those from non‐irradiated mice. In contrast, both non‐irradiated and irradiated adultmelanocytes proliferated and differentiated similarly when they were co‐cultured with irradiated adult keratinocytes. These results suggest that the increased proliferative and differentiative activities of mouse epidermal melanocytes from UVB‐induced pigmented spots are regulated by keratinocytes, rather than melanocytes.     

UV-radiation, apoptosis and skin

Apoptosis or programmed cell death is a key function in regulating skin development, homeostasis and tumorigenesis. The epidermis is exposed to various external stimuli and one of the most important is UV radiation. The UVA and UVB spectra differ in their biological effects and in their depth of penetration through the skin layers. UVB rays are absorbed directly by DNA which results in its damage. UVA can also cause DNA damage but primarily by the generation of reactive oxygen species. By eliminating photodamaged cells, apoptosis has an important function in the prevention of epidermal carcinogenesis. UV-induced apoptosis is a complex event involving different pathways. These include: 1. activation of the tumour suppressor gene p53; 2. triggering of cell death receptors directly by UV or by autocrine release of death ligands; 3. mitochondrial damage and cytochrome C release. The extrinsic pathway through death receptors such as fibroblast-associated, tumour necrosis factor receptor and TNF related apoptosis inducing ligand receptor activate caspase cascade. The intrinsic or mitochondrial pathway of apoptosis is regulated by the Bcl-2 family of proteins, anti-apoptotic (Bcl-2, Bcl-xl, Bcl-w) and the pro-apoptotic (Bax, Bak, Bid). The balance between the pro-apoptotic and anti-apoptotic proteins determines cell survival or death. We discuss recent findings in the molecular mechanisms of UV induced apoptosis.     

Skin mild hypoxia enhances killing of UVB-damaged keratinocytes through reactive oxygen species-mediated apoptosis requiring Noxa and Bim

The naturally occurring skin hypoxia has emerged as a crucial host factor of the epidermal microenvironment. We wanted to systematically investigate how reduced oxygen availability of the epidermis modulates the response of keratinocytes and melanocytes to noxious ultraviolet B radiation (UVB). We report that the exposure of normal human keratinocytes (NHKs) or melanocytes (NHEMs) to mild hypoxia drastically impacts cell death responses following UVB irradiation. The hypoxic microenvironment favors survival and reduces apoptosis of UVB-irradiated NHEMs and their malignant counterparts (melanoma cells). In contrast, NHKs, but not the transformed keratinocytes, under hypoxic conditions display increased levels of reactive oxygen species (ROS) and are significantly sensitized to UVB-mediated apoptosis as compared to NHKs treated under normoxic conditions. Prolonged exposure of UVB-treated NHKs to hypoxia triggers a sustained and reactive oxygen species-dependent activation of the stress kinases p38(MAPK) and JNKs, which in turn, engage the activation of Noxa and Bim proapoptotic proteins. Combined silencing of Noxa and Bim significantly inhibits UVB-mediated apoptosis under hypoxic conditions, demonstrating that hypoxia results in an amplification of the intrinsic apoptotic pathway. Physiologically occurring skin hypoxia, by facilitating the specific removal of UVB-damaged keratinocytes, may represent a decisive host factor impeding important steps of the photocarcinogenesis process.     

Morphine suppresses lymphocyte apoptosis by blocking p53-mediated death signaling

Opiates such as morphine or heroin may promote cell apoptosis and cause dysfunction of immune cells. In simian immunodeficiency virus (SIV)-infected lymphocytic cells, however, morphine may protect the cells from apoptotic lysis and allow the virus to continue to replicate. To further explore this apparently antithetical effect of opiates, we evaluated in the present study the effects of morphine on human lymphocytic CEM x174 cells induced to undergo apoptosis in the presence of actinomycin D. It was found that induction of apoptosis (characterized by DNA laddering) by actinomycin D was accompanied by a stimulation of the expression of active (phosphorylated) form of p53. Pretreatment of the cells with 10nM morphine caused a transient, naloxone-reversible suppression of the appearance of activated p53 and the generation of DNA laddering. Parallel evaluation of the growth of CEM x174 indicated that morphine treatment delays the inception of cell death triggered by actinomycin D. Inasmuch as Bcl-2 suppresses while Bax accelerates apoptosis, treatment of cells with morphine reduced the expression of Bax and enhanced the expression of Bcl-2. Taken together, morphine, through binding at the opioid receptor, may protect lymphocytic cells from apoptotic lysis if cell death is initiated by apoptosis-inducing agents such as human immunodeficiency virus (HIV), SIV or actinomycin D.     

The role of p38 MAPK and JNK in Arsenic trioxide-induced mitochondrial cell death in human cervical cancer cells

Previously, we have shown that the release of AIF from mitochondria is required for As2O3-induced cell death in human cervical cancer cells, and that reactive oxygen species (ROS) is necessary for AIF release from mitochondria. In this study, we further investigated the role of MAPKs in ROS-mediated mitochondrial apoptotic cell death triggered by As2O3. As2O3-induced apoptotic cell death in HeLa cells was associated with activation and mitochondrial translocation of Bax, a marked phosphorylation of Bcl-2, reduction of Bcl-2 and Bax interaction, dissipation of mitochondrial membrane potential. Using small interfering RNA, reduced Bax expression effectively attenuated As2O3-induced mitochondrial membrane potential loss and apoptotic cell death. Moreover, the phosphorylation of Bcl-2 induced by As2O3 diminished its ability to bind to Bax. Treatment of cells with As2O3 activated both the p38 MAPK and JNK pathways. Mitochondrial translocation of Bax was completely suppressed in the presence of p38 MAPK inhibitor PD169316 or si-p38 MAPK. The As2O3-induced Bcl-2 phosphorylation was attenuated largely by JNK inhibition using SP600125 or si-JNK and to some extent by p38 MAPK inhibition with PD169316 or si-p38 MAPK. In addition, N-acetyl-L-cystein (NAC), a thiol-containing anti-oxidant, completely blocked As2O3-induced p38 MAPK and JNK activations, mitochondria translocation of Bax, and phosphorylation of Bcl-2. These results support a notion that ROS-mediated activations of p38 MAPK and JNK in response to As2O3 treatment signals activation of Bax and phosphorylation of Bcl-2, resulting in mitochondrial apoptotic cell death in human cervical cancer cells.     

Effect of low-dose irradiation on induction of an apoptotic adaptive response in the murine system

The aim of the study was to investigate the effect of low doses of irradiation on the induction of an apoptotic adaptive response in the murine system using C3H/HeJ mice bearing 8 mm syngeneic tumors, HCa-I and OCa-I. In OCa-I, the 0.05 Gy priming dose significantly reduced the 25 Gy-induced apoptosis by 30%, whereas this reduction was not seen in HCa-I. The analysis of apoptosis-regulating molecules showed that the application of a priming dose increased the radiation- induced p53 level in both tumors. No other regulators changed in OCa-I. However, in HCa-I, the application of a priming dose increased radiation-induced Bcl-XL and Bcl-XS, but not Bcl-2 or Bax. An apoptotic adaptive response induced by low-dose radiation was shown in one murine tumor, OCa-I and Bcl-XL and Bcl-XS appeared to be implicated. Received: 4 April 2001 / Accepted: 20 September 2001