p53 induction in normal human skin in vitro following exposure to solar simulated UV and UV-B irradiation

doi:10.1016/S1011-1344(99)00053-6

Journal of Photochemistry and Photobiology B: Biology

Volume 49, Issues 2–3, April 1999, Pages 177-186

https://doi.org/10.1016/S1011-1344(99)00053-6 Get rights and content

Abstract

Exposure of normal human breast skin ex vivo to physiological levels of UV-B and solar simulated UV results in a UV dose- and time-dependent increase in epidermal p53, as determined by PAGE analysis. Peak p53 levels are detected 12 to 24 h post irradiation with UV-B (470–1410 mJ cm⁻²) and solar simulated UV (5–12 minimal erythema dose (MED) equivalents). Irradiation with an FS20 UV-B lamp, contaminated with UV-A and UV-C (74–1111 mJ cm⁻²), also induces peak levels after 12 h incubation at 37°C but these levels persist to 36 h post UV irradiation. In all cases p53 levels start to return to normal by 48 h culture. A significant positive correlation is demonstrated between UV-B dose (47–1645 mJ cm⁻²) and p53 level (p < 0.01, R > 0.977) in explants cultured for 24 h at 37°C post irradiation. The FS20 induces a ‘UV-B’ dose-dependent increase in p53 to a maximum from 370 to 1111 mJ cm⁻². Similarly, solar simulated UV induces a plateau of peak p53 induction between 5 and 15 MED equivalents. Immunohistochemical analysis using microwave retrieval on 5 μm sections shows the same pattern of p53 staining with UV-B and solar UV insult, but proves unreliable as a method of quantification. These results suggest that the skin explant model may be a useful tool in the evaluation of UV-induced epidermal cell damage, providing a valuable alternative to in vivo studies.

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Comparison of various methods to analyse toxic effects in human skin explants: Rediscovery of TTC assay
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Human skin explants maintain their 3D structure and contain most of the cell types present in the native skin and also all components of the extracellular matrix, therefore explants represent a suitable model that substitutes for human in vivo skin. Reports suggest the manifold use of skin explants [5–10]. The goal for a screening of a compound or physical factors effect on the tissue is that it should be simple, fast, cheap and reproducible.
Skin explants are a suitable model which can replace dermatological experiments on animals or human volunteers. In this study, we searched for a fast, cheap and reproducible method for screening skin explant viability after treatment with UVA radiation or/and chemical agents. We compared frequently used methods: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), neutral red (NR) and lactate dehydrogenase (LDH) activity assay with a rarely used 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) assay for the evaluation of UVA radiation and/or chlorpromazine and 8-methoxypsoralen effect as model agents. Histological analysis of skin explants was also performed by a simple haematoxylin-eosin method. Only the TTC assay was able to show the toxicity of model agents in a dose- and concentration-dependent manner. LDH assay was partially able to demonstrate results comparable to the TTC method, however, the agents' effect was less pronounced. The MTT and NR assays completely failed in the evaluation. Haematoxylin-eosin staining showed discrete structural changes in samples treated with UVA alone and CPZ + UVA, but only after 48 h. Therefore, the method is not useful for screening of toxic or phototoxic effects either. In conclusion, the TTC assay was the most suitable for the evaluation of toxicity or phototoxicity in ex vivo skin.
Narrow-band ultraviolet B radiation induces the expression of β-endorphin in human skin in vivo
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UV-B irradiation of human foreskin ex vivo showed that virtually every keratinocyte expressed p53 after 1 h of UVR, with maximum expression after 3 h. α-MSH was maximally expressed after 3–6 h of UVR throughout the epidermis [11]. In another study using normal human breast skin ex vivo, p53 was maximally expressed at 12–24 h after UV-B exposure [34]. The p53 expression in our samples could have been even stronger if we took earlier biopsies.
Ultraviolet radiation (UVR) from the sun and solaria has addictive properties that may develop into dependence. In mice, UVR addiction was connected to β-endorphin (β-END) formed in the skin after UVR exposure. In humans, the formation of β-END in skin keratinocytes has not been confirmed in vivo.
To determine with immunohistochemistry if sub-erythematous narrow-band UV-B (NB-UV-B) exposures stimulate p53 mediated expression of pro-opiomelanocortin (POMC), β-END and α-melanocyte stimulating hormone (α-MSH) in human skin keratinocytes in vivo.
Within 12 healthy volunteers, 7 received a single 1 standard erythema dose (SED) of NB-UV-B on their whole body, and 5 volunteers received a cumulative dose of 3 SED delivered on two subsequent days i.e., 1 + 2 SED. Skin biopsies were taken immediately before the first exposure and at 24 h from the last UV-B exposure to assess p53, β-END, POMC, and α-MSH expression.
Nuclear p53 expression increased in all samples taken at 24 h after NB-UV-B exposure. UV-B irradiation also increased epidermal β-END expression in 11 out of 12 samples taken at 24 h after UV-B exposure. The brownish staining was localized in the cytoplasm of keratinocytes and around the nuclei, being more pronounced in the basal cell layers. POMC and α-MSH staining showed no obvious meaningful increase since only one section of each showed any change compared with basal levels.
Our study is the first to show that UV-B exposures increase β-END expression in epidermal keratinocytes of human skin in vivo, which could be the link to proposed UVR addiction.
Graft-versus-Host Disease-Related Cytokine-Driven Apoptosis Depends on p73 in Cytokeratin 15-Positive Target Cells
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Citation Excerpt :
Moreover, restriction of p73 protein to epithelial cells at the tips of epidermal rete ridges of normal human skin was seen in a pattern identical to that within the murine lingual RLP (Figure 2C and F). Immunoreactive p53 and p63 are not preferentially segregated between the rete-associated and non–rete-associated basal cells, as reported previously [24,25] (data not shown). Thus, p73 is preferentially localized to epithelial niches known to undergo apoptosis in experimental GVHD and on exposure to cytotoxic cytokines [2].
Acute graft-versus-host disease (GVHD), a major complication of allogeneic stem cell transplantation, involves cytotoxic soluble and cellular effectors that selectively induce apoptosis in normally apoptosis-resistant, cytokeratin 15 (K15)-expressing epithelial stem cells residing at the tips of rete ridges of human epidermis and in analogous rete-like prominences (RLPs) of murine dorsal lingual epithelium. The mechanisms whereby epithelial stem cells are rendered vulnerable to apoptosis during allostimulation are unknown. We hypothesized that GVHD-induced target cell injury may be related to pathways involving the p53 family that are constitutively expressed by epithelial stem cells and designed to trigger physiological apoptosis as a result of environmental danger signals. Among the p53 family members, we found that p73 protein and mRNA were preferentially expressed in K15⁺ RLPs of murine lingual squamous epithelium. On in vitro exposure to recombinant TNF-α and IL-1 in an organ culture model previously shown to replicate early GVHD-like target cell injury, apoptosis was selectively induced in K15⁺ stem cell regions and was associated with induction of phosphorylated p73, a marker for p73 activation, and apoptosis was abrogated in target tissue obtained from p73-deficient (p73^−/−) mice. Evaluation of early in vivo lesions in experimental murine GVHD disclosed identical patterns of phosphorylated p73 expression that coincided with the onset of effector T cell infiltration and target cell apoptosis within K15⁺ RLPs. This study is the first to suggest that paradoxical apoptosis in GVHD of physiologically protected K15⁺ epithelial stem cells is explainable, at least in part, by cytokine-induced activation of suicide pathways designed to eliminate stem cells after exposure to deleterious factors perceived to be harmful to the host.
Quantification of inducible SOS-like photoprotective responses in human skin
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Citation Excerpt :
p53 also arrests the cell cycle, allowing more time for DNA repair (Petrocelli et al., 1996). Both UVB irradiation and pTT treatment upregulate and activate p53 within 24 hours (Eller et al., 1997; Davenport et al., 1999; Goukassian et al., 2002; Marwaha et al., 2005), including in human skin explants (Arad et al., 2006). Consistent with this, in this study, within 72 hours DNA repair rate following a moderately damaging UV dose was greatly accelerated in explants pretreated with pTT or preirradiated.
To document and quantify inducible photoprotective effects in human skin, explant cultures were treated once with thymidine dinucleotide (pTT) or diluent alone or UV-irradiated. Both pTT and UV increased the melanogenic protein levels on days 1–5 and comparably increased melanocyte dendricity and epidermal melanin content. Explants treated with pTT or UV but not with diluent alone showed initial inhibition of epidermal proliferation followed by mild reactive hyperplasia; melanocyte proliferation was minimal. To determine whether pTT and UV provide comparable protection against subsequent UV-induced DNA damage, explants were pTT- or diluent-treated or UV-irradiated. All explants were then irradiated with the same UV dose 72 hours later. Compared to diluent alone, pTT or UV pretreatment decreased the number of epidermal cells positive for cyclobutane pyrimidine dimers (CPDs) 50% immediately post-irradiation. In pTT- and UV- versus diluent-pretreated explants, the rate of CPD removal was also more rapid, approximately 80 vs 45% of the initial burden within 72 hours. These data confirm and quantify comparable SOS-like responses in human skin after pTT or UV irradiation, attributable to both increased epidermal melanin and increased DNA repair rate, in the case of pTT in the absence of initial damage.
iC3b arrests monocytic cell differentiation into CD1c-expressing dendritic cell precursors: A mechanism for transiently decreased dendritic cells in vivo after human skin injury by ultraviolet B
2003, Journal of Investigative Dermatology
Citation Excerpt :
Punch biopsies and keratomes were taken from normal buttock skin as controls or after a single four minimal erythema dose of UVB irradiation from a bank of Westinghouse FS20 bulbs (PSC Lamps, Pittsford, NY) at different time points. This source has a continuous spectrum from 270 nm to 400 nm, with the predominant UVB emission peaking at 314 nm (Davenport et al, 1999). Six-micron frozen sections of biopsies of normal and UVB-irradiated skin, after thawing and hydration in phosphate-buffered saline (PBS), were first blocked with 10% goat serum/PBS.
Our previous data indicated that C3, its bioactive product iC3b, and the iC3b ligand CD11b are critical for ultraviolet-induced immunosuppression. We thus hypothesized that iC3b is an important skin-based factor regulating CD11b⁺ monocytic cell function in the acute post-ultraviolet period. Although monocytic cell migration peaked at 1–3 d after ultraviolet exposure of skin, dermal CD1c dendritic cells underwent a rapid and prolonged depletion that did not recover until day 7. Because ultraviolet-induced iC3b deposits are reciprocally maximal on day 3, but fade by day 7, we next hypothesized that iC3b can be responsible for the delay in differentiation into dendritic cells of monocytic cells migrating into ultraviolet-exposed skin. Analysis of dermal cells derived from keratome biopsies suggested that iC3b exposure could inhibit the development of CD1c⁺ dermal cells. To model newly immigrating blood monocytes entering ultraviolet-exposed, iC3b-containing dermis, purified monocytes from human blood were induced with granulocyte-macrophage colony stimulating factor to generate a population of dendritic cell precursors expressing CD1c. Incubation with iC3b markedly inhibited the appearance of CD1c⁺ cells (p<0.05) and induced CD1c^–CD14⁺ cells. This inhibition was reversed by coincubation with an anti-CD11b antibody that blocks the iC3b binding site. Other functions associated with dendritic cell maturation were also inhibited by iC3b, such as interleukin-12p70 production as well as CD80 and CD40 expression. Restimulation of monocytes for DC maturation revealed that iC3b induced a temporary inhibition of DC differentiation. Thus, a human skin response in which iC3b is transiently (3–7 d) generated in dermis, such as ultraviolet, can arrest monocytic skin-infiltrating cells from undergoing dendritic cell precursor differentiation.
UV-B induced keratinocyte apoptosis is blocked by 2-selenium-bridged β-cyclodextrin, a GPX mimic
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Cell proliferation and cell death of keratinocytes are tightly regulated to ensure epidermal homeostasis. UV-B induces keratinocyte apoptosis. UV-B also induces lipid peroxidation of keratinocytes to increase their amount of malondialdehyde (MDA). These phenomena can be explained by the production of reactive oxygen species (ROS) induced by UV-B radiation. We synthesized 2-selenium-bridged β-cyclodextrin (2-SeCD) to imitate glutathione peroxidase (GPX), an important antioxidant and established a damage system, in which keratinocytes can be damaged by Ultraviolet B (UV-B) radiation. Using this damage system we studied 2-SeCD protection of keratinocytes against injury induced by UV-B. Experimental results showed that 2-SeCD could protect keratinocytes from apoptosis. Moreover, 2-SeCD inhibits lipid peroxidation of keratinocytes and scavenges ROS. 2-SeCD inhibits the UV-B induced apoptotic signal transduction. This antiapoptotic mechanism may be partly related to the elimination of hydrogen peroxide.

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p53 induction in normal human skin in vitro following exposure to solar simulated UV and UV-B irradiation

Abstract

J. Invest Dermatol.

J. Invest. Dermatol.

Cell

Eur. J. Cancer B: Oral Oncol.

J. Invest. Dermatol.

J. Invest. Dermatol.

J. Photochem. Photobiol. B: Biol.

J. Invest. Dermatol.

Effects of ultraviolet radiation on human epidermal cell alloantigen presentation: initial depression of Langerhans' cell-dependent function is followed by the appearance of T6-Dr+ cells that enhance epidermal alloantigen presentation

J. Immunol.

T antigen is bound to a host protein in SV-40 transformed cells

Nature

Localisation of gene for human p53 tumour antigen to band 17p13

Nature

Abnormalities of p53 protein expression in cutaneous disorders

Arch. Dermatol.

Growth regulation of a cellular tumour antigen p53 in non-transformed cells

Nature

Human papillomavirus type 16 E6 increases the degradation rate of p53 in human keratinocytes

J. Virol.

Distinct conformations of p53 are observed at different stages of keratinocyte differentiation

Oncogene

Wild-type p53 is a cell cycle checkpoint determinant following irradiation