UVA-340 as energy source, mimicking natural sunlight, activates the transcription factor AP-1 in cultured fibroblasts: evidence for involvement of protein kinase-C.

Ultraviolet radiation (UVR) is known to affect a variety of cellular functions, including gene expression. A number of signaling pathways have been suggested to mediate these effects, including the participation of activator protein-1 (AP-1), activator protein-2 (AP-2) and nuclear factor-kappa B (NF-kappa B). The divergent results from previous studies could be explained, at least in part, by the source of UVR with different spectral characteristics as well as the type of cells employed as targets. In this study we have utilized UVA-340 as an energy source with output which closely matches the spectrum of natural sunlight over the range of 295-350 nm for irradiation of cultured fibroblasts. Combination of electrophoretic mobility shift assays and Northern analyses revealed activation of AP-1 but not NF-kappa B or AP-2. Inhibition studies further suggested the participation of protein kinase-C, but not protein kinase-A, and that an inhibitor of mitogen-activated protein kinase (MEK-1/2) did not alter the AP-1 activation. Free radical quenchers, sodium azide and N-acetylcysteine, did not affect the AP-1 binding activity. Finally, UVA-340 was shown to enhance transcriptional expression of the type-VII collagen gene (COL7A1), which is endogenously expressed in dermal fibroblast in an AP-1 dependent manner. Introduction of a mutation into the AP-1 site of the COL7A1 promoter abolished this activation. Thus, our results obtained by utilizing a novel energy source, UVA-340, mimicking natural sunlight at UVB and lower UVA range indicate a role for AP-1 in mediating the enhanced gene expression by UVR. Collectively, these results suggest that AP-1 is an important mediator of UVR action in fibroblasts.

Ultraviolet-filtering properties of commonly used tissue cell culture plasticware.

BACKGROUND: Fluorescent sunlamps are a common source of ultraviolet radiation (UVR) for photobiology research. However, these lamps emit a significant amount of biologically "irrelevant" wavelengths that, if not removed, can drastically skew results and perhaps lead to mistaken conclusions regarding human photobiology. The use of a cellulose triacetate sheet (Kodacel) to filter the shorter ultraviolet wavelengths has become the accepted standard in photobiology. Over time, the transmission characteristics of this sheet may be altered due to photochemical changes. In addition, in vitro experiments utilizing filtered fluorescent sunlamps require the removal of plastic tissue cell culture lids, increasing the possibility of contamination. METHODS: We evaluated the transmission characteristics of various commercially available plastic lids used in tissue cell cultures. In addition, we used a biological system containing the human elastin promoter/chloramphenicol acetyltransferase reporter gene construct to compare the effects of filtering from these plastic lids. RESULTS: Here, we demonstrate that the transmission of UVR and the biological response through plastic culture dish lids is similar to that of Kodacel. CONCLUSION: Although this is an improvement for in vitro experiments, further improvements can be made using more realistic UVR sources, e.g. UVA-340 lamps, which mimic the short wavelengths of sunlight.

Keratinocyte apoptosis induced by ultraviolet B radiation and CD95 ligation -- differential protection through epidermal growth factor receptor activation and Bcl-x(L) expression.

Previous work has shown that activation of the epidermal growth factor receptor by endogenous or exogenous signals markedly enhances survival of cultured keratinocytes upon cellular stress such as passaging. This is due, in part, to epidermal-growth-factor-receptor-dependent expression of Bcl-x(L), an antiapoptotic Bcl-2 homolog. In this study we tested whether epidermal-growth-factor-receptor-dependent signal transduction and attendant Bcl-x(L) expression affected survival of human keratinocytes upon exposure to a frequently encountered apoptotic stimulus, radiation with ultraviolet B. We describe that blocking epidermal-growth-factor-receptor-dependent signal transduction sensitized normal keratinocytes to undergo apoptosis upon ultraviolet B radiation with solar light characteristics. Forced expression of Bcl-x(L) partially but significantly inhibited ultraviolet-B-induced apoptosis of immortalized keratinocytes (HaCaT). Bcl-x(L) overexpression afforded no protection to HaCaT cells against apoptosis induced by binding of an agonist antibody to the death receptor CD95, however. CD95 activation has previously been shown to functionally contribute to apoptosis in ultraviolet-irradiated keratinocytes. These results indicate that epidermal growth factor receptor activation and attendant Bcl-x(L) expression provided a physiologically relevant protective pathway of keratinocytes against ultraviolet-induced but not CD95-dependent apoptosis.

The nitroxide Tempol affords protection against ultraviolet radiation in a transgenic murine fibroblast culture model of cutaneous photoaging.

The generation of reactive oxygen species is among the various mechanisms by which ultraviolet radiation damages skin. Tempol, a superoxide dismutase analogue which readily penetrates cell membranes when administered exogenously, has been shown to provide protection against some forms of oxygen-dependent damage. In this study, we measured the ability of Tempol to protect against ultraviolet A- and ultraviolet B-induced damage, using a previously described transgenic mouse model of cutaneous photoaging. The ability of Tempol to prevent ultraviolet radiation-induced elastin promoter activation was determined in vitro. Tempol provided over 50% protection against ultraviolet B and over 70% protection against ultraviolet A as measured in our in vitro system. These results demonstrate the ability of the superoxide dismutase mimic, Tempol, to protect against ultraviolet induced elastin promoter activation. This compound could be a useful pharmacological agent to prevent cutaneous photoaging.

Stimulation of melanogenesis by DNA oligonucleotides: effect of size, sequence and 5' phosphorylation.

It has been shown that the small DNA fragment thymidine dinucleotide, (pTpT) induces photoprotective responses in cultured cells and intact skin. These responses include increased melanogenesis, enhanced DNA repair, and induction of TNF-alpha, and are accomplished, at least in part, through the induction and activation of the p53 tumor suppressor and transcription factor. Here it is reported that other, but not all, larger oligonucleotides induce the pigmentation response even more efficiently than pTpT. A 9 base oligonucleotide (p9mer) stimulated pigmentation in Cloudman S91 murine melanoma cells to 6-times the level of control cells while a 5 base oligonucleotide (p5mer#1) was inactive. In addition, the p9mer increased p21 mRNA levels and inhibited cell proliferation to a greater degree than did pTpT, consistent with the presumptive mechanism of action involving p53. Smaller, truncated versions of the p9mer also stimulated pigmentation, although to a lesser extent than did the p9mer. The ability of these oligonucleotides to stimulate pigmentation was highly dependent on the presence of a 5' phosphate group on the molecule, which was shown by confocal microscopy and fluorescent activated cell sorter (FACS) analysis to greatly facilitate the uptake of these oligonucleotides into the cells. Although the melanogenic activity of the oligonucleotides was directly related to increased length and 5' phosphorylation, nucleotide sequence is also critical because a p20mer was efficiently internalized yet was a poor inducer of pigmentation.

Common fluorescent sunlamps are an inappropriate substitute for sunlight.

Fluorescent sunlamps are commonly employed as convenient sources in photobiology experiments. The ability of Kodacel to filter photobiologically irrelevant UVC wavelengths has been described. Yet there still remains a major unaddressed issue--the over representation of UVB in the output. The shortest terrestrial solar wavelengths reaching the surface are approximately 295 nm with the 295-320 nm range comprising approximately 4% of the solar UV irradiance. In Kodacel-filtered sunlamps, 47% of the UV output falls in this range. Consequently, in studies designed to understand skin photobiology after solar exposure, the use of these unfiltered sunlamps may result in misleading, or even incorrect conclusions. To demonstrate the importance of using an accurate representation of the UV portion of sunlight, the ability of different ultraviolet radiation (UVR) sources to induce the expression of a reporter gene was assayed. Unfiltered fluorescent sunlamps (FS lamps) induce optimal chloramphenicol acetyltransferase (CAT) activity at apparently low doses (10-20 J/cm2). Filtering the FS lamps with Kodacel raised the delivered dose for optimal CAT activity to 50-60 mJ/cm2. With the more solar-like UVA-340 lamps somewhat lower levels of CAT activities were induced even though the apparent delivered doses were significantly greater than for either the FS or Kodacel-filtered sunlamp (KFS lamps). When DNA from parallel-treated cells was analyzed for photoproduct formation by a radioimmuneassay, it was shown that the induction of CAT activity correlated with the level of induced photoproduct formation regardless of the source employed.

Photodermatology: progress, problems and prospects.

Photodermatology is a sub-specialty of photobiology. As such it includes all aspects of photobiology related to the skin ranging from sun exposure and its consequences (both short term and long term) to the therapeutic effects derived from exposure to natural or artificial radiation. In this review the terms photodermatology and photomedicine are used in a somewhat interchangeable fashion, although the former is really a portion of the latter. Four international journals are the primary sources for information - Photochemistry & Photobiology; Journal of Photochemistry & Photobiology (pt B); Photodermatology, Photomedicine & Photoimmunology; Journal of Investigative Dermatology. This review is structured so as to report on recent progress, existing problem areas and prospects for advances in the near future.

Sunscreens, skin photobiology, and skin cancer: the need for UVA protection and evaluation of efficacy.

Sunscreens are ultraviolet radiation (UVR)-absorbing chemicals that attenuate the amount and nature of UVR reaching viable cells in the skin. They are selected and tested for their ability to prevent erythema. No sunscreen prevents photodamage, as it has been demonstrated that suberythemal doses of UVR cause a variety of molecular changes (including DNA damage) in these cells. Furthermore, the spectrum of UVR reaching viable cells is altered by topically applied sunscreen. In this review, the basic aspects of sunscreens and skin photobiology are reviewed briefly. Although there can be no question concerning the efficacy of sunscreens for the prevention of erythema, questions remain because of the possible cumulative effects of chronic suberythemal doses and the increased exposure of skin cells to longer UVR wavelengths. The current major issue surrounding sunscreens involves their ability to protect skin cells against the effects of UVA radiation. These UVA effects may be direct damage (base oxidations) or effects on the skin immune system, yet there is no uniformly accepted method for the evaluation of UVA protection. This review is focused primarily on the latter topic covering action spectra that implicate the need for UVA protection. In addition, in vivo and in vitro methods proposed for the evaluation of candidate sunscreen formulations of UVA protective ability are reviewed. Finally, revisions in the terminology used to describe the protection afforded by sunscreens are suggested. It is proposed that SPF ("sun" protection factor) be renamed "sunburn" protection factor and that "critical wavelength" be designated "long wave index."

The role of PUVA in the treatment of psoriasis. Photobiology issues related to skin cancer incidence.

Photochemotherapy with methoxsalen (8-methoxypsoralen) and long wavelength ultraviolet (UV) radiation (referred to as 'PUVA' for psoralen plus UVA) is commonly used to treat psoriasis and vitiligo. These vastly different diseases respond to the therapy by different mechanisms even though the immediate effects of the therapy--the photomodification of cellular biomolecules--is the same for each. Because psoriasis is not cured by PUVA, patients receive many treatments over their lifetime and have a significantly increased risk for the development of skin cancers (primarily squamous cell carcinomas). In this article the basic aspects of psoralen photobiology are reviewed briefly. Several recent studies describing the incidence of skin cancer in UVA treated psoriasis cohorts are comparatively reviewed. In addition the impact of the analysis of mutations in the tumor suppressor gene, p53, are summarized. An unexpected mutation spectrum (very few PUVA type T-->A transversions and frequent UVB solar signature C-->T transitions) suggest that effects other than direct DNA photoadduct formation may be at play. These analyses suggest that it may be possible to improve the therapeutic efficacy of PUVA by a careful evaluation of the mode of delivery. In this review the science behind PUVA is summarized. In addition, the incidence of skin cancer as a long term consequence of repeated treatments is surveyed. To relate clinical observations to molecular events, the nature of p53 mutations found in skin cancers from psoriasis patients is also analyzed. Finally some suggestions for improving the delivery of PUVA therapy are presented.

Psoriasis, PUVA, and skin cancer--molecular epidemiology: the curious question of T-->A transversions.

Photochemotherapy with 8-methoxypsoralen and long wavelength ultraviolet radiation (PUVA) is commonly used to treat psoriasis and vitiligo. These vastly different diseases respond to the therapy by different mechanisms even though the immediate effects of the therapy - photoadduct formation - is the same for both. Because psoriasis is not cured by PUVA, patients receive many treatments over their lifetime and develop a significant risk for the development of skin cancers (primarily squamous cell carcinomas). In this review the basic aspects of psoralen photobiology are reviewed briefly. In addition the impact of the analysis of mutations in the tumor suppressor gene, p53, are summarized. An unexpected mutation spectrum (very few T-->A transversions and frequent UVB signature C-->T transitions) suggest that effects other than direct DNA photoadduct formation may be at play. The roles of reactive oxygen species-induced base changes as well as other clastogenic factors are discussed. This analysis suggests that it may be possible to improve the therapeutic efficacy of PUVA by a careful evaluation of the mode of delivery.

Psoralen photobiology and photochemotherapy: 50 years of science and medicine.

In 1998 it is appropriate to commemorate the 50th anniversary of el Mofty's use of purified 8-methoxypsoralen (8-MOP) in the treatment of vitiligo (el Mofty AM. A preliminary clinical report on the treatment of leukoderma with Ammi majus linn. J R Egypt Med Assn 1948,31:651 65. el Mofty AM, el Sawalhy H, el Mofty M. Clinical study of a new preparation of 8-methoxypsoralen in photochemotherapy. Int J Dermatol 1994;8:588 92). Two young American dermatologists (Aaron Lerner and Thomas Fitzpatrick) were intrigued by the potency of this material. After Lerner determined that artificial long wavelength ultraviolet (320-400 nm, UVA) radiation was the most efficient for activating 8-MOP. the development of artificial sources enabled the efficient delivery of these photons to skin containing 8-MOP. Their initial studies for vitiligo led to further development of this therapy for the treatment of psoriasis (Parrish JA, Fitzpatrick TB, Tannenbaum L, et al. Photochemotherapy of psoriasis with oral methoxsalen and long-wave ultraviolet light. New Engl J Med 1974;291:1207-11. Honigsmann H, Fitzpatrick TB, Pathak MA, et al. Oral photochemotherapy with psoralen and UVA (PUVA): principles and practice. In: Fitzpatrick TB, Eisen AZ, Wolf K, editors. Dermatology in General Medicine. New York: McGraw-Hill, 1987:1728-54). This photochemotherapy came to be called 'PUVA' (psoralen + UVA). The position PUVA holds today as one of the most common procedures performed in dermatology can be traced to their initial curiosity and their subsequent ingenuity. Further developments in more recent years capitalized on their seminal work. The therapy met with unprecedented success from the outset, leaving little perceived need to understand underlying science. However, in recent years there has been a new found interest in the basic aspects of psoralen photobiology and molecular mechanistic events contributing to therapeutic responses as well as to the development of skin cancers in PUVA patients. These will be surveyed in this review commemorating the 50 years of modern psoralen photobiology and photomedicine.

A review of sunscreen safety and efficacy.

The use of sunscreen products has been advocated by many health care practitioners as a means to reduce skin damage produced by ultraviolet radiation (UVR) from sunlight. There is a need to better understand the efficacy and safety of sunscreen products given this ongoing campaign encouraging their use. The approach used to establish sunscreen efficacy, sun protection factor (SPF), is a useful assessment of primarily UVB (290-320 nm) filters. The SPF test, however, does not adequately assess the complete photoprotective profile of sunscreens specifically against long wavelength UVAI (340-400 nm). Moreover, to date, there is no singular, agreed upon method for evaluating UVA efficacy despite the immediate and seemingly urgent consumer need to develop sunscreen products that provide broad-spectrum UVB and UVA photoprotection. With regard to the safety of UVB and UVA filters, the current list of commonly used organic and inorganic sunscreens has favorable toxicological profiles based on acute, subchronic and chronic animal or human studies. Further, in most studies, sunscreens have been shown to prevent the damaging effects of UVR exposure. Thus, based on this review of currently available data, it is concluded that sunscreen ingredients or products do not pose a human health concern. Further, the regular use of appropriate broad-spectrum sunscreen products could have a significant and favorable impact on public health as part of an overall strategy to reduce UVR exposure.

Psoralen photochemotherapy, clinical efficacy, and photomutagenicity: the role of molecular epidemiology in minimizing risks.

Photochemotherapy employing 8-methoxypsoralen and ultraviolet radiation (PUVA) is widely used in the treatment of psoriasis. The photoactivation of psoralens in skin cells leads to DNA photoadduct formation which may be responsible for the efficacy of PUVA. Subsequent mutations may lead to the increased incidence of squamous cell carcinoma (SCC). Mutations in the p53 tumor suppressor gene have been detected in many human cancers. In this review, p53 mutation spectra in murine and human SCC are compared to those obtained from murine cells and skin treated with PUVA as well as to the p53 mutation spectrum in human solar SCC. While the expected psoralen-type mutations at alternating AT sites were detected in the treated cells and murine SCC (average frequency > 40%), such mutations were not commonly detected in the human SCC (< 10%). Other common mutations in the human SCC included: CG-->TA transitions (18%) and CG-->AT and TA-->GC transversions (17 and 25%, respectively). In addition, the frequency of UVB-type mutations at dipyrimidine sites (CC-->TT) in the SCC PUVA-treated psoriasis patients was comparable to that in patients with SCC from only solar exposure. A review of therapeutic history of these patients showed that many had also received UVB phototherapy. Furthermore, because sunlight is thought to be beneficial for psoriasis, nontherapeutic, casual UVB exposure cannot be excluded. Thus, the PUVA SCC may have arisen from the solar mutations and PUVA may enhance tumor progression by other epigenetic effects.