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This Perspective briefly reviews the relationship between UV-induced mutations in habitually sun-exposed human skin and subsequent development of actinic keratoses (AKs) and skin cancers. It argues that field therapy rather than AK-selective therapy is the more logical approach to cancer prevention and hypothesizes that treatment early in the process of field cancerization, even prior to the appearance of AKs, may be more effective in preventing cancer as well as more beneficial for and better tolerated by at-risk individuals. Finally, the Perspective encourages use of rapidly advancing DNA analysis techniques to quantify mutational burden in sun-damaged skin and its reduction by various therapies.

A rapid and exciting increase in depth and breadth characterizes dermatologic research in China. Simultaneously, there has been increased interaction between academic dermatologists in China and their colleagues abroad, as well as between clinical and research-oriented dermatologic societies in China and parallel organizations in the United States and Europe. As the official journal of the Society for Investigative Dermatology (SID) in the United States and of the European Society for Dermatological Research (ESDR), the Journal of Investigative Dermatology (JID) has been positively affected by these interactions and generally by the evolution of science and academics in China. At present, three of the JID Associate Editors are Chinese, and a rapidly increasing percentage of total JID submissions originate from Chinese departments of dermatology.

The 63rd annual Montagna Symposium on the Biology of Skin, “Skin Aging: Molecular Mechanisms and Tissue Consequences,” was held October 9–13, 2014, in Gleneden Beach, Oregon. The meeting brought together basic gerontologists, dermatologists and skin biologists working on mechanisms and problems of skin aging, industry scientists attempting to create products to address unmet needs in the field, and trainees wishing to acquire a better understanding of the aging process and its consequences in skin. The many recent advances in both basic and applied aspects of cutaneous aging led to productive exchanges among these participants, broadened everyone's horizons, and stimulated several new collaborations. The Symposium was chaired by Barbara A. Gilchrest with Session Chairs Judith Campisi, Howard Chang, and Gary Fisher.

pBackground: Despite public education efforts, many people at risk for skin cancer do not practice safe sun behaviors./ppObjective: To determine whether machine-based evaluation of UV-induced alterations (VISIA scan) changes self-assessment of facial photoaging, skin cancer risk, and willingness to improve sun protective habits. In addition, to determine whether VISIA scan analysis reveals differences between those with versus without a history of skin cancer, men versus women, those older than 50 versus less than 50 years of age, and Fitzpatrick skin types I-III versus IV-VI./ppMethods: Volunteers attending a health expo were recruited and queried about their perceived risk of skin cancer and degree of skin photoaging. All participants underwent facial skin quality analysis of both sides of the face, and then completed a follow-up survey./ppResults: Participants' scored self-perceptions of overall skin aging were all statistically significantly worse after VISIA scan analysis. There was no change in perceived skin cancer risk, but most participants expressed intent to improve their sun protection habits./ppLimitations: Limitations to this study include selection bias, recall-misclassification bias, and social desirability bias./ppConclusion: Intervention with facial skin analysis can positively affect subjects' stated intent to use sun protection, indicating the importance of appearance in these health decisions./ppemJ Drugs Dermatol. 2017;16(5):453-459./em/p.

Background Many therapeutic options are available for treating keloids, but success rates vary widely, and the keloids often recur. The Food and Drug Administration has recently approved intralesional collagenase for the treatment of Dupuytren's contracture. This medication has not been explored for the treatment of earlobe keloids, a common problem. Objective To evaluate the safety and clinical efficacy of intralesional collagenase followed by compression for the treatment of earlobe keloids. Materials and methods Six earlobe keloids in six patients were injected with a commercial collagenase preparation. Study participants were asked to use compression earrings daily thereafter. Patients were examined and photographed 1 day, 2 weeks, 4 weeks, 10 months, and 12 months after injection. Adverse events were assessed at each visit, and the keloids were measured and photographed. Results All patients had a decrease in the size of their earlobe keloid by an average of 50% (p = .02). Three of the six participants chose to have their earlobe keloids surgically excised for cosmetic reasons 6, 8, and 11 months after enrollment, so measurements for data analysis for these patients were taken after only 1, 1 and 10 months. All participants returned for follow-up at the last study visit 1 year after study commencement. The three patients who completed the study were pleased with the improvement of their earlobe keloid, although complete clearance was not achieved. Side effects included injection site swelling, tenderness, and one ulceration that spontaneously resolved within 2 weeks. Conclusion Intralesional collagenase followed by compression appears to be a safe and modestly effective treatment for earlobe keloids. This approach warrants further investigation in larger studies with longer follow-up in motivated patients who decline surgical excision.

"Vitamin D" is the term commonly used to denote the lipid-soluble hormone critical for calcium homeostasis and skeletal maintenance. A precursor to the active compound is found in many plants and animal tissues and can be absorbed from the gut; it can also be derived from cell membranes in the epidermis during ultraviolet B irradiation. This compound is then hydroxylated sequentially in the liver and kidney to produce the active hormone 1,25(OH)(2)D that binds its nuclear receptor to modulate gene expression. Recently, vitamin D hydroxylases and the nuclear receptor have been identified in many tissues, suggesting previously unrecognized roles for vitamin D. Some epidemiologic studies have also correlated low levels of the inactive storage form 25(OH)D with an increased incidence or prevalence of a variety of diseases, suggesting that large oral supplements and/or increased ultraviolet (UV) exposure might therefore improve individual health. However, randomized, prospective controlled trials comparing vitamin D supplements with placebo have not supported this belief. Moreover, current evidence supports the conclusion that protection from UV radiation does not compromise vitamin D status or lead to iatrogenic disease. In contrast, high vitamin D levels appear to incur a risk of kidney stones and other adverse effects. In the case of true vitamin D deficiency, supplements are a more reliable and quantifiable source of the vitamin than UV exposure.

Cutaneous biopsies often heal with little or no scarring. Prior studies have shown an alarming percentage of patients who incorrectly identify biopsy sites at the time of surgery.To investigate the safety and utility of an ultraviolet (UV)-fluorescent tattoo for biopsy site identification.A preclinical proof of concept was established with skin culture. An UV-fluorescent tattoo was applied to discarded neonatal foreskin in culture medium. The stability of the tattooed skin was examined clinically and histologically. One patient with a recurrent basal cell carcinoma in a difficult-to-identify location underwent tattoo application at the time of biopsy to demarcate the site. The patient was monitored for tattoo reaction and referred for surgical excision.The cultured tissue exhibited stable UV fluorescence with daily washing. Tissue histology demonstrated tattoo particles lining the skin edge under fluorescent microscopy. The patient was reluctant to undergo another surgical procedure and instead returned to our clinic at 3 months and 17 months after the biopsy for management of other tumors. The patient had no symptoms of allergic reaction to the tattoo dye. The fluorescent tattoo remains invisible under visible light and visible only under Wood's light.The present study documents the utility of an UV-fluorescent tattoo to locate a biopsy site.

Tanning is commonly understood as increased melanization of the epidermis observed in skin following UV exposure. It is further understood to represent a host response that protects against future UV-induced damage. Although best studied in humans, tanning can also be observed in other mammals and even in more primitive animals such as sharks. The molecular basis of tanning was poorly understood well into the 1990s, although UV irradiation of murine melanoma cells had been shown to increase the number of cell surface receptors to a-melanocyte–stimulating hormone (a-MSH) and to increase the melanogenic response to a-MSH via increased protein levels and activity of tyrosinase, the rate-limiting enzyme in melanin synthesis (Bolognia et al., 1989; Chakraborty et al., 1991). However, the initiating molecular events were unknown. Identification of the responsible molecules ultimately evolved from studies of the UV action spectrum for critical events in human skin, as well as from a philosophical appreciation of tanning as a genome protective response. In the 1980s, investigators at Harvard’s Wellman Laboratories exposed normal skin of volunteers to a wide range of light doses using narrow band sources across the UV spectrum and observed them for several days to determine the lowest dose at each wavelength capable of producing a delayed tan (Parrish et al., 1982). In other experiments, they used the same approach to define the action spectrum for induction of cyclobutane pyrimidine dimers (CPDs), the most common form of DNA damage following UV exposure (Freeman et al., 1989). The spectra were virtually superimposable, peaking at B300 nm, suggesting a cause-and-effect relationship between the immediate DNA damage and subsequent increased production and dispersion of epidermal melanin. Eller et al. (1996) first documented that agents acting exclusively on DNA, such as DNA restriction enzymes, stimulate melanin production in cultured pigment cells, at least in part by increasing a-MSH binding and the melanogenic response to a-MSH. Although this did not exclude a role for UV-mediated membrane effects, it did directly implicate UV-mediated DNA damage in the tanning response. Similarly, accelerating DNA damage repair by providing UV-irradiated pigment cells with T4 endonuclease V, the bacterial enzyme that catalyzes the first step in CPD resolution, also enhanced the melanization response (Gilchrest et al., 1993). By the 1990s, DNA damage responses were recognized to be largely mediated by the transcription factor and tumor suppressor protein p53, also termed the ‘‘guardian of the genome’’ (Lane, 1992), and two groups independently asked whether p53 was involved in mediating the tanning response. Using p53 null osteosarcoma cells transfected with wild-type p53, Nylander et al. (2000) showed that p53 activation increased read-out from a transfected tyrosinase promoter linked to a reporter gene, implying that p53 could directly or indirectly stimulate tyrosinase transcription. This link between p53 and tanning was expanded and refined using both human melanoma cells and a mouse model. Compared with a p53-null parental melanoma line and to the same line transfected with an empty vector, melanoma cells transfected with wildtype p53 increased their tyrosinase mRNA levels progressively over 72 hours following p53 activation (Khlgatian et al., 1999; Khlgatian et al., 2002). Confirming an earlier report (Kichina et al., 1996), these studies also showed an inverse relationship between total p53 protein (inactive) and tyrosinase levels. The requirement for p53 activation to increase tyrosinase protein level and epidermal melanin content in vivo was confirmed by documenting the tanning ability of wild type versus p53 knockout mice (Khlgatian et al., 2002). The question remained whether p53 directly increased tyrosinase transcription, as no p53 consensus sequence had been identified in its promoter region. The key observation that the UV action spectrum for tanning is virtually identical to that for CPD production, had suggested that thymidine dinucleotide (abbreviated ‘‘TT’’), the obligate substrate for the majority of CPDs, might itself serve as a molecular signal for increased melanogenesis (tanning). Using various models, the responses to UV irradiation versus this DNA fragment were therefore compared. Indeed, TT increased mRNA and protein expression of tyrosinase as well as melanin content of cultured human and murine pigment cells and of intact guinea pig skin-containing melanocytes in the interfollicular epidermis, in a time frame similar to that observed after UV irradiation (Eller et al., 1994). As well, in guinea pig skin, the histological features were virtually identical following either treatment: increased total melanin with prominent

Identification, prevention and reversal of age-related changes in the appearance and function of human skin remain goals of great interest to scientists, dermatologists and patients. In recent decades, the cellular and molecular changes that underlie cutaneous aging have been substantially elucidated. These data suggest that cutaneous aging, like aging in the body generally, results, in part, from a telomere-based genetic program, cumulative DNA damage and repair, oxidative damage, altered proteolysis and energy metabolism and changes in transcription factor-driven gene expression. In animal models, modulation of these events can delay or even reverse aging by many criteria. The resulting insights are likely to revolutionize the prevention and therapy of cutaneous aging.

Overexposure to the sun is associated with an increased risk of melanoma and nonmelanoma skin cancer, but indications of improvements in sun protection behavior are poor. Attempts to identify emerging themes in skin cancer control have largely been driven by groups of experts from a single field. In December 2016, 19 experts from various disciplines convened for Interdisciplinary Perspectives on Skin Cancer, a 2-day meeting hosted by the National Academy of Sciences. The group discussed knowledge gaps, perspectives on sun exposure, implications for skin cancer risk and other health outcomes, and new directions. Five themes emerged from the discussion: (1) The definition of risk must be expanded, and categories for skin physiology must be refined to incorporate population diversities. (2) Risky sun exposure often co-occurs with other health-related behaviors. (3) Messages must be nuanced to target at-risk populations. (4) Persons at risk for tanning disorder must be recognized and treated. (5) Sun safety interventions must be scalable. Efficient use of technologies will be required to sharpen messages to specific populations and to integrate them within multilevel interventions. Further interdisciplinary research should address these emerging themes to build effective and sustainable approaches to large-scale behavior change.

Retinoids are a group of naturally occurring and synthetic compounds with vitamin A-like biological activity. They play an important role in vision, reproduction, growth, and epithelial cell differentiation. Recent discoveries of specific retinoid cellular binding proteins and nuclear receptors have led to a better (although not complete) understanding of the complex mechanisms of retinoid action. Numerous clinical studies have demonstrated beneficial effects of retinoids on skin diseases such as acne, psoriasis, ichthyoses, keratodermas, skin cancers and their precursors, as well as a reversal of the effects of photoaging.

The major differentiated function of melanocytes is the synthesis of melanin, a pigmented heteropolymer that is synthesized in specialized cellular organelles termed melanosomes. Mature melanosomes are transferred to neighboring keratinocytes and are arranged in a supranuclear cap, protecting the DNA against incident ultraviolet light (UV) irradiation. The synthesis and distribution of melanin in the epidermis involves several steps: transcription of melanogenic proteins, melanosome biogenesis, sorting of melanogenic proteins into the melanosomes, transport of melanosomes to the tips of melanocyte dendrites and finally transfer into keratinocytes. These events are tightly regulated by a variety of paracrine and autocrine factors in response to endogenous and exogenous stimuli, principally UV irradiation.

Human B- or T-cell lymphoma lines and primary murine lymphomas were treated with DNA oligonucleotides homologous to the telomere (TTAGGG repeat; "T-oligo"), either alone or in combination with standard, widely-used anticancer chemotherapeutic agents. T-oligo induces cell cycle arrest and apoptosis in cultured human or murine B or T-lymphoma cell lines and primary tumor cells, but exerts no detectable toxicity on normal human or murine primary lymphocytes. Exposure to T-oligo is hypothesized to mimic exposure of the 3' telomere repeat sequence, activating the ataxia telangiectasia mutated kinase, which phosphorylates downstream effectors such as p53, but effects are not dependent solely on functional p53. T-oligo causes early S-phase arrest and cooperates well with G(2)- or M-phase-specific anticancer agents; when combined at 1/10th of the conventional dose, vincristine and T-oligo produce greater-than-additive killing of human or murine lymphoma cells (78% of cells undergoing apoptosis after 6 hr vs. 5% of control cells). In mice, 1/10th of the conventional dose of a standard combination of cyclophosphamide, adriamycin, vincristine and prednisone is twice as effective when used in combination with low dose T-oligo. Thus, T-oligo sensitizes tumors to traditional anticancer agents and represents a potentially important new addition to the therapeutic arsenal for aggressive lymphomas.

Bone Morphogenetic Protein (BMP-4) was shown to down-regulate melanogenesis, in part, by decreasing the level of tyrosinase [Yaar et al. (2006) JBC:281]. Results presented here show that BMP-4 down-regulated the protein levels of TRP-1, PKC-β, and MCI-R. When paired cultures of human melanocytes were treated with vehicle or BMP-4 (25 ng/ml), MAPK/ERK were phosphorylated within one hour of BMP-4 treatment. Then the activated MAPK/ERK caused an acute phosphorylation of MITF, followed by proteosome-mediated degradation of MITF, the key transcription factor for melanogenic proteins [Wu et al. (2000) Gene & Development:14]. However, prolonged exposure of melanocytes to BMP-4 (up to 48 hours) caused a decrease in the level of MITF-M transcript. In addition, BMP-4 decreased the intracellular level of cAMP, the key regulator of MITF expression. These results demonstrate that BMP-4 activates MAPK/ERK signaling pathway to transiently activate MITF; however, chronic treatment of BMP-4 to melanocytes causes a down-regulation of the expression of MITF, possibly in a cAMP-dependent pathway.

Ultraviolet radiation is a carcinogen that also compromises skin appearance and function. Because the ultraviolet action spectra for DNA damage, skin cancer, and vitamin D(3) photosynthesis are identical and vitamin D is readily available from oral supplements, why has sun protection become controversial? First, the media and, apparently, some researchers are hungry for a new message. Second, the controversy is fueled by a powerful special interest group: the tanning industry. This industry does not target the frail elderly or inner-city ethnic minorities, groups for whom evidence of vitamin D(3) insufficiency is strongest, but rather fair-skinned teenagers and young adults, who are at highest risk of ultraviolet photodamage. Third, evolution does not keep pace with civilization. When nature gave humans the appealing capacity for cutaneous vitamin D(3) photosynthesis, life expectancy was

Human skin has developed elaborate defense mechanisms for combating a wide variety of potentially damaging environmental factors; principal among these is UV light. Despite these defenses, short-term damage may include painful sunburn and long-term UV damage results in both accelerated skin aging and skin cancers such as basal cell carcinoma, squamous cell carcinoma and even malignant melanoma. While UV radiation damages many cellular constituents, its most lasting effects involve DNA alteration. The following sections briefly review UV-inducible protective responses in bacteria and in skin, thymidine dinucleotides (pTT) as a powerful probe of DNA damage responses, and potential means of harnessing these inducible responses therapeutically to reduce the now enormous burden of cutaneous photodamage in our society.

Photoageing is the superposition of chronic ultraviolet (UV)-induced damage on intrinsic ageing and accounts for most age-associated changes in skin appearance. It is triggered by receptor-initiated signalling, mitochondrial damage, protein oxidation and telomere-based DNA damage responses. Photodamaged skin displays variable epidermal thickness, dermal elastosis, decreased/fragmented collagen, increased matrix-degrading metalloproteinases, inflammatory infiltrates and vessel ectasia. The development of cosmetically pleasing sunscreens that protect against both UVA and UVB irradiation as well as products such as tretinoin that antagonize the UV signalling pathways leading to photoageing are major steps forward in preventing and reversing photoageing. Improved understanding of the skin's innate UV protective mechanisms has also given rise to several novel treatment concepts that promise to revolutionize this field within the coming decade. Such advances should not only allow for the improved appearance of skin in middle age and beyond, but also greatly reduce the accompanying burden of skin cancer.

It was previously reported that telomere homologue oligonucleotides (T-oligos) can induce a variety of cellular responses in skin including increased melanogenesis. To assess the effects of T-oligos on hair pigmentation, we administered thymidine dinucleotide (pTT), one-third of the TTAGGG telomere repeat sequence, intradermally at distinct time points of the depilation-induced hair cycle in C3H/HeJ mice. Penetration of T-oligos into the hair follicle (HF) was monitored by using FITC-labelled pTT and confocal microscopy. pTT treatment on days 1-5 after depilation, during early anagen, did not significantly alter the number and proliferation of melanocytes (Trp-2-positive cells), compared with vehicle-treated controls. However, pTT treatment on days 5-12 after depilation, during mid- to late anagen, resulted in the formation of darker hairs, that showed a significantly increased eumelanin/total melanin ratio in their sub-apical agouti band region, compared with vehicle-treated controls (P < 0.05). By RT-PCR and western blot, full thickness skin of pTT-treated mice showed increases in Trp-1, Trp-2 and tyrosinase mRNA and protein levels, compared with control mice. Western blot analyses of two receptors that positively regulate eumelanogenesis, melanocortin type 1 receptor (MC-1R) and kit, showed increased expression of MC-1R protein in pTT-treated versus control skin, while the levels of c-kit receptor remained unchanged. These data demonstrate that pTT treatment increases eumelanogenesis in HFs, associated with increased tyrosinase, TRP-1 and MC-1R expression. These data also raise the possibility of using T-oligos to modulate hair pigmentation.

Ultraviolet (UV) radiation is a proven carcinogen, responsible for more than half of all human malignancies. It also compromises skin appearance and function. Since the UV action spectra for DNA damage, skin cancer and Vitamin D(3) (vit D) photosynthesis are identical and vit D is readily available from oral supplements, why has sun protection become controversial, now that some data suggest conventionally "sufficient" levels of vit D may be less than optimal for at least some population groups? First, the media and apparently some researchers are hungry for a new message. Nevertheless, after 50 years, UV exposure is still a major avoidable health hazard. Second, the controversy is fueled by a powerful special interest group: the indoor tanning industry. They target not the frail elderly or inner-city ethnic minorities, groups for whom evidence of vit D insufficiency is strongest, but rather fair-skinned teenagers and young adults, those at highest risk of UV photodamage. Third, evolution does not keep pace with civilization. When nature gave man the appealing capacity for vit D photosynthesis, the expected lifespan was far less than 40 years. Long-term photodamage was not a concern, and vit D was not available at the corner store. The medical community should avoid sensationalism and instead rigorously explore possible cause-and-effect relationships between vit D status and specific diseases while advocating the safest possible means of assuring vit D sufficiency.

We report the synthesis and application of an elastic, wearable crosslinked polymer layer (XPL) that mimics the properties of normal, youthful skin. XPL is made of a tunable polysiloxane-based material that can be engineered with specific elasticity, contractility, adhesion, tensile strength and occlusivity. XPL can be topically applied, rapidly curing at the skin interface without the need for heat- or light-mediated activation. In a pilot human study, we examined the performance of a prototype XPL that has a tensile modulus matching normal skin responses at low strain (

In vivo reflectance confocal microscopy (RCM) provides high-resolution optical sections of the skin in its native state, without needing to fix or section the tissue. Melanin provides an excellent contrast for RCM, giving a bright signal in the confocal images. The pigmented guinea-pig is a common animal model to study human pigment induction and modulation, as its tanning response is comparable to human tanning after exposure to ultraviolet radiation (UVR). We investigated the applicability of RCM to detecting UVR-induced pigmentary changes in this model. Animals were exposed to solar simulator radiation for 7 days. RCM was performed during the irradiation and follow-up period. Compared to non-irradiated skin, an increase in melanocyte size, dendricity, and number, as well as increased pigment in keratinocytes, was seen in the irradiated epidermis. Interestingly, these changes could be detected even before a tanning response was clinically visible. UVR-induced epidermal hyperplasia could also be detected and quantified. In conclusion, in vivo RCM is a sensitive non-invasive imaging technique that can repeatedly measure epidermal pigmentation and thickness, as demonstrated in the guinea-pig model. This technique should greatly enhance our appreciation of dynamic pigmentary changes in human or animal skin over time and in response to specific stimuli.

When I began training in dermatology in the 1970s, I was impressed with the rapidly increasing depth and breadth of cutaneous research and eager to participate in what I imagined would soon be a revolution in the diagnosis and management of skin disease. I was fortunate to train in a department in which recent progress in human photobiology was being translated rapidly into an effective new therapy for psoriasis, mycosis fungoides, and other T cell–mediated dermatoses (Parrish, 2012). I even had the good fortune to spend one year of my residency as the photobiology fellow, participating in clinical research and developing new therapies that became immediately available to patients (Gilchrest et al., 1976, 1977, 1979). The experience was highly gratifying and motivated me to obtain laboratory-based training following my residency to prepare for a career bridging basic discoveries at the cellular and molecular level with improvements in patient care. Limited only by my imagination and work capacity, I embarked on just such a career.

Many older individuals use products and procedures to conceal or delay the signs of aging. For most, this provides a helpful ego boost, but some suffer from pathologies such as eating disorders and body dysmorphic disorder. The impact of aging skin may include social anxiety and social isolation. Poor self-image is associated with chronic illness and fewer preventive health behaviors, such as exercise. Aged appearance, especially in women, is also associated with workplace discrimination. Patients should therefore be offered treatments for aging skin, ensured that society's negative views not be unnecessarily reinforced, and maintain a realistic treatment expectations.

Hair pigmentation is controlled by tightly coordinated programs of melanin synthesis and involves signaling through the melanocortin type 1 receptor (MC-1R) that regulates the switch between pheomelanogenesis and eumelanogenesis. However, the involvement of other signaling systems, including the bone morphogenetic protein (BMP) pathway, in the control of hair pigmentation remains to be elucidated. To assess the effects of BMP signaling on hair pigmentation, transgenic mice overexpressing the BMP antagonist noggin (promoter: keratin 5) were generated. Whereas wild-type C3H/HeJ mice have a subapical yellow band on otherwise black dorsal hairs, K5-Noggin mice are characterized by the absence of a yellow band and near-black pigment in dorsal coat. Noggin overexpression is accompanied by strongly reduced levels of Agouti signal protein and enhanced expression of microphthalmia transcription factor in the midphase of the hair-growth cycle. Wild-type color in K5-Noggin mice is restored by administration of a synthetic MC-1R antagonist resulting in the reappearance of a subapical yellow band. BMP-4 stimulates the expression of Agouti transcripts and protein in primary epidermal keratinocytes, and BMP signaling positively regulates dermal papilla-specific enhancer of the Agouti gene in primary dermal fibroblasts. Taken together, these data suggests that BMP signaling controls the expression of Agouti protein in the hair follicle and provide evidence for interaction between BMP and MC-1R signaling pathways to modulate the balance between pheomelanogenesis and eumelanogenesis during hair growth.

Cellular senescence is a major defense against cancer. In human fibroblasts, suppressing both the p53 and pRb pathways is necessary to bypass replicative senescence as well as senescence induced by ectopic expression of a dominant negative form of the telomere repeat binding factor 2, TRF2DN. We recently reported that exposure to oligonucleotides homologous to the telomere 3′ overhang (T-oligos) activates both the p53 and pRb pathways and leads to senescence in primary human fibroblasts. To further characterize T-oligo-induced senescence, we compared established isogenic fibroblast cell lines lacking functional p53 and/or pRb pathways to the normal parental line. Here, we report that, as in physiologic senescence, inactivation of both the p53 and pRb pathways is necessary to suppress T-oligo-induced senescence. Moreover, T-oligo rapidly induces senescence in a malignant fibroblast-derived cell line, demonstrating the potential of using T-oligo as a novel anticancer therapeutic. Our data support the hypothesis that exposure of the TTAGGG tandem repeat telomere 3′ overhang sequence is the event that initiates signaling through DNA damage response pathways after experimental telomere disruption, serial passage, or acute genomic damage of normal cells.

Melanoma is the most fatal skin cancer, often highly resistant to chemotherapy. Here we show that treatment with an 11-base DNA oligonucleotide homologous to the telomere 3' overhang sequence (T-oligo) induces apoptosis of several established human melanoma cell lines, including the aggressive MM-AN line, whereas normal human melanocytes exposed to the same or higher T-oligo concentrations show only transient cell cycle arrest, implying that malignant cells are more sensitive to T-oligo effects. When MM-AN cells were briefly exposed to T-oligo in culture and injected into the flank or tail vein of SCID mice, eventual tumor volume and number of metastases were reduced 85-95% compared with control mice. Similarly, T-oligos administered intralesionally or systemically selectively inhibited the growth of previously established MM-AN tumor nodules in the flank and peritoneal cavity by 85 to 90% without detectable toxicity. We previously showed that T-oligos act through ATM, p95/Nbs1, E2F1, p16INK4A, p53, and the p53 homologue p73 to modulate downstream effectors and now additionally demonstrate striking down-regulation of the inhibitor of apoptosis protein livin/ML-IAP. We suggest that T-oligo mimics a physiologic DNA damage signal that is frequently masked in malignant cells and thereby activates innate cancer prevention responses. T-oligos may provide a novel therapeutic approach to melanoma.

Protein kinase C (PKC), a family of at least eleven isoforms, mediates numerous cell functions. In human melanocytes, alpha, beta, delta, epsilon and zeta isoforms of PKC are expressed, but uniquely PKC-beta activates tyrosinase, the key and the rate-limiting enzyme in melanogenesis, by phosphorylating specific serine residues on its cytoplasmic domain. To investigate the mechanism by which only PKC-beta phosphorylates tyrosinase, we examined the expression of receptor for activated C-kinase-I (RACK-I), a receptor specific for activated PKC-beta, on the surface of melanosomes, the specialized organelle in which melanogenesis occurs. Immunoblot analysis of purified melanosomes revealed that RACK-I is readily detectable. Immunoprecipitation of RACK-I from purified melanosomes, followed by immunoblot analysis using antibody against PKC-beta, revealed abundant PKC-beta, whereas PKC-alpha was not detected when immunoblot analysis was performed using antibody against PKC-alpha. Activation of PKC in melanocytes increased the level of PKC-beta co-immunoprecipitated with RACK-I, while the level of melanosome-associated RACK-I decreased when melanocytes were treated chronically with the 12-0-tetradecanoyl-phorbol 13-Acetate (TPA), a condition known to deplete PKC and reduce tyrosinase activity. Immunoprecipitation with RACK-I antibody co-precipitated fewer PKC-beta in the presence of UV-activated 1, 1'-decamethylenebis-4-aminoquinaldinium di-iodide (DECA), known to disrupt the interaction between activated PKC-beta and RACK-I. Treatment of intact melanocytes with DECA also decreased tyrosinase activity. Moreover, suppression of RACK-I expression by transfecting melanocytes with siRNA against RACK-I reduced the basal tyrosinase activity and blocked TPA-induced increases in tyrosinase activity. Taken together, these results demonstrate that RACK-I anchors activated PKC-beta on the melanosome membrane, allowing PKC-beta to phosphorylate tyrosinase.

To determine whether inhibition of PKC-beta activity decreases pigmentation, paired cultures of primary human melanocytes were first pretreated with bisindolylmaleimide (Bis), a selective PKC inhibitor, or vehicle alone for 30 min, and then treated with TPA for an additional 90 min to activate PKC in the presence of Bis. Bis blocked the expected induction of tyrosinase activity by activation of PKC. Addition of a peptide corresponding to amino acids 501-511 of tyrosinase containing its PKC-beta phosphorylation site, a presumptive PKC-beta pseudosubstrate, gave similar results. To determine whether Bis reduces pigmentation in vivo, the backs of four shaved and depilated pigmented guinea pigs were UV irradiated with a solar simulator for 2 wk excluding weekends. Compared to vehicle alone, Bis (300 microM), applied twice daily to paired sites for various periods encompassing the irradiation period, decreased tanning. Bis also, although less strikingly, reduced basal epidermal melanin when topically applied twice daily, 5 d per wk, for 3 wk to shaved and depilated unirradiated skin. Moreover, topical application of Bis (100 microM) once daily for 9 d to the freshly depilated backs of 8-wk-old mice markedly lightened the color of regrowing hair. These results demonstrate that inhibiting PKC activity in vivo selectively blocks tanning and reduces basal pigmentation in the epidermis and in anagen hair shafts.

Exposure of skin to solar-simulated irradiation generates a multitude of adaptive responses including cytokine transcription, synthesis and secretion. Interleukin-1 (IL-1) is one of the cytokines induced in epidermal cells in response to UV irradiation. It displays a broad range of mitogenic and inflammatory activities including fibroblast proliferation and T-cell activation. There are two forms, IL-1alpha and IL-1beta; and IL-1alpha is the predominant form secreted by epidermal keratinocytes. UV-induced modulations of IL-1alpha message levels have been extensively studied within the first 48 h after irradiation, but longer term changes and impact on IL-1alpha cellular protein levels are virtually unexplored. We now report that cells of keratinocyte origin (SCC 12F) respond to a single physiologic dose of solar-simulated irradiation with both early (8 h) and late (72 h) peaks of IL-1alpha mRNA induction. UV-stimulated IL-1alpha secretion is increased above sham-irradiated control secretion for at least 96 h after irradiation. Our study provides evidence that UV-induced adaptive cutaneous responses persist for at least several days, and suggests that different mechanisms may mediate the early vs. late inductions.

To the Editor: One major risk factor for cutaneous melanoma is ultraviolet (UV) exposure. Intense intermittent UV exposure and childhood sunburn are linked epidemiologically with melanoma risk, and in mice neonatal UV exposure promotes development of cutaneous melanoma (Noonan et al., 2001; Kannan et al., 2003). Other evidence that UV contributes to melanomagenesis includes increased risk for populations with extensive intense sun exposure, as well as for fair-skinned (Fitzpatrick’s skin type I–II) individuals and patients with xeroderma pigmentosum, who repair photoproducts very poorly (Gilchrest et al., 1999; Kraemer et al., 1987). We have previously shown that telomere homolog oligonucleotides (collectively called T-oligos), but not complementary or unrelated control oligonucleotides, have multiple anti-cancer effects (Eller et al., 1997; Puri et al., 2004; Gilchrest and Eller, 2009). T-oligo treatment prior to UV irradiation accelerates the removal of major UV-induced cyclobutane pyrimidine dimers (CPDs) and 6-4-photoproducts in cultured cells from newborn and adult donors (Goukassian et al., 2002), murine skin in vivo (Goukassian et al., 2004; Arad et al., 2008), and human skin ex-vivo (Arad et al., 2007). T-oligos have also been shown to cause cell cycle arrest, followed in many malignant cell types by apoptosis (Puri et al. 2004; Gilchrest and Eller, 2009). We have previously shown that treatment with T-oligos (specifically, thymidine dinucleotide - pTT) during chronic UV irradiation prevents development of SCC in hairless mice (Goukassian et al., 2004) and of BCC in Ptch-1+/− mice (Arad et al., 2008). In these models, intermittent topical pTT application enhances DNA repair of CPDs and 8-oxo-2′-deoxyguanosine, decreases mutagenesis, and in tumor nodules increases apoptosis and decreases proliferation. pTT also strikingly reduces Cox-2 protein expression in UV-irradiated skin (Arad et al., 2008). Many mutations associated with familial melanoma occur at the CDKN2A locus that encodes two distinct proteins, p16 INK4a and p14 ARF (p19 ARF in mice) (Chudnovsky et al., 2005). Several knockout (KO) and transgenic animal models have been developed to study p16- and p19-dependent molecular mechanisms of melanoma development. Of interest to modeling human melanomagenesis, when p19ARF−/− mice expressing H-ras driven by a tyrosinase promoter (Tyr-Hras/p19KO mice) are UV irradiated on day 2 or 3 after birth, there is a significant increase in melanoma development during early adulthood (Kannan et al., 2003). In this study we evaluated the effect of topical pTT treatment in this model. Newborn mice were treated topically with a 100 µM solution of pTT (Midland Certified Reagent Company, Midland, TX) or the PG/DMSO vehicle alone on days 1 and 2, then UV irradiated on day 3 using FS40 sunlamps (10 mJ/cm2) as metered at 285±5mm, an irradiation protocol known to cause melanomas by week 21 in approximately half the mice (Kannan et al., 2003). pTT-treated mice began to develop melanomas during week 12, while vehicle-treated mice began to develop tumors during week 7; and by week 21, 71% vs 46% of the mice remained tumor free (Fig. 1A). All mice were examined weekly and sacrificed if their tumors were ≥1 cm in diameter or the animals appeared to be in distress. As well, in each treatment group, 4 mice died during weeks 15–17 for unclear reasons without evidence of tumor. Excluding these mice,at the end of 21 weeks, 13 of 24 vehicle-treated mice had tumors and 9 (69% of tumor-bearing mice) had to be sacrificed early, beginning at 11 weeks; but only 7 of the remaining 24 pTT-treated mice developed tumors and only one (14% of tumor-bearing mice) had to be sacrificed early, at 16 weeks (Fig. 1A, 1B and Table). Vehicle-treated mice had an average of 3 times as many tumors per mouse as pTT-treated mice at the end of 21 weeks (p 90% reduction. Figure 1 Effect of pTT on melanomagenesis in UV-irradiated Tyr-Hras/p19KO mice over the 21 week study. Open symbols (rectangles): pTT-treated mice; black symbols (diamonds): vehicle-treated mice. A: Cumulative tumor free survival. Compared to vehicle, pTT markedly ... Table Fate of Tyr-Hras/ p19KO Mice Tumor size and multiplicity for the mice that were sacrificed or died early were included in the calculations as unchanged up to the termination of the experiment on week 21. Hence their contribution to total tumor burden, as well as possibly tumor multiplicity in these mice, is understated, as tumors would have been expected to continue growing. All tumors on surviving animals continued to grow throughout the study, and new tumors continued to appear. Most of the tumors were poorly-differentiated with a spindle-cell morphology; and all harvested tumors stained positively for Mart-1 (data not shown), confirming that they were indeed melanomas, despite being amelanotic in these albino mice. Our data demonstrate that topical pTT application to newborn melanoma-prone mice only twice prior to UV irradiation delays and reduces subsequent melanoma development. This effect is consistent with the previously documented ability of pTT to increase the rate and accuracy of UV-induced DNA damage repair when provided to cultured cells or to intact skin 24–48 hours prior to irradiation. One presumptive pTT target is the melanocytic stem cell that resides in the upper permanent portion of the hair follicle (Nishimura, 2011; Nishikawa-Torikai et al., 2011). These cells are understood to persist after the single irradiation on post-natal day 3 through multiple cycles of cell division and ultimately to give rise, weeks later, to a melanoma. Reducing the number of UV-induced melanocyte mutations, as shown to occur in pTT-treated epidermal keratinocytes in irradiated mouse skin (Goukassian et al., 2004; Arad et al., 2008), should logically reduce the number of Tyr-Hras/p19KO cells further predisposed to give rise to invasive melanoma. In addition, pTT effects on non-melanocytic cells may also have contributed to the >90% reduction in melanoma burden. For example, pTT treatment presumably reduced epidermal expression of Cox-2, the UV-inducible pro-inflammatory enzyme that enhances development of keratinocytic malignancies in irradiated mouse skin (Fischer et al., 1999; Pentland et al., 1999) and whose protein expression is markedly reduced over at least several days by pTT (Arad et al., 2008; Marwaha et al., 2005). Cox-2 inhibitors have been shown in intervention studies to reduce UV-induced non-melanoma skin cancers (Elmets et al., 2010) and more recently also melanomas (Curiel-Lewandrowski et al., 2011) in humans. Finally, pTT is known to have immunomodulatory effects (Cruz et al., 2000; Curiel-Lewandrowski et al., 2003) and may have modified interferon-mediated events recently shown to enhance melanomagenesis in mice following UV irradiation in the newborn period (Zaidi et al., 2011). Of note, in human skin explants pTT treatment enhances epidermal pigmentation within 24 hours and this reduces initial UV-induced DNA damage by approximately 50% (Arad et al., 2007). This potentially additional photoprotective effect of pTT could not be observed in the albino mice used in these experiments. Together, our findings further support a critical role for UV-induced damage in melanoma development and for photoprotection as a key preventive strategy.

Contact of developing sensory organs with the external environment is established via the formation of openings in the skin. During eye development, eyelids first grow, fuse and finally reopen, thus providing access for visual information to the retina. Here, we show that eyelid opening is strongly inhibited in transgenic mice overexpressing the bone morphogenetic protein (BMP) antagonist noggin from the keratin 5 (K5) promoter in the epidermis. In wild-type mice, enhanced expression of the kinase-inactive form of BMPR-IB mediated by an adenovirus vector also inhibits eyelid opening. Noggin overexpression leads to reduction of apoptosis and retardation of cell differentiation in the eyelid epithelium, which is associated with downregulation of expression of the apoptotic receptors (Fas, p55 kDa TNFR), Id3 protein and keratinocyte differentiation markers (loricrin, involucrin). BMP-4, but not EGF or TGF-alpha, accelerates opening of the eyelid explants isolated from K5-Noggin transgenic mice when cultured ex vivo. These data suggest that the BMP signaling pathway plays an important role in regulation of genetic programs of eyelid opening and skin remodeling during the final steps of eye morphogenesis.

The production of immunomodulatory cytokines such as interleukin-10 (IL-10) from keratinocytes and other target cells in the skin plays a crucial role in UV-induced immunosuppression. Substantial evidence supports an association between DNA damage and immunomodulation. It is also known that small DNA fragments such as thymidine dinucleotides (pTpT) can mimic several UV-induced effects, including inhibition of the induction phase of the contact hypersensitivity response and up-regulation of tumor necrosis factor-alpha (TNF-alpha). To determine whether pTpT also induces IL-10 secretion by keratinocytes, and by inference whether IL-10 production after UV irradiation is a response to DNA damage, we compared the effects of pTpT with those of UV irradiation on primary human keratinocyte cultures. Subconfluent cultures of primary human keratinocytes were treated either with 10 micro M or 100 micro M pTpT or diluent alone, or exposed to solar-simulated light (100 J/m2 of UVB) or sham irradiated. An increase in IL-10 mRNA expression was observed 6-24 h after irradiation and at 24-48 h after treatment with pTpT. Detection of secreted IL-10 protein coincided with up-regulation of IL-10 gene expression at 48 and 72 h as determined by ELISA. Conditioned media from human keratinocytes treated with pTpT, like that from irradiated cells, significantly inhibited lymphocyte proliferation in the allogeneic-mixed lymphocyte reaction (MLR) assay. To determine whether pTpT mimics the suppressive influence of UVB on the elicitation phase of contact hypersensitivity, believed to result largely from IL-10 release, we compared the effects of topical application of pTpT with those of UVB irradiation on C57Bl/6 mice sensitized with dinitrofluorobenzene. Sensitized mice treated with pTpT or UVB irradiation showed markedly suppressed elicitation of ear-swelling responses. These results demonstrate that increased keratinocyte IL-10 mRNA level and IL-10 protein release are among the effects of pTpT and support the hypothesis that pTpT treatment triggers many of the biologic effects of UV irradiation by mimicking UV-induced DNA damage. Finally, regardless of mechanism, the data suggest that topical treatment with pTpT may provide a novel means of suppressing contact hypersensitivity or other lymphocyte-mediated reactions in skin.