Melanocyte Precursors Research Articles

Druggable targets in pediatric neurocutaneous melanocytosis: Molecular and drug sensitivity studies in xenograft and ex vivo tumor cell culture to identify agents for therapy.

Neurocutaneous melanocytosis (NCM) is a rare congenital disorder that presents with pigmented cell lesions of the brain or leptomeninges in children with large or multiple congenital melanocytic nevi. Although the exact pathological processes involved are currently unclear, NCM appears to arise from an abnormal development of melanoblasts or melanocyte precursors. Currently, it has an extremely poor prognosis due to rapid disease progression and lack of effective treatment modalities. In this study, we report on an experimental approach to examining NCM cells by establishing subcutaneous tumors in nude mice, which can be further expanded for conducting molecular and drug sensitivity experiments. Analysis of the NRAS gene-coding sequences of an established NCM cell line (YP-MEL) and NCM patient cells revealed heterogeneity in NRAS Q61K that activated mutation and possibly consequential differential sensitivity to MEK inhibition. Gene expression studies were performed to compare the molecular profiles of NCM cells with normal skin fibroblasts. In vitro cytotoxicity screens of libraries of targeted small-molecule inhibitors revealed prospective agents for further evaluation. Our studies provide an experimental platform for the generation of NCM cells for preclinical studies and the production of molecular and in vitro data with which to identify druggable targets for the treatment.

Primary Gastric Melanoma: Wait, I Thought the Stomach Did Not Have Any Melanocytes!

Introduction: We report a case of a patient who presented with abdominal pain and decreased ability to tolerate food, and was found to have melanoma in the gastric mucosa. We highlight the difficulty of diagnosing primary gastric melanoma with certainty, given its rarity and theoretical implausibility, as normal gastric epithelium lacks melanocytes. We also briefly review several pathophysiologic and embryologic hypotheses for this phenomenon. A 50-year-old Hispanic male presented to our clinic with epigastric pain and decreased ability to tolerate food. An EGD revealed a large, irregular ulcerated gastric mass extending 1.5 to 2 cm from the gastroesophageal junction to the antrum. The mass was biopsied, and immunohistochemical stains showed the tumor cells were positive for melan-A, HMB45 and vimentin, confirming melanoma. A thorough search for another possible primary lesion, consisting of a complete skin examination and positron emission tomography (PET), did not identify a clear primary site of melanoma. The PET scan revealed uptake involving the gastric walls and lymph nodes in the perigastric, retroperitoneal and left supraclavicular regions. BRAF analysis was positive for a V600 mutation. The patient subsequently underwent palliative jejunostomy placement and was evaluated by medical oncology. Primary gastric melanoma is extremely rare (with only a handful of cases reported in the literature), likely due to the lack of melanocytes in normal gastric epithelium. Therefore, when melanoma is encountered in the stomach, it is crucial to search for a primary melanocytic lesion that may have metastasized to the stomach. While gastrointestinal involvement by melanoma has been found in up to 60% of autopsies of patients who have died with primary cutaneous melanoma, melanoma located primarily in the gastric mucosa is an uncommon occurrence. Several pathophysiologic mechanisms have been suggested. Ectopic migration of melanocyte precursors or differentiation of the APUD cells to melanocytes have been suggested as possible mechanisms. Spontaneous regression of primary melanoma has also been recognized, and it is possible that our patient’s gastric melanoma may have represented a metastasis from primary lesion that had spontaneously regressed. The median overall survival for primary melanoma of the GI tract is 17 months; for primary melanoma of the stomach, 5 months. There is little consensus on therapy for patients with primary GI melanoma, but treatment with complete excision may provide long-term disease-free survival. Vemurafenib, a BRAF kinase inhibitor, has been shown to improve both overall and progression-free survival in patients with metastatic melanoma with the BRAF V600E mutation

Abstract 5352: Ultraviolet radiation induces carcinogenesis in a novel transgenic mouse model of melanoma

Abstract Melanomagenesis is caused by environmental factors, such as ultraviolet (UV) radiation, as well as genetic factors. The Nucleotide Excision Repair (NER) pathway repairs photoproducts induced by UV radiation. In humans, NER pathway disruption leads to the development of skin cancers, including melanoma. Upon UV exposure, mice deficient in Xpc, an important protein in the NER pathway, develop skin cancer; but not melanoma. The Endothelin 3 (Edn3) signaling pathway is essential for melanocyte precursor cell development. In humans, this pathway has also been associated with melanoma progression and its metastatic potential. The purpose of this study is the development of a UV-dependent melanoma mouse model that combines Xpc deficiency with the over-activation of the Edn3 pathway. Transgenic mice over-expressing Edn3 under the control of the keratin 5 promoter (K5-Edn3) and carrying a targeted mutation in Xpc were exposed to a single suberythemal neonatal dose of UV radiation. Immunostaining and histomorphology were used to confirm the melanocytic origin of primary skin tumors. Melanomas were only found in animals with the K5-Edn3 transgene. High penetrance was observed in Xpc null (67%, n=3) and Xpc heterozygous (67%, n=3) mice in comparison to Xpc wild type (33%, n=3) mice. RNA extracted from melanomas of Xpc null (n=2), Xpc heterozygous (n=1) and Xpc wild type (n=1) mice were screened for UV signature mutations in Hras, Kras and Nras. No mutations were found in any of these genes. These results indicate that UV radiation exposure, in conjunction with over-activation of the Edn3 pathway is sufficient to lead to melanomagenesis in mice independently of mutations in Hras, Kras or Nras. Citation Format: Ana Paula Benaduce, Deannys Batista, Gabriel Grilo, Karen Jorge, Diana Cardero, Clara Milikowski, Lidia Kos. Ultraviolet radiation induces carcinogenesis in a novel transgenic mouse model of melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5352. doi:10.1158/1538-7445.AM2014-5352

Tenascin-C is required for normal Wnt/β-catenin signaling in the whisker follicle stem cell niche

Whisker follicles have multiple stem cell niches, including epidermal stem cells in the bulge as well as neural crest-derived stem cells and mast cell progenitors in the trabecular region. The neural crest-derived stem cells are a pool of melanocyte precursors. Previously, we found that the extracellular matrix glycoproteins tenascin-C and tenascin-W are expressed near CD34-positive cells in the trabecular stem cell niche of mouse whisker follicles. Here, we analyzed whiskers from tenascin-C knockout mice and found intrafollicular adipocytes and supernumerary mast cells. As Wnt/β-catenin signaling promotes melanogenesis and suppresses the differentiation of adipocytes and mast cells, we analyzed β-catenin subcellular localization in the trabecular niche. We found cytoplasmic and nuclear β-catenin in wild-type mice reflecting active Wnt/β-catenin signaling, whereas β-catenin in tenascin-C knockout mice was mostly cell membrane-associated and thus transcriptionally inactive. Furthermore, cells expressing the Wnt/β-catenin target gene cyclin D1 were enriched in the CD34-positive niches of wild-type compared to tenascin-C knockout mice. We then tested the effects of tenascins on this signaling pathway. We found that tenascin-C and tenascin-W can be co-precipitated with Wnt3a. In vitro, substrate bound tenascins promoted β-catenin-mediated transcription in the presence of Wnt3a, presumably due to the sequestration and concentration of Wnt3a near the cell surface. We conclude that the presence of tenascin-C in whiskers assures active Wnt/β-catenin signaling in the niche thereby maintaining the stem cell pool and suppressing aberrant differentiation, while in the knockout mice with reduced Wnt/β-catenin signaling, stem cells from the trabecular niche can differentiate into ectopic adipocytes and mast cells.

Normal pigment production

AbstractMelanin is synthesized in melanocytes and retinal pigment epithelial cells. Two main types of melanin are produced: eumelanin (brown, black) and phaeomelanin (yellow, orange). Pigmentation is a complex process involving various steps from normal development of melanocytes to the synthesis of melanin itself. In the skin and choroid, precursors of melanocytes are melanoblasts which are derived from the neural crest. Retinal pigment epithelium cells are generated directly from the optic neuroepithelium. Melanoblasts migrate and colonize the epidermis, hair follicles, and ocular, ear and nervous structures. The differentiation of melanoblasts into melanocytes is controlled by numerous transcription factors and signalling pathways, and is characterized by the acquisition of Tyrosinase activity, the presence of mature melanosomes, and the formation of dendrites. Melanin production results from catalytic activities by Tyrosinase, TRP‐1 and TRP‐2 and occurs in melanosomes which follow a maturation process involving four stages (I – IV). Melanosomes are transported from the perinuclear region to the end of melanocytic dentrites, where they are eventually transferred to keratinocytes.

Prevention of hair graying by factors that promote the growth and differentiation of melanocytes.

Epidermal melanocyte precursors migrate into developing hair follicles to form the melanocyte stem cell system required to supply pigmented melanocytes necessary for hair pigmentation in repetitive hair cycles. Hair graying is caused by irreversible defects in the self-renewal and/or development of follicular melanocyte stem cells in the hair follicles. To investigate the mechanism(s) of hair graying during the normal aging process, we established a hair graying model in mice by repeatedly plucking or shaving trunk hairs. We repeatedly plucked or shaved trunk hairs to induce and accelerate the hair graying and counted the gray hairs. By using this functional model of hair graying in mice, we assessed the effects of genes known to affect melanocyte development, such as Kitl, hepatocyte growth factor (HGF) and endotheline 3 (ET3). After increasing the total numbers of cumulative hair cycles by plucking or shaving, we observed a significant increase in the gray hair of C57BL/6 mice. Kitl expression in the skin was the most effective for preventing hair graying and a significant effect was also confirmed for HGF and ET3 expression. The repeated hair plucking or shaving led to hair graying without any genetic lesion. Kitl is a more effective factor for prevention of hair graying than HGF or ET3. Our simple model of hair graying may provide a basic tool for screening the molecules or reagents preventing the progression of hair graying.

Ex vivo culture of mouse embryonic skin and live-imaging of melanoblast migration.

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles(1,2). Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture(3-6). Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue(3,6). High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture(6). By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6 cultures in parallel.

The roles of Frizzled-3 and Wnt3a on melanocyte development: In vitro studies on neural crest cells and melanocyte precursor cell lines

BackgroundWnt3a and Frizzled-3 are both expressed in the dorsal neural tube that gives rise to the neural crest in Xenopus, zebrafish and mice. Melanocytes originate from the neural crest (NC) and postnatally, melanocyte stem cells reside in the hair follicle bulge and in the dermis. However, the roles of Wnt3a and Frizzled-3 in melanocyte development have not been clarified. ObjectiveThe aim of this study was to delineate the expression of Frizzled-3 in murine melanocyte lineage and human melanocytes, and to study the effects of Wnt3a on melanocyte development at various stages. MethodsMurine NC explant cultures and three NC-derived melanocyte lineage cell lines, including NCCmelb4M5 (Kit− melanocyte precursors), NCCmelb4 (Kit+ melanoblasts) and NCCmelan5 (differentiated melanocytes), and human epidermal melanocytes were treated with pure recombinant Wnt3a protein and their cell behaviors were analyzed including their proliferation, Kit expression, tyrosinase (Tyr) activity, melanin production, dendrite formation and migration. ResultsFrizzled-3 was expressed in Tyr-related protein (TRP)-1+ cells in NC explant cultures, in all 3 melanocyte precursor cell lines and in human melanocytes. Wnt3a increased the population of TRP-1+ cells, the number of L-3,4-dihydroxyphenylalanine (DOPA)+ cells and dendrite formation in NC explant cultures. Wnt3a stimulated the proliferation of all 3 melanocyte precursor cell lines in a dose-dependent manner and also stimulated human melanocyte proliferation. Moreover, Wnt3a increased Tyr activity and melanin content of differentiated melanocytes, but did not activate Tyr activity in melanoblasts. Wnt3a stimulated dendrite formation in differentiated melanocytes, but not in melanoblasts. Wnt3a did not affect melanoblast or melanocyte migration. Wnt3a did not induce c-Kit expression in Kit− NCCmelb4M5 cells and did not affect c-Kit expression in any cell line tested. ConclusionsFrizzled-3 is constitutively expressed in murine melanocyte precursors, melanocytes and human melanocytes. Wnt3a and Frizzled-3 signalings play important roles in regulating the proliferation and differentiation of murine NCCs and various developmental stages of melanocyte precursors. The effect of Wnt3a on human melanocytes is similar to its effects on murine melanocytes. Therefore Wnt3a/Frizzled-3 signaling is a promising target for human melanocyte regeneration.

Premature Graying as a Consequence of Compromised Antioxidant Activity in Hair Bulb Melanocytes and Their Precursors

Intricate coordinated mechanisms that govern the synchrony of hair growth and melanin synthesis remain largely unclear. These two events can be uncoupled in prematurely gray hair, probably due to oxidative insults that lead to the death of oxidative stress-sensitive melanocytes. In this study, we examined the gene expression profiles of middle (bulge) and lower (hair bulb) segments that had been micro-dissected from unpigmented and from normally pigmented hair follicles from the same donors using quantitative real-time RT-PCR (qPCR) arrays. We found a significant down-regulation of melanogenesis-related genes (TYR, TYRP1, MITF, PAX3, POMC) in unpigmented hair bulbs and of marker genes typical for melanocyte precursor cells (PAX3, SOX10, DCT) in unpigmented mid-segments compared with their pigmented analogues. qPCR, western blotting and spin trapping assays revealed that catalase protein expression and hydroxyl radical scavenging activities are strongly repressed in unpigmented hair follicles. These data provide the first clear evidence that compromised antioxidant activity in gray hair follicles simultaneously affects mature hair bulb melanocytes and their immature precursor cells in the bulge region.

IN VITRO PROPERTIES OF NEURAL CREST-DERIVED MULTIPOTENT STEM CELLS FROM A BULGE REGION OF WHISKER FOLLICLE

71 The neural crest (NC), a transient structure in embryo development, is unique to vertebrates. NC is derived from the ectoderm and formed at the border of neuroectoderm and somatic ectoderm. Originally NC is represented by a cell population lying in the form of epithelial cords emerging from both sides of the neural tube. At the neurulation process end the NC cells undergo an epithelialmesenchymal transition (EMT) and migrate distantly in various tissues and organs of the developing embryo, where they give rise to the multiple differentiated cell types [1, 2]. In adult vertebrate organism the NC derivatives are represented by the following cell types: neurons and glial cells of the peripheral nervous system, the majority of sensory neurons, melanocytes, Merkel cells, odontoblasts, stromal and endothelial cells of cornea, C cells of thyroid gland, the adrenal medulla, a part of thymic stroma [1, 2]. The most plastic are the cells of cranial NC. In addition to the above mentioned differentiated cell types, they form a part of the bone, cartilage and connective tissues of the face, forehead and ventral neck [3, 4]. Unusually wide differentiation potential of the NC cells suggests an existence of multipotent stem cells. Stemple and Anderson firstly revealed the existence of multipotent stem cells in the NC in early embryonic stage of mammalian development [5]. A long time it was thought that, like NC per se, neural crestderived multipotent stem cells (NC-MSCs) have a transient nature. Consequently, the NCMSCs exist only at the premigratory (from the NC specification moment until the beginning of its cell migration) and migratory stages (from the NC cell delamination until their incoming into the sites of final destination), and further, within migration to sites of its final localization, undergo a process of commitment, turning into the progenitor cells with limited proliferative and/or differentiation potential [6, 7]. An example is the transition of NC-MSCs into the melanocyte precursors — melanoblasts. In the last decades, NC-derived cells have been isolated from a number of tissues and organs of the adult mammalians. These cells demonstrate the ability to self-renewal and multilineage differentiation, at least in vitro, i.e., they are multipotent stem cells [8–11]. The origin of these cells from NC was proved by creating special lines of transgenic mice [12, 13], as well as by means of their transplantation in the developing chick embryo, where the NC-MSCs migrated via the UDC 576.3+612.014.2/3

Prevalence and implications of TERT promoter mutation in uveal and conjunctival melanoma and in benign and premalignant conjunctival melanocytic lesions.

Hot-spot mutations in the promoter region of telomerase reverse transcriptase (TERT promoter mutations) occur frequently in cutaneous and conjunctival melanoma and are exceedingly rare in uveal melanoma. No information is available on the presence of these mutations in the conjunctival melanocytic precursor lesion primary acquired melanosis (PAM). We tested a cohort of uveal and conjunctival melanomas as well as conjunctival benign and premalignant melanocytic lesions for TERT promoter mutations in order to elucidate the role of these mutations in tumor progression. TERT promoter mutation analysis on fresh tumor DNA and DNA from formalin-fixed, paraffin-embedded specimens was performed by SNaPshot analysis in 102 uveal melanomas, 39 conjunctival melanomas, 26 PAM with atypia, 14 PAM without atypia, and 56 conjunctival nevi. Mutations of the TERT promoter were not identified in conjunctival nevi or PAM without atypia, but were detected in 2/25 (8%) of PAM with atypia and 16/39 (41%) of conjunctival melanomas. A single TERT promoter mutation was detected in 102 uveal melanomas (1%). We present the second documented case of TERT promoter mutation in uveal melanoma. In comparison with other types of melanoma, TERT promoter mutations occur at extremely low frequency in uveal melanoma. TERT promoter mutations are frequent in conjunctival melanoma and occur at lower frequency in PAM with atypia but were not detected in benign conjunctival melanocytic lesions. These findings favor a pathogenetic tumor progression role for TERT promoter mutations in conjunctival melanocytic lesions.

Behavior of Hair Follicles in Vitiligo: Clinical Presentation and Discussion

The color of the hair is dependent on the existence and function of the melanin producing cells, the melanocytes, in the hair matrix of the bulb. In vitiligo, the loss of color of the affected skin is due to loss of epidermal melanocytes, which may also be accompanied by loss of the bulbar melanocytes with subsequent loss of the hair color. Lymphocytic immune-cytotoxic process maybe involved in the destruction of epidermal or bulbar melanocytes. Hair depigmentation is said to follow surface depigmentation in long standing disease. This delay in development of leukotrichia in vitiligo could be claimed to the difference in melanization nature of hair follicle in comparison to epidermal melanization process. In rare cases, follicular melanocytes can be primarily destroyed in vitiligo without destruction of epidermal melanocytes. Apart from mature melanocytes, the hair follicles possess also immature form of melanocytes residing in the bulge area, outer root sheath, dermal papilla and sometimes the hair matrix. These immature cells usually escape the vitiligo immune destructive process. The existence of leukotrichia used to be a sign of disease recalcitrance and expected treatment failure. In such cases several surgical procedures were introduced to help repigmenting the skin. It was found that activation of the immature melanocytic precursors in hair follicles is responsible for the repigmentation of epidermis and hair follicles due to the supply with new mature melanocytes.

A Newborn With Neurocutaneous Melanocytosis and Dandy-Walker Malformation

BackgroundNeurocutaneous melanocytosis is a rare congenital dysplasia of the neuroectodermal melanocyte precursor cells that leads to proliferation of melanin-producing cells in the skin and leptomeninges. PatientWe describe a newborn with a giant congenital melanocytic nevus on his back, buttocks, and thighs. His brain magnetic resonance imaging study revealed bilateral T1 hyperintense lesions in the cerebellum and in the amygdala, hydrocephalus, and a Blake's pouch cyst, consistent with neurocutaneous melanocytosis and Dandy-Walker malformation. ConclusionNeurocutaneous melanocytosis has a wide clinical spectrum that includes hydrocephalus, epilepsy, cranial nerve palsy, increased intracranial pressure, and sensorimotor deficits.

BMP4 is required for the initial expression of MITF in melanocyte precursor differentiation from embryonic stem cells

Although the differentiation of melanoblasts to melanocytes is known to depend on many distinct factors, it is still poorly understood which factors lead to the induction of melanoblasts. To determine which factors might induce melanoblasts, we examined a set of candidate factors for their ability to induce expression of MITF, a master regulator of melanoblast development, in an ES cell-based melanocyte differentiation system. It appears that BMP4 is capable of inducing MITF expression in stem cells. In contrast, a number of other factors normally implicated in the development of the melanocyte lineage, including WNT1, WNT3a, SCF, EDN3, IGF1, PDGF, and RA, cannot induce MITF expression. Nevertheless, BMP4 alone does not allow MITF-expressing precursors to become differentiated melanocytes, but the addition of EDN3 further promotes differentiation of the precursors into mature melanocytes. Our results support a model in which BMP4 induces MITF expression in pluripotent stem cells and EDN3 subsequently promotes differentiation of these MITF expressing cells along the melanocyte lineage.

Ectopic KIT Copy Number Variation Underlies Impaired Migration of Primordial Germ Cells Associated with Gonadal Hypoplasia in Cattle (Bos taurus)

Impaired migration of primordial germ cells during embryonic development causes hereditary gonadal hypoplasia in both sexes of Northern Finncattle and Swedish Mountain cattle. The affected gonads exhibit a lack of or, in rare cases, a reduced number of germ cells. Most affected animals present left-sided gonadal hypoplasia. However, right-sided and bilateral cases are also found. This type of gonadal hypoplasia prevails in animals with white coat colour. Previous studies indicated that gonadal hypoplasia is inherited in an autosomal recessive fashion with incomplete penetrance. In order to identify genetic regions underlying gonadal hypoplasia, a genome-wide association study (GWAS) and a copy number variation (CNV) analysis were performed with 94 animals, including 21 affected animals, using bovine 777,962 SNP arrays. The GWAS and CNV results revealed two significantly associated regions on bovine chromosomes (BTA) 29 and 6, respectively (P=2.19 x 10-13 and P=5.65 x 10-6). Subsequent cytogenetic and PCR analyses demonstrated that homozygosity of a ~500 kb chromosomal segment translocated from BTA6 to BTA29 (Cs29 allele) is the underlying genetic mechanism responsible for gonadal hypoplasia. The duplicated segment includes the KIT gene that is known to regulate the migration of germ cells and precursors of melanocytes. This duplication is also one of the two translocations associated with colour sidedness in various cattle breeds.

Optimization of the method for the culture of melanocyte precursors from hair follicles and their activation by 1,25-dihydroxyvitamin D3

The melanocytes in vitiligo repigmentation are derived predominantly from melanocyte precursors (MPs) present in the outer root sheath (ORS) of hair follicles. The methods currently used for culturing MPs are unstable, and the cultured cells have the capacity to produce melanin. These factors are problematic when conducting in vitro studies to investigate the mechanism of repigmentation. Although 1,25-dihydroxyvitamin D3 (VID) has been demonstrated to be highly effective in the treatment of vitiligo in the clinic, its precise mode of action has yet to be elucidated. In the present study, the method for the culture of MPs from the ORS of hair follicles was optimized and the ability of VID to activate MPs was investigated. The results suggested that the MPs cultured using the optimized method mainly exhibited bipolar morphology. The cells proliferated well and were negative for 3,4-dihydroxy-L-phenylalanine (DOPA) staining. Transmission electron microscopy revealed that the cytoplasm of the MPs contained numerous stage I and stage II melanosomes; however, stage III and IV melanosomes were not observed. Following VID treatment, the MPs showed increased dendritic morphology, the cells stained positive for DOPA and stage III and IV melanosomes appeared in the cells. Western blotting revealed that microphthalmia-associated transcription factor (MITF), tyrosinase (TYR), tyrosinase-related protein-1 (TRP-1) and TRP-2 were expressed in the MPs and that VID increased the expression levels of MITF, TYR and TRP-1. However, the levels of MITF, TYR and TRP-1 in the MPs prior to and following VID treatment were significantly lower compared with those in cultured epidermal melanocytes, while the levels of TRP-2 in these three groups were not significantly different. Subsequent to VID treatment, the TYR activity in the MPs increased significantly, as did the corresponding melanin levels. In conclusion, the present study successfully optimized the method for MP culture. The MPs demonstrated no significant TYR activity or melanin synthesis; therefore, the MP cultures exhibited the features of MPs in vivo. In addition, VID significantly promoted the differentiation of MPs.

Controlled insertional mutagenesis using a LINE-1 ( ORFeus ) gene-trap mouse model

A codon-optimized mouse LINE-1 element, ORFeus, exhibits dramatically higher retrotransposition frequencies compared with its native long interspersed element 1 counterpart. To establish a retrotransposon-mediated mouse model with regulatable and potent mutagenic capabilities, we generated a tetracycline (tet)-regulated ORFeus element harboring a gene-trap cassette. Here, we show that mice expressing tet-ORFeus broadly exhibit robust retrotransposition in somatic tissues when treated with doxycycline. Consistent with a significant mutagenic burden, we observed a reduced number of double transgenic animals when treated with high-level doxycycline during embryogenesis. Transgene induction in skin resulted in a white spotting phenotype due to somatic ORFeus-mediated mutations that likely disrupt melanocyte development. The data suggest a high level of transposition in melanocyte precursors and consequent mutation of genes important for melanoblast proliferation, differentiation, or migration. These findings reveal the utility of a retrotransposon-based mutagenesis system as an alternative to existing DNA transposon systems. Moreover, breeding these mice to different tet-transactivator/reversible tet-transactivator lines supports broad functionality of tet-ORFeus because of the potential for dose-dependent, tissue-specific, and temporal-specific mutagenesis.

Fascin 1 is transiently expressed in mouse melanoblasts during development and promotes migration and proliferation

Fascins, a family of actin-bundling proteins, are expressed in a spatially and temporally restricted manner during development and often in cancer. Fascin 1 has a clear role in cell migration in vitro, but its role in vivo in mammals is not well understood. Here, we investigate the role of fascin 1 in the melanocyte lineage and in melanoma cells. Fascin 1 knockout causes hypopigmentation in adult mice owing to migration and cell cycle progression defects in melanoblasts, the melanocyte precursor cell. Study of live embryo skin explants reveals that E14.5 fascin 1-null melanoblasts migrate slower, and generate fewer and thinner pseudopods. By contrast, fascin 1 expression drives faster migration and lamellipodia protrusion in melanocytes in vitro. In addition, fascin 1 depletion retards melanoblast proliferation in vivo and melanoma cell growth in vitro. These data indicate that fascin 1 not only promotes cell migration in mouse melanocytes but it also has a role in growth and cell cycle progression.

Fascin 1 is transiently expressed in mouse melanoblasts during development and promotes migration and proliferation

Fascins, a family of actin-bundling proteins, are expressed in a spatially and temporally restricted manner during development and often in cancer. Fascin 1 has a clear role in cell migration in vitro, but its role in vivo in mammals is not well understood. Here, we investigate the role of fascin 1 in the melanocyte lineage and in melanoma cells. Fascin 1 knockout causes hypopigmentation in adult mice owing to migration and cell cycle progression defects in melanoblasts, the melanocyte precursor cell. Study of live embryo skin explants reveals that E14.5 fascin 1-null melanoblasts migrate slower, and generate fewer and thinner pseudopods. By contrast, fascin 1 expression drives faster migration and lamellipodia protrusion in melanocytes in vitro. In addition, fascin 1 depletion retards melanoblast proliferation in vivo and melanoma cell growth in vitro. These data indicate that fascin 1 not only promotes cell migration in mouse melanocytes but it also has a role in growth and cell cycle progression.

Abstract 1276: UV radiation interacts with Endothelin 3 and loss of nucleotide excision repair pathway to promote melanomagenesis.

Abstract Melanomagenesis is influenced by the interaction of environmental (such as UV radiation) and genetic factors. The Xpa gene is important in the Nucleotide Excision Repair (NER) pathway. Similar to humans afflicted with Xeroderma Pigmentosum, which results from mutations in genes that play a role in NER, Xpa deficient mice show high sensitivity to UV light, leading to skin cancer development. However, Xpa deficient mice do not develop melanoma even upon UV exposure. The Endothelin 3 (Edn3) signaling pathway is essential for proliferation, survival and migration of melanocyte precursor cells. In humans, this pathway has also been implicated in melanoma progression and its metastatic potential. The purpose of this study is to develop a UV induced melanoma mouse model that combines Xpa deficiency with the over-activation of the Edn3 pathway. To this end, transgenic mice over-expressing Edn3 under the control of the keratin 5 promoter (K5-Edn3) and carrying a targeted mutation in Xpa were exposed to a single suberythemal neonatal dose of UV radiation. A subgroup of mice was also exposed to a second dose of UV radiation at 6 weeks of age. Histomorphology and immunostaining were used to confirm the melanocytic origin of primary skin tumors and metastases. Melanoma was only found in animals with the K5-Edn3 transgene. High penetrance was observed in animals exposed to one dose of UV radiation that were Xpa null (100%, n=3) when compared to Xpa heterozygous (73%, n=11) or Xpa wild type (43%, n=7). Enlarged and hyperpigmented lymph nodes were present in animals with melanoma skin lesions and these were diagnosed as local metastases. These results indicate that UV radiation exposure, in conjunction with over-activation of the Edn3 pathway is sufficient to lead to melanomagenesis in mice. Citation Format: Ana Paula Benaduce, Deannys Batista, Gabriel Grilo, Karen Jorge, Lidia Kos. UV radiation interacts with Endothelin 3 and loss of nucleotide excision repair pathway to promote melanomagenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1276. doi:10.1158/1538-7445.AM2013-1276