Ultraviolet Protection From a Patented Amino Acid Complex Technology.

OBJECTIVE:   This study aimed to investigate the ultraviolet (UV) protection/repair benefits of a patented Amino Acid Complex (AAComplex). METHODS: I) AAComplex was incubated with dermal fibroblasts, with/without UVA, and collagen I was measured with a GlasBoxPlus device. II) A lotion, with/without AAComplex (1%) was applied topically to skin explants, following UVA irradiation, and quantified for health-related biomarkers (TNFalpha, histamine, and MMP-1). III) A broad spectrum sunscreen with SPF 46 and a skincare serum containing AAComplex (2%) were assessed using epidermal equivalents, in the presence of UV irradiation, for effects on IL-1alpha, thymine dimers, Ki-67, filaggrin and Nrf2. RESULTS: I) Collagen I synthesis in dermal fibroblasts was significantly decreased after UVA compared to without UV. The presence of AAComplex prevented this decrease. II) UVA irradiation of skin explants increased histamine, TNFα, and MMP-1. Hydrocortisone aceponate cream significantly decreases all 3 biomarkers. AAComplex contained lotion also significantly decreased all 3 biomarkers, the no AAComplex control lotion only reduced histamine. III) With the regimen of sunscreen + AAComplex contained skincare serum, the significant reduction in IL-1alpha was observed along with a complete recovery of Ki-67 and stimulation of filaggrin and Nrf2T. No thymine dimer positive cell was observed indicating the most positive skin impact from the regiment.  Conclusion: This research using different human skin models demonstrated that AAComplex can provide protection and damage repair caused by UV, at the ingredient level also when formulated in a serum or lotion formula. Skin may be best protected from UV damage when the regimen is used.   J Drugs Dermatol. 2024;23(5):366-375. doi:10.36849/JDD.7916.

Microbiome and lipidomic analysis reveal the interplay between skin bacteria and lipids in a cohort study.

Human skin acts as a protective barrier between the body and the external environment. Skin microbiome and intercellular lipids in the stratum corneum (SC) are essential for maintaining skin barrier function. However, the interplay between skin bacteria and the lipids is not fully understood. In this study, we characterized the skin microbiome and SC lipid profiles from the forearm and face in a cohort of 57 healthy participants. 16S rRNA gene sequencing showed the skin microbial composition is significantly different between body locations and genders. Female forearm samples have the highest microbial diversity. The relative abundance of Staphylococcus hominis, Micrococcus luteus, Corynebacterium tuberculostearicum, Finegoldia magna, and Moraxellaceae sp. are significantly higher in the forearm than the face. The predictive functional analysis of 16S rRNA gene sequencing by Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) and ANCOM-BC showed different bacterial metabolic pathway profiles between body locations or genders, and identified 271 differential pathways, including arginine and polyamine biosynthesis, chorismate biosynthesis pathways, which are more abundant in the female forearm, and sulfur oxidation pathway, which is more abundant in the male face. The SC lipid profiles differ between the body locations as well. Total free fatty acids (FFA), cholesterol sulfate and sphingosine are more abundant in the face. Dihydro-/6-hydroxy/phyto-ceramides are more abundant in the forearm. The correlation analysis of 16S rRNA gene sequencing and lipids revealed novel interplay between the bacteria and skin lipids. Shannon entropy and S. hominis negatively correlated with FFA, cholesterol sulfate and sphingosine; while positively correlated with dihydro-/6-hydroxy/phyto-ceramides. The correlation of predictive pathway profiles and lipids identified pathways involved in amino acids metabolism, carbohydrates degradation, aromatic compounds metabolism and fatty acid degradation metabolism are positively correlated with dihydro-/6-hydroxy/phyto-ceramides and negatively correlated with FFA, cholesterol sulfate and sphingosine. This study provides insights on the potential correlation between skin microbiome and lipids.

Amino acid complex (AAComplex) benefits in cosmetic products: In vitro and in vivo clinical studies.

BACKGROUND: Amino acids are major components of skin's natural moisturizing factors and play a role in regulating skin hydration and skin pH. OBJECTIVE: This research examines a proprietary amino acid complex technology (AAComplex) designed to help reduce skin irritation and repair skin damage. METHODS: In- vitro Scratch Assay HaCaT cells are scratched, and the wounds are imaged at different time points until the closure of the scratch wound is detected. In-vitro 3D Reconstructed Human Tissue Evaluation The concentration of heat shock protein 27 (HSP-27) extracted from 3D reconstructed human skin equivalent tissues and IL-1a released to the media was determined using enzyme-linked immunosorbent assays. In Vivo Clinical Study 37 subjects were enrolled in a split-face study design. On test days 1, 2, 4, and 8, subjects visited the test facility to have their face assessed by facial swabbing and bio-instrumentation measurements. RESULTS: In- vitro Scratch Assay The AAComplex demonstrated a strong cell renewal benefit in the HaCaT (human) cells scratch assay. In Vitro 3D Reconstructed Human Tissue Evaluation AAComplex demonstrated a significant skin repair benefit by quantifying Heat Shock Protein, HSP-27. Induced skin irritation was significantly reduced by quantifying interleukin-1 alpha biomarker, IL-1a. In Vivo Clinical Study The test products delivered skin benefits by reducing visual redness and skin irritation while increasing moisturization. CONCLUSION: The in vitro and in vivo clinical studies demonstrated that the AAComplex technology formulated in the commercially available Skin Recovery System effectively reduced skin irritation and redness as well as accelerating the skin repair process.

Efficacy Evaluation of a Topical Hyaluronic Acid Serum in Facial Photoaging.

INTRODUCTION: Hyaluronic acid (HA) acts as a biologic humectant, thus retaining water in the skin, making HA useful as a topical moisturizing ingredient. The goal of the research was to evaluate the ability of a HA facial serum to deliver skin benefits. METHODS: Forty females 30-65 years of age with Fitzpatrick skin types I-VI who exhibited photoaging used the HA facial serum twice daily with sunscreen. The dermatologist investigator evaluated smoothness, plumping, hydration, fine lines/wrinkles, and global appearance issues on a 5-point ordinal scale. The subjects assessed product tolerability in terms of stinging, itching, and burning. Corneometry was undertaken, with assessments performed at baseline, immediately after application, and at weeks 2, 4, and 6. Facial swabbing and photography were performed at the same intervals on a subset of 15 subjects. RESULTS: The HA serum demonstrated excellent tolerability and produced an increase in skin hydration (as measured by corneometry) immediately after application of 134% (p < 0.001), with a sustained increase of 55% (p < 0.001) at week 6. At week 6, there was also improvement (p ≤ 0.001) in all evaluated attributes: smoothness (64%), plumping (60%), hydration (63%), fine lines (31%), wrinkles (14%), and overall global assessment (43%). Facial swabbing confirmed an increase in topical HA at week 6 (p = 0.04), accounting for the enhanced skin appearance, but there was no statistically significant increase in IL-1a, indicating no product irritation. CONCLUSION: Topical HA in a serum formulation provides excellent skin hydration, as demonstrated through clinical, photographic, chemical, and instrumental assessments.

Prognostic role of the beta-2 adrenergic receptor in clear cell renal cell carcinoma.

The beta-2 adrenergic receptor (ADRB2) regulates the proliferation, apoptosis, angiogenesis, migration, and metastasis of cancer cells. However, its function in the progression of clear cell renal cell carcinoma (ccRCC) is unknown. Here, we report that ADRB2 can be a novel prognostic factor for patients with ccRCC. The differential expression of ADRB2 in low-stage (stages I and II), high-stage (stages III and IV), low-grade (grades I and II), and high-grade (grades III and IV) ccRCC was identified in cohorts of patients from The Cancer Genome Atlas and the International Cancer Genome Consortium. We evaluated ADRB2 expression as a prognostic factor using the Kaplan-Meier survival curve, multivariate analysis, time-dependent area under the curve (AUC) of Uno's C-index, and AUC of the receiver operating characteristics (ROC) at five years. Kaplan-Meier analysis revealed that reduced ADRB2 expression is associated with poor prognosis in ccRCC patients. Analysis of C-indices and AUC-ROC further confirmed this result. Moreover, multivariate analysis confirmed the prognostic significance of ADRB2 expression. Collectively, these findings suggest that ADRB2 is a potential prognostic factor for ccRCC.

Assessment of Human Skin Gene Expression by Different Blends of Plant Extracts with Implications to Periorbital Skin Aging.

Since the skin is the major protective barrier of the body, it is affected by intrinsic and extrinsic factors. Environmental influences such as ultraviolet (UV) irradiation, pollution or dry/cold air are involved in the generation of radical oxygen species (ROS) and impact skin aging and dermal health. Assessment of human skin gene expression and other biomarkers including epigenetic factors are used to evaluate the biological/molecular activities of key compounds in cosmetic formulas. The objective of this study was to quantify human gene expression when epidermal full-thickness skin equivalents were exposed to: (a) a mixture of betaine, pentylene glycol, Saccharomyces cerevisiae and Rhodiola rosea root extract (BlendE) for antioxidant, skin barrier function and oxidative stress (with hydrogen peroxide challenge); and (b) a mixture of Narcissus tazetta bulb extract and Schisandra chinensis fruit extract (BlendIP) for various biomarkers and microRNA analysis. For BlendE, several antioxidants, protective oxidative stress biomarkers and many skin barrier function parameters were significantly increased. When BlendE was evaluated, the negative impact of the hydrogen peroxide was significantly reduced for the matrix metalloproteinases (MMP 3 and MMP 12), the skin aging and oxidative stress biomarkers, namely FBN2, ANXA1 and HGF. When BlendIP was tested for cell proliferation and dermal structural components to enhance the integrity of the skin around the eyes: 8 growth factors, 7 signaling, 7 structural/barrier function and 7 oxidative stress biomarkers were significantly increased. Finally, when BlendIP was tested via real-time RT-PCR for microRNA expression: miR-146a, miR-22, miR155, miR16 and miR21 were all significantly increased over control levels. Therefore, human skin gene expression studies are important tools to assess active ingredient compounds such as plant extract blends to advance dermal hypotheses toward validating cosmetic formulations with botanical molecules.

Protective effects of a novel nutritional and phytonutrient blend on ultraviolet radiation-induced skin damage and inflammatory response through aging defense mechanisms.

BACKGROUND: The human body relies on several aging defense mechanisms (ADMs) to limit damage induced from pro-aging stressors (aging aggressors). However, such protective mechanisms can be compromised, leading to accelerated aging. The skin provides a model to probe the effects of an oral nutritional intervention on ADMs in response to ultraviolet radiation (UVR)-induced damage. OBJECTIVE: To determine whether supplementation with a novel nutritional and phytonutrient blend could protect against UVR-induced skin damage and positively influence facial skin attributes and characteristics by bolstering ADMs. METHODS: Thirty-six healthy, nonsmoking women (40-75 years) with Fitzpatrick skin types I and II were recruited. UVR-induced erythema and the number of apoptotic cells were determined before (pre-) and after 8-week (post-) supplementation. Other clinical variables included skin carotenoid concentrations, facial skin attributes and characteristics. RESULTS: Eight-week supplementation led to protection against UVR-induced skin damage as evidenced by reductions in erythema at all three minimal erythema doses (MEDs) (9.1 to 7.4 [P = 0.10]; 15.8 to 13.6 [P = 0.02]; and 19.6 to 17.3 [P = 0.01] for one, two, and three MEDs and a reduction in the average number of apoptotic cells [11.3 to 5.3, P < 0.0001] pre- and post-supplementation, respectively). Skin carotenoid concentrations increased from 28 111 Raman intensity units to 38 472 (P < 0.0001) along with noticeable improvements in facial skin attributes and characteristics: elasticity, transepidermal water loss, radiance, texture, and overall appearance (all P < 0.05) following supplementation. CONCLUSION: Eight weeks of oral supplementation positively impacted ADMs resulting in protection against UVR-induced skin damage and improvements in facial skin attributes and characteristics.

Glutamatergic pathway targeting in melanoma: single-agent and combinatorial therapies.

PURPOSE: Melanoma is a heterogeneous disease where monotherapies are likely to fail due to variations in genomic signatures. B-RAF inhibitors have been clinically inadequate but response might be augmented with combination therapies targeting multiple signaling pathways. We investigate the preclinical efficacy of combining the multikinase inhibitor sorafenib or the mutated B-RAF inhibitor PLX4720 with riluzole, an inhibitor of glutamate release that antagonizes metabotropic glutamate receptor 1 (GRM1) signaling in melanoma cells. EXPERIMENTAL DESIGN: Melanoma cell lines that express GRM1 and either wild-type B-RAF or mutated B-RAF were treated with riluzole, sorafenib, PLX4720, or the combination of riluzole either with sorafenib or with PLX4720. Extracellular glutamate levels were determined by glutamate release assays. MTT assays and cell-cycle analysis show effects of the compounds on proliferation, viability, and cell-cycle profiles. Western immunoblotting and immunohistochemical staining showed apoptotic markers. Consequences on mitogen-activated protein kinase pathway were assessed by Western immunoblotting. Xenograft tumor models were used to determine the efficacy of the compounds in vivo. RESULTS: The combination of riluzole with sorafenib exhibited enhanced antitumor activities in GRM1-expressing melanoma cells harboring either wild-type or mutated B-RAF. The combination of riluzole with PLX4720 showed lessened efficacy compared with the combination of riluzole and sorafenib in suppressing the growth of GRM1-expressing cells harboring the B-RAF(V600E) mutation. CONCLUSIONS: The combination of riluzole with sorafenib seems potent in suppressing tumor proliferation in vitro and in vivo in GRM1-expressing melanoma cells regardless of B-RAF genotype and may be a viable therapeutic clinical combination.

Oncogenic activities of metabotropic glutamate receptor 1 (Grm1) in melanocyte transformation.

Previously, we reported a transgenic mouse line, TG-3, that develops spontaneous melanoma with 100% penetrance. We demonstrated that ectopic expression of Grm1 in melanocytes was sufficient to induce melanoma in vivo. In this present study, the transforming properties of Grm1 in two cultured immortalized melanocytes were investigated. We showed that, in contrast to parental melanocytes, these Grm1-clones have lost their requirement of TPA supplement for proliferation and have acquired the ability to form colonies in semi-solid medium. Xenografts of these cells formed robust tumors in both immunodeficient nude and syngeneic mice with a short latency (3-5 days). The malignancy of these cells was demonstrated by angiogenesis and invasion to the muscle and the intestine. The requirement of Grm1 expression for the maintenance of transformation was demonstrated by an inducible siRNA system. Induction of expression of siRNA for Grm1 reduced the number of proliferating/viable cells in vitro and suppressed in vivo xenografted tumor growth in comparison with control. Taken together, these results showed that expression of exogeneously introduced Grm1 is sufficient to induce full transformation of immortalized melanocytes.

Curcumin downregulates the constitutive activity of NF-kappaB and induces apoptosis in novel mouse melanoma cells.

Melanoma, the most deadly form of skin cancer, is very aggressive and resistant to present therapies. The transcription factor nuclear factor-kappa B (NF-kappaB) has been reported to be constitutively active in many types of cancer. Constitutively active NF-kappaB seen in melanoma likely plays a central role in cell survival and growth. We have established and characterized novel cell lines from our murine melanoma model. Here we report the constitutive activity of NF-kappaB in these melanoma-derived cells, as shown by electrophoretic mobility shift assay and reporter assays. We hypothesized that agents that inhibit NF-kappaB may also inhibit cell proliferation and may induce apoptosis in such melanoma cells. Curcumin has been shown to inhibit NF-kappaB activity in several cell types. In our system, curcumin selectively inhibited growth of melanoma cells, but not normal melanocytes. Curcumin induced melanoma cells to undergo apoptosis, as shown by caspase-3 activation, inversion of membrane phosphatidyl serine, and increases in cells in the sub-G1 phase. A curcumin dose-dependent inhibition of NF-kappaB-driven reporter activity correlated with decreased levels of phospho-IkappaBalpha, and decreased expression of NF-kappaB-target genes COX-2 and cyclin D1. This study demonstrates that the use of cells from our model system can facilitate studies of signaling pathways in melanoma. We furthermore conclude that curcumin, a natural and safe compound, inhibits NF-kappaB activity and the expression of its downstream target genes, and also selectively induces apoptosis of melanoma cells but not normal melanocytes. These encouraging in-vitro results support further investigation of curcumin for treatment of melanoma in vivo.

Metabotropic glutamate receptor 1 and glutamate signaling in human melanoma.

Recently, several laboratories have started to investigate the involvement of glutamate signaling in cancer. In previous studies, we reported on a transgenic mouse model that develops melanoma spontaneously. Subsequent studies in these mice identified that the aberrant expression of metabotropic glutamate receptor 1 (GRM1) in melanocytes played a critical role in the onset of melanoma. Confirmation of the etiologic role of GRM1 in melanoma development was shown in a second transgenic line with GRM1 expression under the regulation of a melanocyte-specific dopachrome tautomerase promoter. Ectopic expression of GRM1 was also detected in a subset of human melanoma cell lines and biopsies, suggesting that aberrant expression of GRM1 in melanocytes may contribute to the development of human melanoma. GRM1, a seven-transmembrane domain G protein-coupled receptor, is normally expressed and functional in neuronal cells, and its ligand, glutamate, is the major excitatory neurotransmitter. Human melanoma cells are shown here to release elevated levels of glutamate, implying a possible autocrine loop. Treatment of GRM1-expressing human melanoma cells with a GRM1 antagonist (LY367385 or BAY36-7620) or a glutamate release inhibitor (riluzole) leads to a suppression of cell proliferation as well as a decrease in levels of extracellular glutamate. Treatment of human melanoma cell xenografts with riluzole for 18 days via p.o. gavage or i.v. injection leads to inhibition of tumor growth by 50% in comparison with controls. These data suggest the importance of glutamate signaling in human melanoma and imply that the suppression of glutamate signaling may be a new target for melanoma therapy.

From existing therapies to novel targets: a current view on melanoma.

Identifying new drugs and targets for melanoma therapy is critical, considering that melanoma, the most dangerous form of skin cancer, is resistant to currently available therapeutics. Much work has been focused on finding novel drugs and exploring different treatment options that could increase the overall survival of patients. In our laboratory we have developed mouse models to study melanoma. We discovered that aberrant expression of metabotropic glutamate receptor 1 (Grm1) in melanocytes promotes melanoma development in vivo. Grm1 is a seven transmembrane domain G-protein coupled receptor that is normally expressed and functional in the central nervous system. The natural ligand of Grm1 is glutamate. Signaling by the major neurotransmitter glutamate has been well characterized in neuronal cells; however glutamate signaling in other tissues is not well understood. We demonstrated that Grm1 signaling in melanoma cells is mediated by the Ras/Raf/MEK/ERK pathway, one of the major pathways previously shown to be activated in human melanoma cells. Based on these earlier studies and results from our recent work, we predict that inhibition of Grm1 signaling and its downstream cascade may potentially provide new, effective therapies for melanoma patients. In this review, we propose several attractive targets.

Stimulation of oncogenic metabotropic glutamate receptor 1 in melanoma cells activates ERK1/2 via PKCepsilon.

Metabotropic glutamate receptor 1 (Grm1, formerly mGluR1) is a G protein coupled receptor (GPCR) normally expressed and functional in the central nervous system. Studies of our transgenic mouse melanoma model (TG-3) revealed that ectopic expression of Grm1 in melanocytes is sufficient to induce melanoma development in vivo [P.M. Pollock, K. Cohen-Solal, R. Sood, J. Namkoong, J.J. Martino, A. Koganti, H. Zhu, C. Robbins, I. Makalowska, S.S. Shin, Y. Marin, K.G. Roberts, L.M. Yudt, A. Chen, J. Cheng, A. Incao, H.W. Pinkett, C.L. Graham, K. Dunn, S.M. Crespo-Carbone, K.R. Mackason, K.B. Ryan, D. Sinsimer, J. Goydos, K.R. Reuhl, M. Eckhaus, P.S. Meltzer, W.J. Pavan, J.M. Trent, S. Chen, Nat. Genet. 34 (2003) 108-112.]. We have established and characterized several cell lines in vitro from independent mouse melanoma tumors [Y.E. Marín, J. Namkoong, S.S. Shin, J. Raines, K. Degenhardt, E. White, S. Chen, Neuropharmacol. 49 (2005) 70-79.]. These cell lines are useful tools in the studies of signaling events that may be mediated by Grm1 in transformed melanocytes. Here we show that stimulation of Grm1 by l-quisqualate, a group I metabotropic glutamate receptor agonist, results in inositol triphosphate (IP3) accumulation, and the activation of ERK1/2 in these cell lines. IP3 accumulation and ERK1/2 activation were inhibited by pretreatment of the tumor cells with a Grm1-specific antagonist (LY367385) or by dominant negative mutants of Grm1, demonstrating the specificity of these events. We also show that ERK1/2 activation by Grm1 was PKC-dependent, but cAMP and PKA-independent. PKCepsilon was shown to play a pivotal role in Grm1-mediated ERK1/2 phosphorylation. Insights into the signaling cascades mediated by Grm1 in melanoma cells may aid in the identification of key molecular targets for the future design of combined therapies for melanoma.

Grm5 expression is not required for the oncogenic role of Grm1 in melanocytes.

Melanoma is the aberrant proliferation of melanocytes, the cells in the skin responsible for pigment (melanin) production. In its early stages, melanoma can be surgically removed with great success, however, advanced stages of melanoma have a high mortality rate due to the lack of responsiveness to currently available therapies. We have previously characterized a mouse melanoma model, TG-3, which has implicated the ectopic expression of metabotropic glutamate receptor 1 (Grm1, formerly mGluR1), in melanomagenesis and metastasis [Pollock et al., 2003. Melanoma mouse model implicates metabotropic glutamate signaling in melanocytic neoplasia. Nat Genet. 34, 108-112.]. Here we report the characterization of several in vitro cell lines derived from independent mouse melanoma tumors. These cell lines show characteristic phenotypes of transformed melanocytes, and express Grm1, and Grm5 (another metabotropic glutamate receptor), as well as melanocyte-specific protein markers. To investigate the possible role of Grm5 in vivo during melanoma development in our mice, we have crossed Grm5 null mice with TG-3, generating a new line of transgenic mice, TGM. TGMs, which are homozygote knockouts for Grm5 and carry the TG transgene, develop tumors with onset, progression, and metastasis very similar to that described for TG-3. Taken together, these results indicate that Grm1 can act as an oncogene in melanocytes independently of Grm5 expression.

Cloning and characterization of a novel gene, SHPRH, encoding a conserved putative protein with SNF2/helicase and PHD-finger domains from the 6q24 region.

Here we report the identification of a novel transcript containing SNF2, PHD-finger, RING-finger, helicase, and linker histone domains mapping to the q24 band region of human chromosome 6. These domains are characteristic of several DNA repair proteins, transcription factors, and helicases. We have cloned both human and mouse homologs of this novel gene using interexon PCR and RACE technologies. The human cDNA, termed SHPRH, is 6018 bp and codes for a putative protein of 1683 amino acids. The mouse cDNA, termed Shprh, is 7225 bp and codes for a putative protein of 1616 amino acids. The deduced amino acid sequences of the two proteins share 86% identity. Both genes are expressed ubiquitously, with a transcript size of approximately 7.5 kb. Mapping of this gene to 6q24, a region reported to contain a tumor suppressor locus, prompted us to evaluate SHPRH by mutation analysis in tumor cell lines. We have identified one truncating and three missense mutations, thus suggesting SHPRH as a possible candidate for the tumor suppressor gene.

Melanoma mouse model implicates metabotropic glutamate signaling in melanocytic neoplasia.

To gain insight into melanoma pathogenesis, we characterized an insertional mouse mutant, TG3, that is predisposed to develop multiple melanomas. Physical mapping identified multiple tandem insertions of the transgene into intron 3 of Grm1 (encoding metabotropic glutamate receptor 1) with concomitant deletion of 70 kb of intronic sequence. To assess whether this insertional mutagenesis event results in alteration of transcriptional regulation, we analyzed Grm1 and two flanking genes for aberrant expression in melanomas from TG3 mice. We observed aberrant expression of only Grm1. Although we did not detect its expression in normal mouse melanocytes, Grm1 was ectopically expressed in the melanomas from TG3 mice. To confirm the involvement of Grm1 in melanocytic neoplasia, we created an additional transgenic line with Grm1 expression driven by the dopachrome tautomerase promoter. Similar to the original TG3, the Tg(Grm1)EPv line was susceptible to melanoma. In contrast to human melanoma, these transgenic mice had a generalized hyperproliferation of melanocytes with limited transformation to fully malignant metastasis. We detected expression of GRM1 in a number of human melanoma biopsies and cell lines but not in benign nevi and melanocytes. This study provides compelling evidence for the importance of metabotropic glutamate signaling in melanocytic neoplasia.

Progressive appearance of pigmentation in amelanotic melanoma lesions.

We previously described a transgenic mouse line (TG-3) that spontaneously develops pigmented cutaneous melanoma. The generation of several albino mice that developed amelanotic melanoma has also been reported. In this report, we describe an unanticipated result with crosses between C57BL/6-c2j and TG-3 mice. C57BL/6-c2j has the same genetic background as TG-3 (C57BL/6), except for a single base mutation (nucleotide 291) in the tyrosinase locus, resulting in albino coat colour. Only albino F2 mice generated from (TG-3 x C57BL/6-c2j) F1s were selected for further studies. Mice that contained the transgene showed a very high incidence of tumor development as early as 4-6 weeks of age. Raised amelanotic tumors developed on the ear pinnae and perianal region in young F2 albino mice, similar phenotypes as those described earlier for the other albino inbred strains. However, with time, these amelanotic tumors not only increased in size, but unexpectedly developed foci of dark pigmentation. DNA sequence analysis on reverse transcriptase-polymerase chain reactions (RT-PCRs) of tyrosinase mRNA showed that the original tyrosinase mutation was still present in the tumors, indicating that no reversion at this nucleotide had occurred in the tumors. Two different tyrosinase activity assays were used and tyrosinase activity was detected in most tumor samples. Furthermore, Western blot analysis demonstrated various levels of tyrosinase protein in ear tumor samples. These results suggest that tyrosinase and/or melanin are not directly involved in the establishment of melanoma, but that late events occurring within the tumors may generate some tyrosinase activity and production of melanin.