Diss. (sammanfattning) Linköping : Linköpings universitet, 2007. / Härtill 4 uppsatser.

Melanoblasts are the embryonic precursors of melanocytes, the pigment producing cells of the skin and hair. Melanoblasts are of key interest to developmental biologists for numerous reasons, including their ability to migrate throughout the body from a single origin in the neural crest (NC). Current methods for the study of the melanocyte lineage are limited by the heavy reliance on animal models. To challenge this, a platform of in vitro tools were designed to replace and complement current studies. A major obstacle is the transition from 2D cultures, which provide only limited behavioural information, to 3D models which are able to recapitulate the environmental conditions. 3D cultures are regularly created using tissue samples and synthetic matrices for attachment, but building a model from cell lines only has not been achieved. A co-culture model using immortalised keratinocyte (COCA) and melanoblast cell lines proved unsuitable for observing developmental processes, due to lack of movement at high cell densities, but may be practical in pigmentation research. Other methods were explored to examine melanoblast behaviour, including the use of cell derived matrices (CDMs) integrated with melanoblast cell lines, and aggregates formed by hanging drop (HD) culture. CDMs were successfully generated from the COCA line, as well as NIH3T3 fibroblasts which has been shown previously. These structures are denuded of cells to leave the deposited extracellular matrix (ECM) components intact, representative of the dermal (fibroblast) and epidermal (keratinocyte) layers of the skin. HDs were prepared from cultured melanoblast cell lines, and form tight aggregates which disseminate when plated, in a manner similar to the dissemination of cells from the NC in explant cultures. The receptor tyrosine kinase KIT and its ligand (KITL), are vital for melanoblast development. Previous study of this signalling complex has often focussed on the haematopoietic lineage and spermatogenesis, where they perform essential roles. KITL is expressed in a membrane localised form found on the surface of keratinocytes thought to promote melanoblast/melanocyte survival, and a soluble isoform found sequestered in the ECM which promotes cell migration. Cell lines expressing fluorescently tagged KIT and KITL were created to visualise their interactions using live-cell confocal imaging. Firstly, cell lines were generated to perform co-culture experiments with KIT and KITL, and we showed that these constructs are able to interact by uptake of KITL into KIT cells. Secondly, tandem fluorescent protein timers of KIT and KITL were generated which were used to observe protein kinetics. We showed that these protein timers can be manipulated using cycloheximide to block protein production, or by increasing ligand availability. These protein timers reveal that soluble KITL (sKITL) has a faster turnover than membrane bound KITL (mKITL), and that in all three proteins, there is distinct change in spatial localisation as the proteins age. Using a novel melanoblast reporter mouse, Pmel-CMN, primary mouse melanoblasts between E12.5 and E14.5 were isolated for RNA sequencing. This time period is the earliest reported for melanoblast isolation for use in gene expression analysis. We show that within this time course, there are significant changes in the RNA expression profiles, including decreasing expression of other NC cell markers, and huge increasing expression of pigmentation genes. To assess the biological relevance of using in vitro assays, cells of the immortalised melanoblast cell line, melb-a, were cultured under different conditions and examined via RNA sequencing. Results reveal differences in several areas between primary cells and those in culture, including loss of melanocyte specificity. The different tools described in this thesis provide a platform on which to study various aspects of cell behaviour, including migration, morphology and cell adhesion at both the individual cell and population levels.

University of Macau / Institute of Chinese Medical Sciences

The embryonic source and chemical nature of those factor/s directing the in vivo differentiation of melanocytes from crest cells are as yet unknown. To begin to address this issue, it is important to establish exactly when and where these signa/s first exert their effects. Therefore, in the present study, overtly differentiated melanocytes containing melanin were quantitated in developing Black Australorp X New Hampshire Red chick embryos. In contrast to previous studies, it was found that embryos synthesize melanin from as early as Day 5 of development, and that at this stage, the melanocytes are predominantly dermally located. Between 5 and 8 days, the numbers of both dermal and epidermal melanocytes increase, after which the dermal melanocyte population declines rapidly while the number of epidermal melanocytes continues to increase. These findings suggest that premelanocytes do not have to be epidermally located to initiate terminal differentiation and implicate the dermis as a possible source of melanocyte inducing factor/s. The next step was to examine stages of development prior to the onset of pigment production. For this reason, tyrosinase was purified for use as antigen in the production of a polyclonal antibody. The antibody was tested for specificity by western blotting, - immunocytochemistry and immunoinhibition procedures. Lack of specificity was demonstrated, rendering it unsuitable as an antibody marker for early melanocytes. Fowl melanocytes are thought to differentiate into either eumelanosome- or pheomelanosome synthesizing cells. To test the validity of this concept, embryonic skin of the red/black cross breed were screened for possible mixed type melanocytes by electron microscopy. The melanocytes contained melanosomes with a matrix of irregularly arranged filaments amongst typical eumelanogenic melanosomes. This suggests that these chick melanocytes may synthesize both eumelanosomes and pheomelanosomes in single cells. In a further study on pure breeding New Hampshire Reds, it was found that the melanocytes contained a mixture of typical and less typical pheomelanosomes. Outer membrane indentations in the latter melanosome type suggest that tyrosinase may enter these pheomelanosomes by a mechanism related to that proposed for the melanosomes of goldfish.

Melanoma is among the most aggressive and treatment-resistant cancers. Currently, available therapies for melanoma are not satisfactory, and the prognosis for patients with metastatic melanoma is still poor. Vemurafenib, a BRAF kinase inhibitor (BRAFi), provides an approximately 50% response rate in patients with metastatic melanoma, but eventually, relapse occurs due to acquired resistance to the drug. Novel therapeutics and BRAFi adjuvants for treating melanoma are needed. Si-Jun-Zi-Tang (SJZT) is a traditional Chinese medicine formula used to treat chronic and debilitating diseases including melanoma. SJZT-based therapies alone or in combination with chemotherapies have achieved good clinical outcomes in melanoma management. However, the pharmacological basis of SJZT for its clinical use in melanoma treatment is not fully understood. c-Met is a receptor tyrosine kinase (RTK), and hepatocyte growth factor (HGF) is the only known ligand of c-Met. Abnormal activation of HGF/c-Met has been implicated in melanoma progression. HGF/c-Met has been proposed as a therapeutic target for melanoma. Some bioactive constituents in SJZT have been shown to inhibit c-Met signaling. In this study, we investigated the anti-melanoma effects and c-Met signaling-related action mechanisms of SJZT. Our in vivo study showed that SJZT-A, an ethanolic extract of SJZT, inhibited B16 tumor growth in mice without overt toxicity. Mechanistic investigations revealed that SJZT-A elevated miR-34b (a tumor-suppressing miRNA), and lowered c-Met (a miR-34b target gene) and β-catenin (a downstream molecule of c-Met signaling) expression levels in the B16 tumors. But SJZT-A failed to reduce the viability of B16 and A375 melanoma cells. Active component-enriched SJZT-B, which was prepared from SJZT-A using macroporous resin column chromatography, exhibited potent anti-proliferation effect and inhibited miR-34b/c-Met/β-catenin signaling in cultured melanoma cells. Overexpression of constitutively active β-catenin partially diminished the inhibitory effect of SJZT-B on cell proliferation. SJZT-B also exerted anti-proliferative effects, inhibited c-met signaling, and induced ER stress in vemurafenib resistance melanoma cells. In vivo experiments showed that intragastric administration of SJZT-B for consecutive 14 days overcame vemurafenib resistance in melanoma-bearing mice without observable toxicities. Overall, these results indicate that SJZT has anti-melanoma effects and is relatively safe. Also, we found that licochalcone A, a flavonoid presented in SJZT-B, overcame vemurafenib resistance both in vitro and in vivo, as well as inhibited c-Met signaling and induced ER stress in A375-VR cells, which is in line with the effects of SJZT-B in melanoma. The role of triggering ER stress in licochalcone A's effects in overcoming vemurafenib resistance effects has also been established. Overall, these results suggest that licochalcone A is one of the active compounds responsible for the anti-melanoma effects of SJZT-B. In conclusion, our results demonstrated that SJZT has anti-melanoma effects and is safe in cell and mouse melanoma models. Licochalcone A has been identified to be one of the active components responsible for the anti-melanoma effects of SJZT. This study provides a pharmacological and chemical basis for the traditional use of the formula SJZT in treating melanoma, and suggests that SJZT and SJZT-derived compounds have the potential to be developed as modern alternative and/or complementary agents for melanoma management.

Melanoma is among the most aggressive and treatment-resistant cancers. Currently, available therapies for melanoma are not satisfactory, and the prognosis for patients with metastatic melanoma is still poor. Vemurafenib, a BRAF kinase inhibitor (BRAFi), provides an approximately 50% response rate in patients with metastatic melanoma, but eventually, relapse occurs due to acquired resistance to the drug. Novel therapeutics and BRAFi adjuvants for treating melanoma are needed. Si-Jun-Zi-Tang (SJZT) is a traditional Chinese medicine formula used to treat chronic and debilitating diseases including melanoma. SJZT-based therapies alone or in combination with chemotherapies have achieved good clinical outcomes in melanoma management. However, the pharmacological basis of SJZT for its clinical use in melanoma treatment is not fully understood. c-Met is a receptor tyrosine kinase (RTK), and hepatocyte growth factor (HGF) is the only known ligand of c-Met. Abnormal activation of HGF/c-Met has been implicated in melanoma progression. HGF/c-Met has been proposed as a therapeutic target for melanoma. Some bioactive constituents in SJZT have been shown to inhibit c-Met signaling. In this study, we investigated the anti-melanoma effects and c-Met signaling-related action mechanisms of SJZT. Our in vivo study showed that SJZT-A, an ethanolic extract of SJZT, inhibited B16 tumor growth in mice without overt toxicity. Mechanistic investigations revealed that SJZT-A elevated miR-34b (a tumor-suppressing miRNA), and lowered c-Met (a miR-34b target gene) and β-catenin (a downstream molecule of c-Met signaling) expression levels in the B16 tumors. But SJZT-A failed to reduce the viability of B16 and A375 melanoma cells. Active component-enriched SJZT-B, which was prepared from SJZT-A using macroporous resin column chromatography, exhibited potent anti-proliferation effect and inhibited miR-34b/c-Met/β-catenin signaling in cultured melanoma cells. Overexpression of constitutively active β-catenin partially diminished the inhibitory effect of SJZT-B on cell proliferation. SJZT-B also exerted anti-proliferative effects, inhibited c-met signaling, and induced ER stress in vemurafenib resistance melanoma cells. In vivo experiments showed that intragastric administration of SJZT-B for consecutive 14 days overcame vemurafenib resistance in melanoma-bearing mice without observable toxicities. Overall, these results indicate that SJZT has anti-melanoma effects and is relatively safe. Also, we found that licochalcone A, a flavonoid presented in SJZT-B, overcame vemurafenib resistance both in vitro and in vivo, as well as inhibited c-Met signaling and induced ER stress in A375-VR cells, which is in line with the effects of SJZT-B in melanoma. The role of triggering ER stress in licochalcone A's effects in overcoming vemurafenib resistance effects has also been established. Overall, these results suggest that licochalcone A is one of the active compounds responsible for the anti-melanoma effects of SJZT-B. In conclusion, our results demonstrated that SJZT has anti-melanoma effects and is safe in cell and mouse melanoma models. Licochalcone A has been identified to be one of the active components responsible for the anti-melanoma effects of SJZT. This study provides a pharmacological and chemical basis for the traditional use of the formula SJZT in treating melanoma, and suggests that SJZT and SJZT-derived compounds have the potential to be developed as modern alternative and/or complementary agents for melanoma management.

No / The human epidermis holds the full machinery for cholinergic signal transduction. However, the presence of the vesicular transporter (vesicular acetylcholine (ACh) transporter (VAChT)) for both choline and ACh has never been shown in this compartment. The results of this study confirm the presence of VAChT in cutaneous nerves and in both epidermal melanocytes and keratinocytes as well as in their nuclei using immunofluorescence labelling in situ and in vitro, Western blot analysis of cellular and nuclear extracts and reverse transcription-PCR. These results underline that ACh/choline transport in the non-neuronal epidermis is no different from the neuronal pathway. However, the function of VAChT in the nucleus remains to be shown.

No / The present invention relates to compositions and methods useful in studying or modulating melanin pigmentation in the skin. Particularly, the invention relates to compositions comprising a substance capable of modulating the activity or expression of ALK6 (SEQ ID 2) or Cdc42 which in turn are capable of modulation of the transfer of melanin from melanocytes to keratinocytes and potentially from keratinocytes to keratinocytes. The invention also relates to assays for identifying such compositions, and methods of modulating skin pigmentation.

No / Canities or senile hair graying, a universally recognized sign of aging, remains unresolved in terms of physiological causes, although a strong genetic contribution is understood (Gunn et al., 2009). As the hair fiber continues to grow long after melanin production ceases, we suggest that melanocytes in the hair follicle may be more sensitive to the impact of chronological aging than are keratinocytes. Moreover, follicular melanocytes also age more markedly than those in the overlying epidermis. The hair follicle provides a unique opportunity to decouple the impact of age on two hair follicular tissue functions: hair formation and hair pigmentation. ... This study provides analysis of race, age, and anatomically matched cultures of adult human epidermal and hair follicle melanocytes (HFMs).

Um eine verbesserte Therapieoption mit weniger Schmerzen und Nebenwirkun-gen für Patienten mit Depigmentierungsstörungen wie Vitiligo zu entwickeln, wurde eine Methode zur nichtinvasiven Gewinnung von autologen Melanozyten aus der Haarwurzel genutzt. Die Haarwurzel als einfach zugängliches Stammzell-reservoir bietet die Möglichkeit, Vorläufermelanozyten aus der äußeren Haar-wurzelscheide zu isolieren, differenzieren und zu proliferieren. Für zukünftige autologe Transplantationsversuche wurden in dieser Arbeit die kultivierten hu-manen Melanozyten aus der äußeren Haarwurzelscheide (Human Melanocytes from the Outer Root Sheath, HUMORS) auf drei unterschiedlichen Scaffolds getes-tet. Hinsichtlich mitochondrialer Aktivität (Marker für Zellproliferation), mela-nozytenspezifischer Markerexpression und ihrer Funktionalität (Tyrosinase-Enzymaktivität und Melaningehalt) wurden die Zellen auf Collagen Cell Carrier® (CCC), Poly-ε-Caprolacton-Scaffolds (PCL) und kollagen basierten Hydrogelen (cGEL) kultiviert und charakterisiert. Alle Scaffolds waren biokompatibel, immu-nologisch nur gering aktiv und wiesen eine dreidimensionale Struktur auf, die der extrazellulären Matrix nachempfunden war. Einen positiven Effekt auf die Prolife-ration wiesen die HUMORS auf den Collagen Cell Carrier® auf. Bei Untersuchun-gen der melanotischen Aktivität überzeugten die HUMORS auf dem cGEL Typ4 durch einen signifikant höheren Melaningehalt. Da Melanin das entscheidende Produkt der Repigmentierung bei Vitiligoläsionen ist, stellte sich damit das cGEL Typ4 als vielversprechender Zellträger für die Kultivierung und vorgesehene Transplantation der Melanozyten heraus.