New Melanogenesis and Photobiological Processes in Activation and Proliferation of Precursor Melanocytes after UV-Exposure: Ultrastructural Differentiation of Precursor Melanocytes from Langerhans Cells

Photobiological processes involving new melanogenesis after exposure to ultraviolet (UV) light were experimentally studied in C57 black adult mice by histochemistry, cytochemistry, and autoradiography. The trunk and the plantar region of the foot, where no functioning melanocytes were present before exposure, were exposed to UV-A for 14 consecutive days. Both regions revealed a basically similar pattern for new melanogenesis which involved an activation of precursor melanocytes. Essentially all of “indeterminate” cells appeared to be precursor melanocytes, the fine structure of which could be differentiated even from poorly developed Langerhans cells. New melanogenesis was manifested by 4 stages of cellular and subcellular reactions of these cells as indicated by histochemistry of dihydroxyphenylalanine (dopa) and autoradiography of thymidine incorporation: (a) an initial lag in the activation of precursor melanocytes with development of Golgi cisternae and rough endoplasmic reticulum followed by formation of unmelanized melanosomes (day 0 to 2); (b) synthesis of active tyrosinase accumulated in Golgi cisternae and vesicles with subsequent formation of melanized melanosomes in these cells (day 3 to 5); (c) mitotic proliferation of many of these activated cells, followed by an exponential increase of new melanocytes (day 6 to 7); and (d) melanosome transfer with differentiation of 10nm filaments and arborization of dendrites, but without any significant change in the melanocyte population (day 8 to 14). The melanosome transfer was, however, not obvious until after 7days of exposure. The size of newly synthesized melanosomes was similar to that of tail skin where native melanocytes were present before exposure

Cells Containing Langerhans Granules in Human Lymph Nodes of Dermatopathic Lymphadenopathy**From the Department of Dermatology, Sapporo Medical College, Sapporo, Japan

Cells containing Langerhans granules are found in human lymph nodes of dermato-pathic lymphadenopathy. The ultrastructural characteristics of these cells are identical to the epidermal Langerhans cells of the same patients. The possible development of Langerhans cells from mesenchymal tissues is discussed

ヒト悪性黒色腫における細胞接着分子とインターロイキン2受容体の発現

This study investigated the expression of intercellular adhesion molecule-1 (ICAM-1), interleukin-2 receptor (IL-2R), integrins and E-cadherin (E-CD) in vivo and in vitro melanoma cells and their adhesion to extracellular matrix (ECM) as well as endothelial cells (HUVEC) in order to characterize the biological processes of melanoma invasion and metastasis. IL-2R was expressed in 21% of primary cutaneous melanoma, whereas 43% were positive in metastatic melanomas, suggesting that IL-2 may increase the growth of melanoma cells in certain subtypes. Integrin α2β1 was expressed highly in SK-MEL-118, and α2β1 was increased in a time- and dose-dependent manner by TPA treatment. Furthermore, the phorbol ester, TPA-induced α2β1 was mediated by calmodulin kinase, but not by protein kinase C (PKC). However, the adherence of SK-MEL-118 to HUVEC treated with TPA was decreased by 40%, suggesting that TPA affects a signaling mechanism. E-CD expression in primary melanoma did not show any correlation with the tumor invasion indexes. It was, however, indicated that primary melanoma cells adhere to each other through E-CD and that its expression correlates with invasion

正常ヒト表皮メラノサイト(NHEM)とヒト異色腫細胞株SK-MEL23の細胞増殖と分化に対する線維芽細胞培養液上清(FCM)の影響

Our goal is to study the role of dermal fibroblasts in the proliferation and differentiation of epidermal melanocytes using a reconstituted epidermal melanin unit (EMU). In order to achieve this goal, we have investigated the effect of fibroblast-conditioned medium (FCM) on the growth and melanogenesis of normal human epidermal melanocytes (NHEM) from neonatal foreskin using SK-MEL23 human melanoma cells as controls. NHEM and SK-MEL23 cells grown on type-I collagen gel alone revealed short dendrites. The culture on type-I collagen gel and fibroblasts resulted in prominent dendricities of NHEM. The FCM, which was likely to contain extracellular matrix (ECM) proteins and cytokines derived from fibroblasts, remarkably enhanced the dendricity of NHEM and their attachment to petri dishes/culture plates, but did so to a lesser degree with control SK-MEL23 cells. However, it did not affect the proliferation of both NHEM and SK-MEL23 cells. The FCM treatment decreased the tyrosinase activity of NHEM, while this decrease was not seen in melanoma cells. These studies suggest that cytokines and ECM proteins from dermal fibroblasts are important in regulating the functional and morphological differentiation of epidermal human melanocytes, and that this biological effect is much smaller in their neoplastic counter parts, melanoma cells

Some Aspects Of Melanin Biology: 1950–1975

Recent advances in the biology of mammalian pigmentation are reviewed. The multicellular epidermal melanin unit (melanocyte and associated pool of keratinocytes) rather than the melanocyte alone forms the focal point for melanin metabolism within mammalian epidermis. Within an epidermal melanin unit, melanosomes are synthesized by melanocytes and transferred to keratinocytes where they are degraded as they ascend to the epidermal surface. During the past 25 years, technical advances in biology and biochemistry have fostered a multi- disciplinary approach to research on mammalian pigmentation. Emphasizing this perspective, we have examined the current state of knowledge of the form and function of epidermal melanin units from the levels of biologic organization ranging from the molecules relevant to melanin synthesis through the skin as a totally integrated system. To an unusual degree, advances in melanin pigmentation have resulted from the integration of clinical medicine and basic science

N-propionyl-4-S-Cysteaminylphenolのアポトーシスを介した色素細胞障害効果

Phenolic thioether amines, 4-S-cysteaminylphenol (4-S-CAP) and its N-acetyl derivative, N-acetyl-4-S-CAP, are tyrosinase substrates and potent depigmenting agents for skin and hair follicles. They also possess an antitumor effect on melanoma. However, it is not clear if their biological activity derives from a cytostatic or a cytocidal effect on melanocytes. We have recently synthesized a new derivative of 4-S-CAP, N-propionyl-4-S-CAP (NPr-CAP), in order to develop a better targeted chemotherapeutic or depigmenting agent. Structurally, NPr-CAP is more lipophilic than N-acetyl-4-S-CAP, hence it is expected to have better penetration into the cells and a more potent pharmacological effect. This study is aimed to identify the selectivity and potency of the biological effect of NPr-CAP on melanocytotoxicity. NPr-CAP was found to be a good tyrosinase substrate. The i. p. administration of NPr-CAP caused dose-dependent depigmentation of black hair follicles in C57 mice. However, the black hair follicles replaced the depigmented ones in the next hair cycle, suggesting the existence of dormant or drug-resistant melanocytes. Follicular melanocytes stained positively with fluorescein isothiocyanate by TUNEL (TdT-mediated dUTP-biotin nick end labeling) method, and by using the electron microscope, showed apoptotic changes as early as 12 h post-administration of NPr-CAP. However, surrounding keratinocytes and fibroblasts revealed no changes. NPr-CAP treatment induced irreversible inhibition of cell proliferation of melan-a2, an immortal melanocyte line of C57 black mice, but this did not occur in the control albino melanocyte line, melan-c. The agarose gel electrophoresis of DNA from drug-treated melan-a2 cells showed the nucleosomal DNA ladder pattern. The MTT assay and trypan blue test indicated that NPr-CAP can cause cell death of tyrosinase-positive melan-a2 cells, whereas, it transiently inhibits the proliferation of tyrosinase-negative melan-c cells. Thus, we propose that the melanocytotoxicity of NPr-CAP is selective and causes apoptosis in melanocytes through the interaction with active tyrosinase. (Received February 27, 1998 and accepted March 10, 1998

Biological and Biochemical Studies on Melanogenesis and M elanoma Cells

For the previous ten years, we have been studying intracellular transport of melanosoma1 proteins and their biological and biochemical functions in melanoma cells. Melanosoma1 proteins,tyrosinase, tyrosinase-related protein 1 (TYRP1) and TYRP2/DOPAchrome tautomerase (DCT), gp100/Pme117 are transported from the trans-Golgi network (TGN) to earlystage melanosomes via endosoma1 compartments. We suggest that Rab7 is involved in the vesicular transport of tyrosinase and TYRP1 and in the melanogenesis through the regulation of gp100/Pme117 maturation. TYRP1 and TYRP 2/DCT were shown to play an essential role in suppressing TYR-mediated cytotoxicity in melanocytic cells, possibly through interaction with TYR in melanosomes. We also studied apoptotic cell death of melanoma cells and death mediators. Among p53 family members, p51A (p63) induced apoptosis in both wild-type and mutant p53-expressing melanoma cells more significantly than p53 and 73β. Interferon(IFN) exerts anti-tumor activities possibly by regulating IFN-stimulated genes. Caspase-2 activation was commonly associated with induction of apoptosis in IFN一β一sensitive melanoma cells. The diacylglycero1 kinase (DGK) , expressed in several human melanoma cell lines but not in melanocytes, was a novel positive regulator of NF-KB, which suppresses TNF一α一induced melanoma cell apoptosis

Growth Inhibition of Re-Challenge B16 Melanoma Transplant by Conjugates of Melanogenesis Substrate and Magnetite Nanoparticles as the Basis for Developing Melanoma-Targeted Chemo-Thermo-Immunotherapy

Melanogenesis substrate, N-propionyl-cysteaminylphenol (NPrCAP), is selectively incorporated into melanoma cells and inhibits their growth by producing cytotoxic free radicals. Magnetite nanoparticles also disintegrate cancer cells and generate heat shock protein (HSP) upon exposure to an alternating magnetic field (AMF). This study tested if a chemo-thermo-immunotherapy (CTI therapy) strategy can be developed for better management of melanoma by conjugating NPrCAP on the surface of magnetite nanoparticles (NPrCAP/M). We examined the feasibility of this approach in B16 mouse melanoma and evaluated the impact of exposure temperature, frequency, and interval on the inhibition of re-challenged melanoma growth. The therapeutic protocol against the primary transplanted tumor with or without AMF exposure once a day every other day for a total of three treatments not only inhibited the growth of the primary transplant but also prevented the growth of the secondary, re-challenge transplant. The heat-generated therapeutic effect was more significant at a temperature of 43°C than either 41°C or 46°C. NPrCAP/M with AMF exposure, instead of control magnetite alone or without AMF exposure, resulted in the most significant growth inhibition of the re-challenge tumor and increased the life span of the mice. HSP70 production was greatest at 43°C compared to that with 41°C or 46°C. CD8+T cells were infiltrated at the site of the re-challenge melanoma transplant

N-Propionyl-Cysteaminylphenol-Magnetite Conjugate (NPrCAP/M) Is a Nanoparticle for the Targeted Growth Suppression of Melanoma Cells

A magnetite nanoparticle, NPrCAP/M, was produced for intracellular hyperthermia treatment of melanoma by conjugating N-propionyl-cysteaminylphenol (NPrCAP) with magnetite and used for the study of selective targeting and degradation of melanoma cells. NPrCAP/M, like NPrCAP, was integrated as a substrate in the oxidative reaction by mushroom tyrosinase. Melanoma, but not non-melanoma, cells incorporated larger amounts of iron than magnetite from NPrCAP/M. When mice bearing a B16F1 melanoma and a lymphoma on opposite flanks were given NPrCAP/M, iron was observed only in B16F1 melanoma cells and iron particles (NPrCAP/M) were identified within late-stage melanosomes by electron microscopy. When cells were treated with NPrCAP/M or magnetite and heated to 43°C by an external alternating magnetic field (AMF), melanoma cells were degraded 1.7- to 5.4-fold more significantly by NPrCAP/M than by magnetite. Growth of transplanted B16 melanoma was suppressed effectively by NPrCAP/M-mediated hyperthermia, suggesting a clinical application of NPrCAP/M to lesional therapy for melanoma. Finally, melanoma cells treated with NPrCAP/M plus AMF showed little sub-G1 fraction and no caspase 3 activation, suggesting that the NPrCAP/M-mediated hyperthermia induced non-apoptotic cell death. These results suggest that NPrCAP/M may be useful in targeted therapy for melanoma by inducing non-apoptotic cell death after appropriate heating by the AMF